Kit Granby

Nikoline Juul Nielsen

Rikke V Hedegaard

Tue Christensen

Mette Kann

Leif H Skibsted

Granby

Läntmannen Schulstad

A / S Skibsted

Acrylamide in relation to asparagine in baked

Keywords: Acrylamide, asparagine, bread, rye, wheat, toast, dietary, intake 15 16

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 17

The presence of acrylamide in heat treated food was recognized in 2002 [START_REF] Tareke | Analysis of acrylamide, a 419 carcinogen formed in heated foodstuffs[END_REF] and the 18 same year it was clear that acrylamide is formed mainly from asparagine and a carbonyl compound 19 through Maillard browning reactions during food processing (Mottram et al. 2002;Stadler et al. 20 2002). Several pathways and intermediates for acrylamide formation have been suggested including 21 the intermediates: Schiffs bases, decarboxylated Amadori products (Yaylayan 2003), Strecker 22 aldehydes (Mottram et al. 2002) and the deamination product 3-aminopropionamide which also 23 generate acrylamide under aqueous conditions (Granvogl and Schieberle, 2006). Besides the main 24 reaction routes through the Maillard reactions, acrylamide may be formed in bread after protein 25 pyrolysis of added gluten (Claus et al. 2006). Also addition of cheese to bread has been found to 26 increase the acrylamide formation, probably formed with 3-aminopropionamide as an intermediate 27 in the cheese, which has been found to generate acrylamide [START_REF] Deleted ; Granvogl | Influence of sulfur fertilization on the amounts of free amino acids in Wheat. Correlation with baking properties as well as with 3-aminopropionamide and acrylamide generation during baking[END_REF]Schieberle 2006, 28 Hedegaard et al. 2008). 29 Acrylamide is known to be carcinogenic in rodents and is classified by the International Agency for 30

Research on Cancer as probably carcinogenic to humans (IARC 1994). The dietary acrylamide 31 intake have been estimated to e.g. 0.3-0.8µg (kg bw) -1 day -1 (WHO 2002); appr. 0.5µg (kg bw) -1 32 day -1 [START_REF] Svensson | Dietary 413 intake of acrylamide in Sweden[END_REF]. The dietary intake of acrylamide from bread is about 11% of the total 33 dietary acrylamide intake of 31µg day -1 as estimated for the Swedish population. A similar 34 contribution from bread related foods such as crisp bread and biscuits are estimated (Svensson et al. 35 2003). The acrylamide level in baked bread is relatively low, but due to high consumption of bread 36 the contribution of acrylamide from bread is still important. 37

Toasting the bread, which is frequently used e.g. for breakfast meals, increases acrylamide 38 significantly. While untoasted bread contains <50 µg kg -1 the acrylamide content may increase to a 39 few to several hundreds micrograms per kilo bread after toasting (Konings et al. 2003;Becalski et 40 al. 2003;Ahn et al. 2002). The acrylamide levels have been found to increase with increasing 41 toasting time and whole meal rye bread was found to form more acrylamide than white wheat bread 42 (Ahn et al. 2002). 43

The occurrence of acrylamide in bread may depend on several factors. [START_REF] Surdyk | Effects of asparagine, fructose, and baking 410 conditions on acrylamide contents in yeast-leavened wheat bread[END_REF] showed 44 that acrylamide in baked wheat bread correlated with the amount of asparagine added to dough, 45 while the added reducing sugar fructose did not show a similar correlation, hence the asparagine 46 was a limiting substrate for the acrylamide formation. The clear relationship between asparagine 47 Deleted: 7a 1 (dw) compared to whole grain wheat flour i.e. 0.5 g kg -1 (dw) or wheat bran. Rye generally contains 59 more asparagine: sifted rye flour 0.6 g kg -1 (dw) and whole grain rye flour: 1.1 g kg -1 (dw). Not only 60 milling has been found to influence the levels of the limiting substrate asparagine. It was found that 61 yeast fermentation of wheat dough depleted a major part of the asparagine (>80%), while 62 sourdough fermentation of rye dough reduced the asparagine content to a smaller extent 63 (Fredriksson et al. 2004). During baking, parameters such as water activity, heating time and 64 temperature, surface to volume ratio, the presence of additives and pH also influence acrylamide 65 levels [START_REF] Surdyk | Effects of asparagine, fructose, and baking 410 conditions on acrylamide contents in yeast-leavened wheat bread[END_REF][START_REF] Rydberg | Investigations 400 of factors that influence the acrylamide content of heated foodstuffs[END_REF]. Acrylamide formation may increase when 66 increasing pH e.g. by using baking soda or ammonium carbonate for bread leavening (Amrein et al. 67 2005) as it will decrease when lowering pH (Jung et al. 2003;Pedreschi et al. 2004Pedreschi et al. , 2005)). 68

Fermentation will cause pH in dough to drop due to release of carbon dioxide [START_REF] Raccach | Natural 398 fermentation of wheat flours[END_REF]) 69 and it will reach pH~5 using yeast and pH~4.4-4.8 using sourdough [START_REF] Suhr | Effect of weak acid preservatives on growth of bakery product 407 spoilage fungi at different water activities and pH values[END_REF]. Comment [k1]: Left out to shorten the introduction Deleted: Favorable light and temperature conditions one year gave higher acrylamide in bread compared to another year probably by enhancing amino acid and protein contents. Flour derived from sulphur deprived wheat was also found to contain high levels of asparagine [START_REF] Muttucumaru | ). 120 121 202 203 Acrylamide in baked and toasted bread in relation to asparagine 204 Baked wheat bread slices contained <5 µg kg -1 acrylamide (<LOD), medium toasted 11-130 µg kg -1 205 acrylamide and hard toasted 15-161 µg kg -1 acrylamide for asparagine levels in flour of 0.14-0.60 g 206 kg -1 (natural background content to content after highest asparagine addition level). The 207 corresponding figures for the rye bread slices were: baked 7-23 µg kg -1 acrylamide, medium toasted 208 27-155 µg kg -1 acrylamide; hard toasted 68-205 µg kg -1 acrylamide for asparagine levels in flour of 209 0[END_REF], Granvogl & Schieberle 2007), so it is important that the cereals are grown at a sufficient sulfur level Deleted: . The aim of the present study was to find the relationship between asparagine levels and acrylamide 71 formation in wheat bread and rye bread during baking and toasting in order to be able to reduce the 72 acrylamide levels. Furthermore to assess the acrylamide levels in bread on the Danish market and to 73 estimate the dietary intake af acrylamide from wheat and rye bread including toasted bread. 74 75

Materials and methods 76 77

Baking wheat bread and rye bread with asparagine additions 78 An industrial bakery (Lantmännen Schulstad A/S, Hvidovre, Denmark) made the wheat and rye 79 breads in their laboratory bakery with different amounts of aparagine added (in August 2004). The 80 experimental design appears from Figure 1. Four batches of wheat dough were prepared and two 81 breads selected from each batch: One batch of dough was made without adding asparagine 82 representing the natural background asparagine level and three batches were added asparagine at 83 concentration levels of 0.05, 0.13 and 0.27 g kg -1 dough. As the dough contains 60% wheat flour, 84 the added amounts of asparagine based on flour were 0.09, 0.22 and 0.44 g kg -1 corresponding to 85 addition at approximately 0.6 -3 times the actual background level of asparagine. The recipe for a 86 wheat bread (appr. 800 g dough) included sifted wheat flour, yeast, salt, sugar vegetable oil and 87 water. Kneading time was 7 min., holding time 20 min. and resting time at 32°C was 45 min. 88

Baking was performed initially at 230°C for 2 min. and subsequently at 200°C for 30 min. 89 Rye breads were made using the same experimental design. The asparagine was added to the dough 90 at concentrations based on rye flour of 0.17, 0.43 and 1.75 g kg -1 respectively corresponding to 91 addition at 0.2 -2 times the natural background level. The rye bread (based on appr. 900 g dough) 92

was made from half and half mixing of sourdough and rye flour and addition of yeast, salt and 93 water. Kneading time was 10 min, holding time 20 min, and resting time at 37°C was 77 min. 94 The chemical analyses were performed using a recently developed LC-MS/MS method for 120 simultaneous analysis of acrylamide, asparagine and reducing sugars (Nielsen et al. 2005) and in 121 addition some of the acrylamide analyses were made with another LC-MS/MS method as described 122 in Pedreschi et al. (2005). 123 Sliced toasted or untoasted wheat or rye bread was weighed, dried in an oven at 50°C over night 124 and homogenized using a Braun handheld mixer (type 4169) fitted with a blender like sample 125 compartment (type 4297) (Braun AG, Frankfurt/m, Germany). The drying temperature was set 126 relatively low in order to avoid acrylamide formation during the drying process. The wet (semidry) 127 and dry weight was determined from weighing before and after drying of the baked and toasted 128 bread slices. Aliquots of 4 g of homogenate was extracted with MilliQ water after adding 129 internalstandards: 200 µL 10 µg mL -1 D 3 -acrylamide, 100 µL 880 µg mL -1 15 N 2 -asparagine. The 130 sample was extracted by an Ultra Turrax mixer. After centrifuging, SPE cleanup was performed by 131 an automated sampler using LiChroLut RP-C18 SPE-cartridges LC-MS/MS detection was 132 performed on a triple quadrupole instrument (Micromass Quattro Ultima) after separation on a 133 hypercarb LC-column (dimensions 2.1 mm x 100 mm, particle size 5 µm) eluted with 0.1% 134 aqueous formic acid at 0.2 mL min -1 . The mass spectrometer was operated in ESI + for acrylamide 135 and saccharides and in ESI -for asparagine. Capillary voltages of 3 kV (ESI + ) and 2 kV (ESI -) were 136 applied but for simultaneous detection in positive and negative mode a lower capillary voltage, 137 e.g.1 kV is recommended to prevent sparkling from the capillary. 138 Some of the acrylamide analysis was performed after the methods were optimized to detect low 139 concentrations (e.g. by increasing the HPLC injection volume and by optimizing the MS 140 performance before analysing the bread samples), so the method was able to quantify down to 5 µg 141 kg -1 . The repeatability and reproducibility of the acrylamide analyses at 50 and 250 µg kg -1 (n=24) 142 were 6-17% and 9-29% respectively. The laboratory has participated in proficiency tests for 143 acrylamide in food and all results obtained have been with satisfactory test scores, including the 144 results obtained in an EU validation study on acrylamide analyses in food with 22 samples (11 145 matrices including bread and toasted bread: assigned value 38µg kg -1 , obtained results 41 and 44µg 146 kg -1 ) [START_REF] Wenzl | Collaborative trial 421 validation study of two methods, one based on high performance liquid chromatography-tandem 422 mass spectrometry and on gas chromatography-mass spectrometry for the determination of 423 acrylamide in bakery and potato products[END_REF]. The detection limit of the asparagine analyses was 0.002 g kg -1 , the 147 repeatability and reproducibility measured at 0.02, 0.1 and 0.4 g kg -1 0-5% and 10-18% repectively. The levels of acrylamide and asparagine in wheat and rye bread slices after baking and medium or 160 hard toasting appear from Table I. The results presented are all means of six bread slices, each 161 wheat bread slice weighing 34±2 g and each rye bread slice weighing 32±1 g. 162 163

Asparagine in flour and bread slices after asparagine additions to dough and subsequent 164 fermentation, baking and toasting 165

The background content of asparagine in sifted wheat flour was 0.14 ± 0.004 g kg -1 (based on three 166 determinations and with the precision measured as 95% confidence levels) and in whole grain rye 167 flour 0.76 ± 0.02 g kg -1 , figures comparable to those reported by Frederiksson et al. (2004) of 0.14-168 0.17 g kg -1 (dw) in sifted wheat flour and 1.1 g kg -1 (dw) in whole grain rye flour. 169

The levels of asparagine added to the wheat dough were 0.6, 1.6 and 3.1 times the natural 170 background level and to the rye dough 0.2, 0.6 and 2.3 times the natural background level. The 171 highest concentration after addition correspond to 0.6 g kg -1 asparagine in wheat flour and 2.5 g kg -1 172 asparagine in rye flour; hence levels likely to be present in cereal products used for bread 173 production. 174

In Table II the reacted amounts of asparagine in the batches with different asparagine levels upon 175 fermentation, baking and toasting are presented. Some asparagine had reacted during wheat bread 176 production as can be seen from data determined on a dry weight basis (Table I), where differences 177 from evaporation of water during toasting does not have to be considered. In the wheat batch with a 178 natural background level of asparagine 12% was left after fermentation and baking. Upon hard 179 toasting all asparagine in the wheat bread slices with the natural background content and the lowest 180 addition level of asparagine had reacted. 181 182 Table I 183 Table II 184 185 In rye bread the relative reduction in asparagine during sourdough fermentation and baking was less 186 than for the yeast fermented wheat bread, e.g. 18% at the natural background level and 9% for the 187 highest addition level of asparagine. As the results are based on subtracting two large percentages, 188 they may be more uncertain. The results are in accordance with those of Frederiksson et al. (2004) A general conclusion may be that rye flour contains more asparagine than wheat flour and 193 sourdough fermentation and baking of rye dough reduces asparagine to a smaller extent than yeast 194 fermentation and baking of wheat dough. Prolonging or optimizing the fermentation process, 195 especially the yeast fermentation of wheat bread may be an efficient tool to reduce the acrylamide 196 formation in bread products. 197 198 199 Acrylamide in baked and toasted bread in relation to asparagine 200 Baked wheat bread slices contained <5 µg kg -1 acrylamide (<LOD), medium toasted 11-130 µg kg -1 201 acrylamide and hard toasted 15-161 µg kg -1 acrylamide for asparagine levels in flour of 0.14-0.60 g 202 kg -1 (natural background content to content after highest asparagine addition level). The 203 corresponding figures for the rye bread slices were: baked 7-23 µg kg -1 acrylamide, medium toasted 204 27-155 µg kg -1 acrylamide; hard toasted 68-205 µg kg -1 acrylamide for asparagine levels in flour of 205 0.76-2.5 g kg -1 . 206

In Figure 2 acrylamide concentrations in wheat and rye bread, initially baked with different 207 asparagine addition levels, and after baking toasted to medium or hard level are plotted and marked 208 with correlation coefficients. Due to the low acrylamide contents in the baked wheat bread slices no 209 correlation was found between asparagine content in dough and acrylamide content in baked wheat 210 bread. However, acrylamide contents in wheat bread correlated well with the asparagine levels 211 added to the dough for medium (r 2 = 0.86) and hard toasted bread slices (r 2 = 0.87). As smaller 212 proportions of asparagine were consumed during yeast fermentation and baking for the higher 213 asparagine additions the acrylamide contents increased exponentially with increasing asparagine 214 levels (quadratic equations). 215

The untoasted rye bread slices contained naturally 0.62 g kg -1 (dw) asparagine , about 36 times 216 more than the wheat bread slices. As for the wheat bread quadratic relationships between the 217 In the present study, a major part of the asparagine added to the wheat dough had reacted during 223 preparation of the bread. It is known, that especially yeast fermentation promote elimination of 224 asparagine, and that the reduction depends on e.g. time of fermentation, the amount of yeast added 225 and pH (Frederiksson et al. 2004). When comparing the asparagine levels left in baked bread before 226 toasting with the acrylamide content in toasted bread slices good linear relationships are present for 227 both medium toasted (r 2 = 0.87) and hard toasted (r 2 = 0.94) wheat bread and for asparagine levels 228 of 0.017-0.29 g kg -1 (dw) bread. The asparagine levels in baked rye bread also showed good linear 229 correlations with acrylamide formed after medium toasting (r 2 = 0.90) and hard toasting (r 2 = 0.76). 230 231 Figure 2 232 233 Although it is not possible directly to compare between acrylamide formation in wheat and rye 234 bread having different slice thicknesses, water contents and asparagine levels it seems as the rate of 235 acrylamide formation is somewhat higher in wheat bread compared to rye bread. This may be 236 explained by lower pH conditions in sourdough fermented rye bread compared to yeast fermented 237 wheat bread (pH appr. 0.5 units lower in sourdough). A better retention of water in the bread when 238 toasting rye bread compared to wheat bread may also reduce the acrylamide formation. The average 239 water content in hard, medium and untoasted rye bread slices were 24±2%, 26±2% and 41±1% 240 while the average water content in hard, medium and untoasted wheat bread slices were somewhat 241

Deleted: sourdough Deleted: 91 Deleted: 5 Deleted: In the initial setup of the experiment, the natural asparagine level in rye was estimated too low, why the two lowest addition levels are close to the background level i.e. at only 1.4 and 1.6 times the actual asparagine background level in flour. For untoasted rye bread the asparagine additions all showed significantly higher acrylamide content compared to the background (p < 0.05) but the correlation between asparagine addition and acrylamide in baked bread is weaker (r 2 = 0.45). The medium and hard toasted rye bread were significantly higher in acrylamide for all asparagine additions (p < 0.005).

Deleted: formed

Deleted: Acrylamide content in baked bread was found to be proportional to the asparagine content in dough and the acrylamide content in toasted bread was likewise proportional to the asparagine content in bread before toasting. However Deleted: is different Deleted: In wheat and rye bread toasted for 3.1 min., representing the hard toasted wheat bread and the medium toasted rye bread, the ratio of acrylamide units in the toasted bread per unit asparagine in the untoasted bread was found to be higher in wheat bread (6.7 * 10 -4 ) compared to rye bread (0.89 * 10 -4 ) that is the ratio of acrylamide formation in wheat bread was seven times higher than in rye bread.

Deleted: Lindenmeier & Hofmann (2004) showed that due to their lower pH value (~4.0) sourdough fermented breads contained higher amounts of the key antioxidant in bread crust pronyl-L-lysine than breads that have been yeastfermented only. Hence besides the lower acrylamide formation rate at lower pH, the increased antioxidant content in the crust of rye bread may further reduce acrylamide formation, as some antioxidants have an inhibitory effect on the acrylamide formation. Addition of the lysine-rich casein (lysine reduces the acrylamide yield) to the bread recipe also increased the amounts of antioxidant [START_REF] Hedegaard | Influence of baking conditions and precursor supplementation on the amounts of the oxidant pronyl-L-lysine in bakery products[END_REF][START_REF] Pedreschi | Effect of amino acids on acrylamide formation and elimination kinetics[END_REF]. Hence the effect of lysine may be from increasing the amounts of antioxidants with inhibitory effects. ¶ Deleted: contribute to the lower reaction rate of acrylamide from asparagine when toasting rye bread in comparison to wheat bread, as higher water content will reduce the acrylamide formation. which may explain the exponential increase in acrylamide contents (as the water content are 249 reduced) generally found during thermally processing of food at a fixed temperature. It was also 250 found in other model systems that acrylamide formation increased when lowering the water content 251 [START_REF] Schieberle | New aspects on the formation and analysis of 403 acrylamide[END_REF], Blank et al. 2005) . 252

Deleted

The proportions of asparagine in wheat bread reacting to give acrylamide were also estimated 253 (Table III). Generally the ratio is higher in medium toasted bread suggesting that at higher 254 temperatures and/or longer duration of toasting precursors are depleted faster, reacting faster in 255 competing reactions and/or acrylamide is eliminated to a larger extent by subsequent reaction. For 256 the hard toasted bread is seen, that as depletion of the asparagine pool approaches, the formation of 257 acrylamide diminishes, resulting in lower ratios at lower asparagine addition levels. At the low 258 asparagine levels in wheat bread asparagine was depleted after hard toasting while at higher 259 addition levels some asparagine was left. This may be interpreted as if asparagine was the limiting 260 precursor at low asparagine levels while at higher levels other factors also influenced the 261 acrylamide formation. However the other precursor the reducing sugars was not depleted (data not 262 shown). 2005) found in a model system that the amount of water had a significant effect on the yields of acrylamide, however the formation of acrylamide needs a certain amount of water to form acrylamide, and that 25% water content gave higher acrylamide yields than 10% water content. However the optimum could be between the two water percentages. Blank et al. (2005) In order to estimate the level of acrylamide in rye bread, in 2004 13 sifted and whole meal rye 273 breads produced for or sold on the Danish market were sampled and analysed. They contained 12 ± 274 5 µg kg -1 acrylamide (range <5-19 µg kg -1 , n=13). 275

In 2005 ten samples of wheat breads, produced for or sold on the Danish market were analysed, 276 containing 10 ± 6 µg kg -1 acrylamide (range <5-22 µg kg -1 ). 277

The contents of acrylamide in bread found in the present study are comparable with the results 278 found in other studies. Becalski et al. (2003) found 290 µg kg -1 of acrylamide in toasted wheat bread 279 compared to 19 µg kg -1 in untoasted wheat bread (n=2). Light toasted rye bread contained 28 µg kg - 280 1 acrylamide and untoasted rye bread 17 µg kg -1 . Eerola et al. (2007) found that toasted wheat bread 281 contained 111±10 µg kg -1 acrylamide (n=5) and untoasted wheat bread <68 µg kg -1 acrylamide, 282 which was the limit of detection (n=5). Ahn et al. (2002) found that acrylamide increased with 283 longer toasting time. After 3.1 min. of toasting when the toasts were still considered eatable wheat 284 bread contained appr. 60 µg kg -1 and rye bread contained appr. 80 µg kg -1 acrylamide. After 6 285 minutes wheat bread contained appr. 206 µg kg -1 and rye bread contained appr. 220 µg kg -1 286 acrylamide. Konings et al. (2003) reported acrylamide contents in toasted bread of 183 ± 336 µg kg - 287 1 (range < 30-1430 µg kg -1 , n=17). The contents in dark rye bread were 44 ± 13 µg kg -1 (range 30-288 60 µg kg -1 , n=5) and in wheat bread < 30 µg kg -1 (n=6). The average dietary intake of wheat bread in Denmark is 125 g day -1 and in addition Danes has a 292 tradition for eating whole meal sourdough fermented rye bread as part of nearly every lunch meal 293 comprising an average daily intake of 59 g rye bread day -1 (Lyhne et al. 2005) In case all bread is 294 consumed without toasting and the mean figures for acrylamide in bread is used: 10 µg kg -1 in 295 wheat bread and 12 µg kg -1 in rye bread, the daily intake comprises 1.3 µg from wheat bread and 296 0.7 µg from rye bread, respectively. The daily intake of acrylamide from wheat and rye bread 297 comprising 2.0 µg is about 8 % of the average daily acrylamide intake of appr. 25 µg day -1 298 estimated for Danes (Figure 4). The acrylamide intake from bread is relatively low compared to 299 other surveys, estimating e.g. 3.4 µg [START_REF] Svensson | Dietary 413 intake of acrylamide in Sweden[END_REF]) and 3.6 µg (Matthys et al. 2005). An 300 explanation for the low Danish dietary acrylamide intake from bread may be that most Danish bread 301 are produced using either yeast fermentation or sourdough fermentation, hence the contents of the 302 precursor asparagine are reduced during preparation of the breads. Another explanation for the low 303 acrylamide intake from bread may be that in previous studies on acrylamide in bread the limit of 304 detection (LOD) was higher and the levels in bread often below the LOD. Hence in many cases 305 LOD was used as the acrylamide level in bread. Finally in the present estimate, as the Danish 306 Nationwide dietary survey do not have information on how frequently people eat toasted bread, the 307 acrylamide intake from toasted bread is not included in the dietary acrylamide intake from bread. 308 However, in case the average daily bread intake includes two hard toasted wheat bread slices 309 (acrylamide 161 µg kg -1 (ww)) each of 28 g and with the high pre-processing asparagine level of 0.6 310 g kg -1 (dw) which is about the concentration found in wholegrain wheat flour i.e. 0.5 g kg -1 311 (Frederiksson et al. 2004), the daily acrylamide intake is estimated to 9 µg (36 % of the average 312 Danish acrylamide intake). If a consumer eats an average amount of bread of which two slices of 25 313 g rye bread are hard toasted with asparagine and acrylamide concentrations of 1.2 g kg -1 and 123 µg 314 kg -1 respectively, the daily acrylamide intake from bread comprises 7 µg (28 % of the average The contents of acrylamide in baked rye bread and in toasted wheat and rye bread are correlated to 325 the contents of asparagine in the cereal product used for the bread production and at asparagine 326 concentration ranges that may occur in grain products for bread production. The content of 327 asparagine in the dough for wheat bread preparation is reduced considerably during bread 328 production. After 65 min. raising time and subsequent baking, the asparagine content in the baked 329 wheat bread was reduced to 12% compared to the content in the flour before yeast fermentation. 330

The sourdough fermentation of rye bread showed a modest asparagine reduction to 82% of the 331 content in the flour. This is probably due to the more acidic conditions in sourdough fermented rye 332 bread compared to yeast fermented wheat dough. The contents of acrylamide formed in toasted 333 wheat bread slices and toasted rye bread slices are proportional to the asparagine concentration of 334 the untoasted bread. The dietary intake of acrylamide from untoasted bread of 2.0 µg day -1 is 335 relatively low but the dietary acrylamide intake increases somewhat for people eating toasted bread. 336 337

Ackowledgement 338

Deleted: s Deleted: s Deleted: also Deleted: The formation of acrylamide in toasted bread per unit of asparagine in the untoasted bread was appr. seven times higher in wheat bread than in rye bread. Acrylamide in baked and toasted wheat and rye bread was studied in relation to the content of 5 asparagine in flour, dough, bread and toasted bread. Asparagine was consumed during bread 6 preparation resulting in a reduced acrylamide contents in the products. In wheat bread 12% of the 7 asparagine initially present in the flour (0.14 g kg -1 ) was left after yeast fermentation and baking, 8 while for rye bread 82% of the asparagine was left after sourdough fermentation and baking. The 9 asparagine present in the untoasted wheat bread had all reacted after hard toasting. Toasted wheat 10 and rye bread slices contained 11-161 µg kg -1 and 27-205 µg kg -1 acrylamide compared to untoasted 11 wheat and rye bread containing <5 µ g kg -1 and 7-23 µg kg -1 acrylamide. The dietary intake of 12 acrylamide from bread (untoasted) of 2 µg/day is relatively low, however the acrylamide exposure 13 from bread increases several times for people eating toasted bread. 14 15 Keywords: Acrylamide,asparagine,bread,rye,wheat,toast,dietary,intake 16 17

Introduction 18
The presence of acrylamide in heat-treated food was recognized in 2002 [START_REF] Tareke | Analysis of acrylamide, a 419 carcinogen formed in heated foodstuffs[END_REF] and 19 the same year it was clear that acrylamide is formed mainly from asparagine and a carbonyl 20 compound through Maillard browning reactions during food processing (Mottram et al. 2002;21 Stadler et al. 2002). Several pathways and intermediates for acrylamide formation have been 22

suggested including the intermediates: Schiffs bases, decarboxylated Amadori products (Yaylayan 23 2003), Strecker aldehydes (Mottram et al. 2002) and the deamination product 3-aminopropionamide 24 which also generate acrylamide under aqueous conditions (Granvogl and Schieberle, 2006) the main reaction routes through the Maillard reactions, acrylamide may be formed in bread after 26 protein pyrolysis of added gluten (Claus et al. 2006). Also addition of cheese to bread has been 27

found to increase the acrylamide formation, probably formed with 3-aminopropionamide as an 28 intermediate in the cheese, which has been found to generate acrylamide (Granvogl and Schieberle 29 2006, Hedegaard et al. 2008). 30 Acrylamide is known to be carcinogenic in rodents and is classified by the International Agency for 31

Research on Cancer as probably carcinogenic to humans (IARC 1994). The dietary acrylamide 32 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 33 day -1 [START_REF] Svensson | Dietary 413 intake of acrylamide in Sweden[END_REF]. The dietary intake of acrylamide from bread is about 11% of the total 34 dietary acrylamide intake of 31 µg day -1 as estimated for the Swedish population. A similar 35 contribution from bread related foods such as crisp bread and biscuits are estimated (Svensson et al. 36 2003). The acrylamide level in baked bread is relatively low, but due to high consumption of bread 37 the contribution of acrylamide from bread is still important. 38

Toasting the bread, which is frequently used e.g. for breakfast meals, increases acrylamide 39 significantly. While untoasted bread contains <50 µg kg -1 the acrylamide content may increase to a 40 few to several hundreds micrograms per kilo bread after toasting (Konings et al. 2003;Becalski et 41 al. 2003;Ahn et al. 2002). The acrylamide levels have been found to increase with increasing 42 toasting time and whole meal rye bread was found to form more acrylamide than white wheat bread 43 (Ahn et al. 2002). 44 45The occurrence of acrylamide in bread may depend on several factors. [START_REF] Surdyk | Effects of asparagine, fructose, and baking 410 conditions on acrylamide contents in yeast-leavened wheat bread[END_REF] showed 46 that acrylamide in baked wheat bread correlated with the amount of asparagine added to dough, 47 while the added reducing sugar fructose did not show a similar correlation, hence the asparagine 48 was a limiting substrate for the acrylamide formation. The clear relationship between asparagine 49 content in the dough and acrylamide formation has also been demonstrated in other wheat and rye 50 bread model systems (Elmore et al. 2005;Bråthen et al. 2005). One of the strategies to reduce 51 acrylamide in cereal products may be to choose raw materials low in asparagine by selecting 52 optimum cultivars and growth conditions. However, variations in free asparagine in cereals are 53 modestly researched. Claus et al. 2006 investigated agronomic factors related to the acrylamide 54 formation in cereal products. They showed that nitrogen fertilization resulted in elevated amino acid 55 levels resulting in increased acrylamide levels. The asparagine contents of selected European wheat 56 varieties ranged from 0.07-0.66 g kg -1 , that is a factor of five between varieties and a factor of two 57 within varieties [START_REF] Taeymans | A review of acrylamide: An industry perspective on research, analysis, 417 formation and control[END_REF]. 58 59 Milling will differentiate the asparagine levels in the different milling fractions. Fredriksson et al. 60 (2004) found that the contents of free asparagine are lower in sifted wheat flour i.e. 0.14-0.17 g kg - 61 1 (dw) compared to whole grain wheat flour i.e. 0.5 g kg -1 (dw) or wheat bran. Rye generally contains 62 more asparagine: sifted rye flour 0.6 g kg -1 (dw) and whole grain rye flour: 1.1 g kg -1 (dw). Not only 63 milling has been found to influence the levels of the limiting substrate asparagine. It was found that 64 yeast fermentation of wheat dough depleted a major part of the asparagine (>80%), while 65 sourdough fermentation of rye dough reduced the asparagine content to a smaller extent 66 (Fredriksson et al. 2004). During baking, parameters such as water activity, heating time and 67 temperature, surface to volume ratio, the presence of additives and pH also influence acrylamide 68 levels [START_REF] Surdyk | Effects of asparagine, fructose, and baking 410 conditions on acrylamide contents in yeast-leavened wheat bread[END_REF][START_REF] Rydberg | Investigations 400 of factors that influence the acrylamide content of heated foodstuffs[END_REF]. Acrylamide formation may increase when 69 increasing pH e.g. by using baking soda or ammonium carbonate for bread leavening (Amrein et al. 70 2005) as it will decrease when lowering pH (Jung et al. 2003;Pedreschi et al. 2004Pedreschi et al. , 2005)). 71 Fermentation will cause pH in dough to drop due to release of carbon dioxide [START_REF] Raccach | Natural 398 fermentation of wheat flours[END_REF]) 72 and it will reach pH~5 using yeast and pH~4.4-4.8 using sourdough [START_REF] Suhr | Effect of weak acid preservatives on growth of bakery product 407 spoilage fungi at different water activities and pH values[END_REF]. 73 74The aim of the present study was to find the relationship between asparagine levels and acrylamide 75 formation in wheat bread and rye bread during baking and toasting in order to be able to reduce the 76 acrylamide levels. Furthermore to assess the acrylamide levels in bread on the Danish market and to 77 estimate the dietary intake of acrylamide from wheat and rye bread including toasted bread. 78 79

Materials and methods 80

Baking wheat bread and rye bread with asparagine additions 81 An industrial bakery (Lantmännen Schulstad A/S, Hvidovre, Denmark) made the wheat and rye 82 breads in their laboratory bakery with different amounts of asparagine added (in August 2004). The 83 experimental design appears from Figure 1. Four batches of wheat dough were prepared and two 84 breads selected from each batch: One batch of dough was made without adding asparagine 85 representing the natural background asparagine level and three batches were added asparagine at 86 concentration levels of 0.05, 0.13 and 0.27 g kg -1 dough. As the dough contains 60% wheat flour, 87 the added amounts of asparagine based on flour were 0.09, 0.22 and 0.44 g kg -1 corresponding to 88 addition at approximately 0.6 -3 times the actual background level of asparagine. The recipe for a 89 wheat bread (appr. 800 g dough) included sifted wheat flour, yeast, salt, sugar vegetable oil and 90 water. Kneading time was 7 min., holding time 20 min. and resting time at 32°C was 45 min. 91

Baking was performed initially at 230°C for 2 min. and subsequently at 200°C for 30 min. 92 93 Rye breads were made using the same experimental design. The asparagine was added to the dough 94 at concentrations based on rye flour of 0.17, 0.43 and 1.75 g kg -1 respectively corresponding to 95

Chemical analysis 122

The chemical analyses were performed using a recently developed LC-MS/MS method for 123 simultaneous analysis of acrylamide, asparagine and reducing sugars (Nielsen et al. 2005) and in 124 addition some of the acrylamide analyses were made with another LC-MS/MS method as described 125 in Pedreschi et al. (2005). 126 Sliced toasted or untoasted wheat or rye bread was weighed, dried in an oven at 50°C over night 127 and homogenized using a Braun handheld mixer (type 4169) fitted with a blender like sample 128 compartment (type 4297) (Braun AG, Frankfurt/m, Germany). The drying temperature was set 129 relatively low in order to avoid acrylamide formation during the drying process. The wet (semidry) 130 and dry weight was determined from weighing before and after drying of the baked and toasted 131 bread slices. Aliquots of 4 g of homogenate was extracted with MilliQ water after adding 132 internalstandards: 200 µL 10 µg mL -1 D 3 -acrylamide, 100 µL 880 µg mL -1 15 N 2 -asparagine. The 133 sample was extracted by an Ultra Turrax mixer. After centrifuging, SPE cleanup was performed by 134 an automated sampler using LiChroLut RP-C18 SPE-cartridges LC-MS/MS detection was 135 performed on a triple quadrupole instrument (Micromass Quattro Ultima) after separation on a 136 hypercarb LC-column (dimensions 2.1 mm x 100 mm, particle size 5 µm) eluted with 0.1% 137 aqueous formic acid at 0.2 mL min -1 . The mass spectrometer was operated in ESI + for acrylamide 138 and saccharides and in ESI -for asparagine. Capillary voltages of 3 kV (ESI + ) and 2 kV (ESI -) were 139 applied but for simultaneous detection in positive and negative mode a lower capillary voltage, 140 e.g.1 kV is recommended to prevent sparkling from the capillary. 141 142 Some of the acrylamide analysis was performed after the methods were optimized to detect low 143 concentrations (e.g. by increasing the HPLC injection volume and by optimizing the MS 144 performance before analysing the bread samples), so the method was able to quantify down to 5 µg 145 kg -1 . The repeatability and reproducibility of the acrylamide analyses at 50 and 250 µg kg -1 (n=24) 146 were 6-17% and 9-29% respectively. The laboratory has participated in proficiency tests for 147 acrylamide in food and all results obtained have been with satisfactory test scores, including the 148 results obtained in an EU validation study on acrylamide analyses in food with 22 samples (11 149 matrices including bread and toasted bread: assigned value 38µg kg -1 , obtained results 41 and 44µg 150 kg -1 ) [START_REF] Wenzl | Collaborative trial 421 validation study of two methods, one based on high performance liquid chromatography-tandem 422 mass spectrometry and on gas chromatography-mass spectrometry for the determination of 423 acrylamide in bakery and potato products[END_REF]. The detection limit of the asparagine analyses was 0.002 g kg -1 , the 151 repeatability and reproducibility measured at 0.02, 0.1 and 0.4 g kg -1 0-5% and 10-18% repectively. The background content of asparagine in sifted wheat flour was 0.14 ± 0.004 g kg -1 (based on three 169 determinations and with the precision measured as 95% confidence levels) and in whole grain rye 170 flour 0.76 ± 0.02 g kg -1 , figures comparable to those reported by Frederiksson et al. (2004) of 0.14-171 0.17 g kg -1 (dw) in sifted wheat flour and 1.1 g kg -1 (dw) in whole grain rye flour. 172

The levels of asparagine added to the wheat dough were 0.6, 1.6 and 3.1 times the natural 173 background level and to the rye dough 0.2, 0.6 and 2.3 times the natural background level. The 174 highest concentration after addition correspond to 0.6 g kg -1 asparagine in wheat flour and 2.5 g kg -1 175 asparagine in rye flour; hence levels likely to be present in cereal products used for bread 176 production. 177

In Table II the reacted amounts of asparagine in the batches with different asparagine levels upon 178 fermentation, baking and toasting are presented. Some asparagine had reacted during wheat bread 179 production as can be seen from data determined on a dry weight basis (Table I), where differences 180 from evaporation of water during toasting does not have to be considered. In the wheat batch with a 181 natural background level of asparagine 12% was left after fermentation and baking. Upon hard 182 toasting all asparagine in the wheat bread slices with the natural background content and the lowest 183 addition level of asparagine had reacted. 184 185 Table I 186 Table II 187 188 In rye bread the relative reduction in asparagine during sourdough fermentation and baking was less 189 than for the yeast fermented wheat bread, e.g. 18% at the natural background level and 9% for the 190 highest addition level of asparagine. As the results are based on subtracting two large percentages, 191 they may be more uncertain. The results are in accordance with those of Frederiksson et al. (2004), 192 who also found sourdough fermentation to reduce asparagine less than yeast fermentation and at 193 toasting rye bread compared to wheat bread may also reduce the acrylamide formation. The average 243 water content in hard, medium and untoasted rye bread slices were 24±2%, 26±2% and 41±1% 244 while the average water content in hard, medium and untoasted wheat bread slices were somewhat 245 lower: 17±2%, 19±1% and 32±1%, respectively. However, the actual water contents at the toasted 246 surfaces were probably somewhat lower. Hedegaard et al. (2007) found that at high water activity 247 the rate determining step of acrylamide formation from the Schiffs base is the elimination of water 248 from an intermediate, while at lower water activity, water elimination becomes easier and 249 decarboxylation of a reaction intermediate leading to acrylamide becomes the rate determining step. 250

Hence the acrylamide formation rate increases when the water activity decreases. Hence if different 251 reactions are rate determining, the overall reaction rate may differ for different water activities, 252 which may explain the exponential increase in acrylamide contents (as the water content are 253 reduced) generally found during thermally processing of food at a fixed temperature. It was also 254 found in other model systems that acrylamide formation increased when lowering the water content 255 [START_REF] Schieberle | New aspects on the formation and analysis of 403 acrylamide[END_REF], Blank et al. 2005) . 256 257The proportions of asparagine in wheat bread reacting to give acrylamide were also estimated 258 (Table III). Generally the ratio is higher in medium toasted bread suggesting that at higher 259 temperatures and/or longer duration of toasting precursors are depleted faster, reacting faster in 260 competing reactions and/or acrylamide is eliminated to a larger extent by subsequent reaction. For 261 the hard toasted bread is seen, that as depletion of the asparagine pool approaches, the formation of 262 acrylamide diminishes, resulting in lower ratios at lower asparagine addition levels. At the low 263 asparagine levels in wheat bread asparagine was depleted after hard toasting while at higher 264 addition levels some asparagine was left. This may be interpreted as if asparagine was the limiting 265 precursor at low asparagine levels while at higher levels other factors also influenced the 266 The average dietary intake of wheat bread in Denmark is 125 g day -1 and in addition Danes has a 295 tradition for eating whole meal sourdough fermented rye bread as part of nearly every lunch meal 296 comprising an average daily intake of 59 g rye bread day -1 (Lyhne et al. 2005) In case all bread is 297 consumed without toasting and the mean figures for acrylamide in bread is used: 10 µg kg -1 in 298 wheat bread and 12 µg kg -1 in rye bread, the daily intake comprises 1.3 µg from wheat bread and 299 0.7 µg from rye bread, respectively. The daily intake of acrylamide from wheat and rye bread 300 comprising 2.0 µg is about 8 % of the average daily acrylamide intake of appr. 25 µg day -1 301 estimated for Danes (Figure 4). The acrylamide intake from bread is relatively low compared to 302 other surveys, estimating e.g. 3.4 µg [START_REF] Svensson | Dietary 413 intake of acrylamide in Sweden[END_REF]) and 3.6 µg (Matthys et al. 2005). An 303 explanation for the low Danish dietary acrylamide intake from bread may be that most Danish bread 304 are produced using either yeast fermentation or sourdough fermentation, hence the contents of the 305 precursor asparagine are reduced during preparation of the breads. Another explanation for the low 306 acrylamide intake from bread may be that in previous studies on acrylamide in bread the limit of 307 detection (LOD) was higher and the levels in bread often below the LOD. Hence in many cases 308 LOD was used as the acrylamide level in bread. Finally in the present estimate, as the Danish 309 Nationwide dietary survey do not have information on how frequently people eat toasted bread, the 310 acrylamide intake from toasted bread is not included in the dietary acrylamide intake from bread. 311 However, in case the average daily bread intake includes two hard toasted wheat bread slices 312 (acrylamide 161 µg kg -1 (ww)) each of 28 g and with the high pre-processing asparagine level of 0.6 313 g kg -1 (dw) which is about the concentration found in wholegrain wheat flour i.e. 0.5 g kg -1 314 (Frederiksson et al. 2004), the daily acrylamide intake is estimated to 9 µg (36 % of the average 315 Danish acrylamide intake). If a consumer eats an average amount of bread of which two slices of 25 316 g rye bread are hard toasted with asparagine and acrylamide concentrations of 1.2 g kg -1 and 123 µg 317 kg -1 respectively, the daily acrylamide intake from bread comprises 7 µg (28 % of the average 318 Danish acrylamide intake). Hence dietary habits with consumption of toasted bread, which are 319 common throughout at least Europe, may increase the acrylamide intake from bread considerable. 320

Especially intake of toasted wholemeal wheat bread and bread produced without leavening by 321 fermentation may further increase the acrylamide exposure. The contents of acrylamide in baked rye bread and in toasted wheat and rye bread are correlated to 327 the contents of asparagine in the cereal product used for the bread production and at asparagine 328 concentration ranges that may occur in grain products for bread production. The content of 329 asparagine in the dough for wheat bread preparation is reduced considerably during bread 330 production. After 65 min. raising time and subsequent baking, the asparagine content in the baked 331 wheat bread was reduced to 12% compared to the content in the flour before yeast fermentation. 332

The sourdough fermentation of rye bread showed a modest asparagine reduction to 82% of the 333 content in the flour. This is probably due to the more acidic conditions in sourdough fermented rye 334 bread compared to yeast fermented wheat dough. The contents of acrylamide formed in toasted 335 wheat bread slices and toasted rye bread slices are proportional to the asparagine concentration of 336 the untoasted bread. The dietary intake of acrylamide from untoasted bread of 2.0 µg day -1 is 337 relatively low but the dietary acrylamide intake increases somewhat for people eating toasted bread.

  Baking was performed initially at 280°C for 5 min., followed by 210°C for35 min. and 190°C for 95 12 min. 96 97 Toasting wheat bread and rye bread with asparagine additions 98 Toasting of the bread slices was done in a pushdown toaster (Tefal Delfini Compact Toaster 5396, 99 800 W) with room for two slices. The four batches of bread (background level and the three 100 addition levels) were analyzed either untoasted or after medium or hard toasting, for each level 101 using three slices from each of two breads (n=18). Medium toasting of wheat bread slices of 13 mm 102 was performed 1.9 min. and hard toasting 2.8 min.. Medium toasting of the rye bread slices of 8 103 mm was done 2.8 min. and the hard toasting 3.1 min.. All the toasted bread slices were medium to 104 dark brown and were considered eatable. The thicknesses of the bread slices were chosen from what 105 is common practice among bakers and consumers. .5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA), asparagine 113 monohydrate (>99%) from Acros Organics (Geel, Belgium), D 3 -acrylamide (>98%) from Polymer 114 Source (Dorval, Quebec, Canada), 15 N 2 -asparagine monohydrate (>98%) from Cambridge Isotope 115 Labs. (MA, USA), methanol (>98%), formic acid (>98%) and acetonitrile (HPLC-grade) Baking small wheat breads (baguettes): ¶ The industrial bakery produced small wheat breads (baguettes) in their laboratory bakery. The recipe for the wheat breads (appr. 3000 g dough ~ 60 breads) included sifted wheat flour, yeast, salt, sucrose, margarine and water. Kneading time was 3+5 min., holding time 20+10 min., and resting time at 37°C was 50 min. Baking was performed at 220°C for 10 min. or 15 min. in a ventilated oven and with water spray added for 30 s.. ¶

148 149

 148 Estimation of dietary acrylamide intakes 150 Exposure estimates were accomplished using data from the Danish Nationwide Dietary Survey 151 made from 2000-2002 by the National Food Institute (Lyhne et al. 2005). Information on food 152 consumption was collected from a representative sample of 4120 individuals aged 4-75 years. The 153 participants in the survey kept a food record for seven consecutive days. The monitoring results of 154 acrylamide in Danish foods (appr. 330 samples) were made 2002-2005 by the National Food 155 Institute (data not published elsewhere)

Formatted

  in the rye flour and acrylamide content in bread after medium (r 2

  Figure 3 265 266

  In order to study the acrylamide formation in industrial produced breads, the industrial bread producer made small wheat breads (baguettes) of 50 g dough in their laboratory bakery. The baguettes were baked in a vented oven at 220 °C for 10 min. (to yellow colour of crust) and 15 min. (to light brown colour of crust). As the baguettes are produced and sold frozen for final preparation by the consumer, the baguettes were also baked at 220 °C for 10 min. and reheated from frozen for 5 min. (to light yellow-brow colour). The crumb of all breads (15 analyses, 5 x 15 breads) only contained a few µg kg -1 acrylamide (<5 µg kg -1

  Figure 4. 321 322 Conclusions 323 324

  This research was supported by the Danish Ministry of Food, Agriculture and Fisheries as a part of 339 the project "Reduction of the formation and the occurrence of acrylamide in food" and The Nordic 340 Innovation Centre Project NORDACRYL (Acrylamide precursors; Limiting substrates and in vivo 341 effects). Lone Hertz is acknowledged for performing parts of the chemical analyses. 342 Bamiro T, Clinch J, Combs G, Gierczynski A, Karam R. 2004. Natural fermentation of wheat flours. Eriksson S, Tareke E, Karlsson P, Ehlenberg L, Törnqvist M. 2003. Investigations of factors that influence 390 the acrylamide content of heated foodstuffs. Journal of Agricultural and Food Chemistry 51:7012-7018. 391 Schieberle P, Köhler P, Ganvogl M. 2005. New aspects on the formation and analysis of acrylamide. Advanced 392 experimental medicine and Biology 561: 191-203. 393 Stadler, RH, Blank I, Varga N, Robert F, Hau J, Guy PA, Robert MC, Riediker S. 2002. Acrylamide from Maillard 394 reaction products. Nature 419:449. 395 Suhr KI, Nielsen PV. 2004. Effect of weak acid preservatives on growth of bakery product spoilage fungi at different 396 water activities and pH values. International Journal of Food Microbiology 95:67-78. 397 Surdyk N, Rosén J, Andersson R, Åman P. 2004. Effects of asparagine, fructose, and baking conditions on acrylamide 398 contents in yeast-leavened wheat bread. Journal of Agricultural and Food Chemistry 52:2047-2051. 399 Svensson K, Abramsson L, Becker W, Glynn A, Hellenäs KE, Lind Y, Rosén J. 2003. Dietary intake of acrylamide in 400 Sweden. Food and Chemical Toxicology 41:1581-1586. 401 Taeymans D, Wood J, Ashby P, Blank I, Studer A, Stadler RH, Gondé P, Van Eijck P, Lalljie S, Lingnert H, Lindblom 402 M, Matissek R, Müller D, Tallmadge D, O'brian J, Thompson S, Silvani D, Whitmore T. 2004. A review of 403 acrylamide: An industry perspective on research, analysis, formation and control. Critical Review in Food Sciences 404 and Nutrition 44:323-347. 405 Tareke E, Rydberg P, Karlsson P, Eriksson S, Törnqvist M. 2002. Analysis of acrylamide, a carcinogen formed in 406 heated foodstuffs. Journal of Agricultural and Food Chemistry 50:4998-5006. 407 Wenzl T, Karasek L, Rosen J, Hellenaes K-E, Castle L, Anklam E. 2006. Collaborative trial validation study of two 408 methods, one based on high performance liquid chromatography-tandem mass spectrometry and on gas 409 chromatography-mass spectrometry for the determination of acrylamide in bakery and potato products. Journal of 410 Chromatography A 1132: 211-218.

  Fig.1

Fig

  Fig. 3.

  fried

  the natural background level. The rye bread (based on appr. 900 g dough) 96was made from half and half mixing of sourdough and rye flour and addition of yeast, salt and 97 water. Kneading time was 10 min, holding time 20 min, and resting time at 37°C was 77 min. 98Baking was performed initially at 280°C for 5 min., followed by 210°C for 35 min. and 190°C for 99 12 min. 100 101 Toasting wheat bread and rye bread with asparagine additions 102 Toasting of the bread slices was done in a pushdown toaster (Tefal Delfini Compact Toaster 5396, 103 800 W) with room for two slices. The four batches of bread (background level and the three 104 addition levels) were analyzed either untoasted or after medium or hard toasting, for each level 105 using three slices from each of two breads (n=18). Medium toasting of wheat bread slices of 13 mm

152 153

 152 Estimation of dietary acrylamide intakes 154Exposure estimates were accomplished using data from the Danish Nationwide Dietary Survey 155 made from 2000-2002 by the National Food Institute(Lyhne et al. 2005). Information on food 156 consumption was collected from a representative sample of 4120 individuals aged 4-75 years. The 157 participants in the survey kept a food record for seven consecutive days. The monitoring results of 158 acrylamide in Danish foods (appr. 330 samples) were made 2002-2005 by the National Food 159 Institute (data not published elsewhere). 160 161Results and discussion 162The levels of acrylamide and asparagine in wheat and rye bread slices after baking and medium or 163 hard toasting appear from TableI. The results presented are all means of six bread slices, each 164 wheat bread slice weighing 34±2 g and each rye bread slice weighing 32±1 g. 165 166Asparagine in flour and bread slices after asparagine additions to dough and subsequent 167 fermentation, baking and toasting 168

  rate i.e. >80% for yeast fermentation of wheat bread and 17% for sourdough 194 fermentation of rye. 195 196 A general conclusion may be that rye flour contains more asparagine than wheat flour and 197 sourdough fermentation and baking of rye dough reduces asparagine to a smaller extent than yeast 198 fermentation and baking of wheat dough. Prolonging or optimizing the fermentation process, 199 especially the yeast fermentation of wheat bread may be an efficient tool to reduce the acrylamide

  Figure 3 270 271 272TableIII. 273 274

  Figure 4. 324 325 Conclusions 326

  This research was supported by the Danish Ministry of Food, Agriculture and Fisheries as a part of 341 the project "Reduction of the formation and the occurrence of acrylamide in food" and The Nordic 342 Innovation Centre Project NORDACRYL (Acrylamide precursors; Limiting substrates and in vivo 343 effects). Lone Hertz is acknowledged for performing parts of the chemical analyses. Amount of acrylamide formed and present [µg] from asparagine reacted [mg] when 436 toasting wheat bread (ratios calculated by subtracting analyte concentrations determined in medium 437 or hard toasted bread from analyte concentrations determined in untoasted bread). 438 439 Figure1. Experimental design for baking wheat and rye bread with addition of asparagine and 440 subsequent medium or hard toasting of bread slices. All bread slices were analysed for acrylamide 441 and asparagine. 442 443 Figure 2. Correlation between naturally occurring and added asparagine in flour and acrylamide in 444 baked and toasted bread slices of a) wheat or b) rye. 445 446 Figure 3. Correlation between asparagine in untoasted bread and acrylamide in toasted bread slices 447 of a) wheat or b) rye. 448 449 Figure 4. The proportions from different foods to the daily mean acrylamide intake for Danish 450

, 189 who also found sourdough fermentation to reduce asparagine less than yeast fermentation and at 190

			Deleted: 3
			Deleted: 1
		F o r
		P e e r	Deleted: 88 Deleted: had reacted during Deleted: , and additional 1% had reacted during medium toasting while additional 12% (until depletion) had reacted during hard toasting. At the highest asparagine level added 51% had reacted during fermentation and baking, additional 10% had reacted during
			medium toasting and additional 16%
		R	during hard toasting. Deleted: were depleted in asparagine. The findings provide evidence for
		e	asparagine as the limiting factor in acrylamide formation in wheat bread, especially in toasted wheat bread.
		v i e	Deleted: ¶ Deleted: ¶
		w
	191 192	about the same rate i.e. >80% for yeast fermentation of wheat bread and 17% for sourdough fermentation of rye. O n l y	Deleted: ings Deleted: o Deleted: a

  : 17±2%, 19±1% and 32±1%, respectively. However, the actual water contents at the toasted 242 surfaces were probably somewhat lower.Hedegaard et al. (2007) found that at high water activity 243 the rate determining step of acrylamide formation from the Schiffs base is the elimination of water 244 from an intermediate, while at lower water activity, water elimination becomes easier and 245 decarboxylation of a reaction intermediate leading to acrylamide becomes the rate determining step. 246 Hence the acrylamide formation rate increases when the water activity decreases. Hence if different 247 reactions are rate determining, the overall reaction rate may differ for different water activities, 248

	F o r
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	267
	268	Table III.
	269
	270
	271	Acrylamide contents in wheat breads and rye breads produced in Denmark and comparison with
	272	other surveys.
		F o r
		P
		e e r	Deleted: although Deleted: the formation rate drops again below a certain water content
		R
		e v i e	Deleted: s Deleted: Because much smaller proportions of the asparagine had reacted upon toasting of the rye bread the fractions reacted were more uncertain and are therefore not measured. ¶
		w
		O n l
		y

Table III

 III

	293
	294	Dietary acrylamide intake from bread
	273	.
	274
	275	Acrylamide contents in wheat breads and rye breads produced in Denmark and comparison with
	276	other surveys. In 2004, in order to estimate the level of acrylamide in rye bread, 13 sifted and whole meal rye F o r
		P
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Violette ; Ducruet
email: violette.ducruet@agroparistech.fr

Olivier Vitrac

Philippe Saillard

Elisabeth Guichard

Alexandre Feigenbaum

Nicole Fournier

COMPOUNDS IN PET AND PVC DURING THE STORAGE OF A STRAWBERRY SYRUP

Keywords: aroma, PET, PVC, packaging, sorption, strawberry, syrup

The sorption of 14 aroma compounds into PET and PVC was monitored during storage of a strawberry syrup for 1 year. Concentrations in the syrup and in the polymer were determined during storage and compared with previously published results obtained with glass bottles.

Apparent partition coefficients between the polymer and the syrup (noted K app ) were estimated from experimental kinetics without reaching equilibrium K app values were optimally identified from the kinetic data obtained between 30 and 90 days. They exhibited a similar behaviour for both polymers with values were between 2•10 -5 and 2•10 -3 , 4•10 -5 and 3•10 -2 respectively for PET and PVC. The variation of K app values in PET was mainly correlated to the polarity of tested compounds as assessed by their logP values. By contrast, the variations in K app values for PVC were mainly related to their chain lengths. Due to slightly higher partition coefficients and diffusion coefficients in PVC compared to PET, the amount of absorbed aroma was 4 times higher in PVC than in PET, however the amount of absorbed aroma compounds was less than 0.1% of the initial amount present into the syrup, except for octyl butanoate.. The variation in concentration in the syrup was interpreted as a combination of a degradation process and a transport process into the packaging material. Both effects were particularly noticeable for both PET and unstable aroma compounds.

Introduction

The absorption of aroma compounds by plastic materials may induce both a weakening of flavour and changes to the organic profile of packaged food products. This has mainly been reported for aqueous food, such as fruit juices, or model solutions, where the partition coefficient of aromas between food and packaging strongly favours sorption into the plastic material [START_REF] Kwapong | Comparative sorption of aroma compounds by polyethylene and ionomer food-contact plastics[END_REF][START_REF] Sizer | Maintaining flavor and nutrient quality of aseptic orange juice[END_REF]. However, there are many discrepancies in reported partition coefficient values [START_REF] Kutty | Oxidation of D-limonene in presence of low-density polyethylene[END_REF][START_REF] Hernandez-Munoz | Food aroma partition between packaging materials and fatty food simulants[END_REF][START_REF] Tehrany | Partition coefficients in food packaging systems : a review[END_REF]).

The possible reasons include: i) sorption kinetic studies performed without reaching the thermodynamic equilibrium [START_REF] Gavara | Methods to determine partition coefficient of organic compounds in water/polystyrene systems[END_REF][START_REF] Lebossé | Interactions between reactive aroma compounds from model citrus juice with polypropylene packaging film[END_REF], Feigenbaum et al. 1998), ii) aroma used beyond their concentrations limits, which yield to deviations from Henry sorption equilibrium. Besides, partitioning results obtained with model solutions are difficult to extrapolate to the behaviour of aroma compounds in real foods, which are generally multi-constituent and possibly multi-phase. Recent papers have shown that in complex solutions simulating food matrices, the partition coefficients between aroma compounds and polymers depend also on the interactions with macromolecules (lipids, polyoligosides or proteins). In particular, lipids compete with polymers concerning the transfer of aroma compounds into polyolefins (Van Willige et al. 2000a,b). Moreover, during long periods of storage of up to a year, changes in aroma composition were detected in the food matrix without any contact with the polymer [START_REF] Ducruet | Influence of packaging on the aroma stability of strawberry syrup during shelf life[END_REF][START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF]. In this case it is important to understand how the reactivity of the aroma compound in the matrix can influence the absorption of the aroma compounds by the packaging material.

Although PET and PVC are known to be good barriers to oxygen, in comparison with polyolefins, PET (one of the most common packaging polymers) and PVC (which is not widely used to package flavoured drinks), have seldom been tested (Ducruet et al. 2001, Van [START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF] for their barrier properties to aroma. The objective of the present study was to provide additional results on the sorption of aroma compounds in glassy polymers such as PET and PVC during long storage periods and to compare the kinetic sorption process with the degradation process, which also can reduce the amount of aroma. To make the results valuable for industry, the sorption and degradation kinetics were assessed on real food products and real packaging materials. Since the thermodynamic equilibrium cannot be obtained when the rate constants of both processes have similar orders of magnitude, the apparent partition coefficients were derived from an approach based on a new approximated solution proposed by [START_REF] Vitrac | Identification of diffusion transport properties from desorption/sorption[END_REF] to identify transport properties (including the partition coefficient) from censored kinetics (i.e. without reaching the thermodynamic equilibrium).

Materials and methods

Packaging materials

All the bottles were supplied from Teisseire S.A France. Strawberry syrups were packaged in rigid PVC bottles (PVC1, PVC2: average thickness: 1 mm) and in amorphous PET bottles (PET1 and PET2; average thickness: 0.75 mm). PVC1 was made using GFG 52 D resin from Dorlyl; it is suitable for contact with oils and is stabilized with an organotin stabilizer. PVC2 was manufactured by Teisseire. PET1 was manufactured from Ramapet, and PET2 from Shinpet. The surface area / volume in dm 2 l -1 ratio was approximately = 6. Packaging in glass bottles (GL) was used as a reference to test the stability of the syrup without the influence of polymer contact.

Strawberry syrup

Strawberry syrup was obtained by mixing a sucrose syrup (°Brix: 64.5 +/-1) and fruit juices (strawberry, elder and lemon juices). Nature-identical flavouring substances were also included.

The pH of the strawberry syrup was 2.65 +/-0.2 and its shelf life was 18 months. temperature under aseptic conditions. All bottles (PET, PVC and glass) were sealed after filling with aluminium foil in order to avoid a cap effect. The packaged syrups were stored at 20°C Solvent extraction of the syrup 200 ml of syrup, 200 ml of Milli Q water and 500 µl of tridecane solution (used as an internal standard, 500 µl l -1 ) were mixed in a 1 litre flask. Volatile compounds in the strawberry syrup were extracted three times using distilled dichloromethane, with 80, 30 and 30 ml for 30 min, 15 min and 15 min respectively, at 0° C while stirring. Organic phases were separated from the aqueous phase and pooled. The dichloromethane extracts were dried over anhydrous Na 2 SO 4 and concentrated using a Kuderna-Danish column. Analysis were done in triplicate.

Solvent extraction of polymers

At the end of each period of contact, the strawberry syrup and its packaging were separated. The plastic bottle (PET or PVC) was carefully and rapidly rinsed twice with ultra pure water, then with ethanol, and finally wiped before cutting. Because of the heterogeneous thickness of bottles, samples were taken by cutting pieces from three sections of a bottle (top, bottom and middle) and then pooled. Approximately 7g of each pooled sample was then plunged immediately into a 100 ml flask containing distilled diethyl ether (95 ml). 2 ml of a solution of ethyl pentanoate in diethyl ether (100 µl l -1 ) were added as an internal standard. The flasks were tightly plugged with Teflon caps and extraction was carried out for 2 hours at 15 °C with magnetic stirring. Solvent extracts were dried with sodium sulfate and filtered over glass wool. Diethyl ether extracts were concentrated using a Kuderna-Danish apparatus.

Two plastic bottles were analyzed for each contact period. The extraction process was repeated twice for each bottle so as to enable analysis in duplicate of the plastic material after each period of contact.

Mass spectra

Identification of the constituents was achieved using gas chromatography-mass spectrometry (GC-MS). The GC-MS system consists of a Fisons GC-8000 chromatograph and a MD 800 mass spectrometer (Fisons Instruments, Les Ulis, France). Separations were performed on a Supelcowax fused silica capillary column (0.32 mm x 30 m; 0.5 µm; Supelco, Bellefonte, PA).

The linear flow velocity of helium was 32 cm sec -1 . The column was maintained at 30°C for 10 min and then programmed at 240°C for SPME analysis and 260°C for solvent extracts at 5°C min - 1 . The split-splitless injector was at 230°C. Solvent extracts were injected into the splitless/split system (the split valve was closed after 30 sec). Electron ionization mass spectra were recorded under the following conditions: capillary direct interface, 250°C; ion source, 200°C; ionization voltage, 70eV; mass range, 29-300 m/z; electron multiplier voltage, 450 V; scan rate 3 scans sec -1 .

Mass spectral matches were made by comparison with NIST and INRAMASS mass spectra libraries. Kovat's Indices of authentic compounds compiled in the INRAMASS library were used to confirm identification.

Estimation of apparent partition coefficients

The partition coefficient is defined from the ratio of concentrations of aroma at thermodynamic equilibrium, noted P eq C and F eq C respectively in the packaging material (P) and in Food (F):

P eq F eq C K C = (1)
It is emphasized that equation (1) is valid whatever the considered equilibrium. Since the equilibrium was not reachable due to insufficient contact time (even after one year) or due to aroma degradation during storage, the equilibrium was extrapolated from the kinetic phase diagram corresponding to the experimental sorption kinetic, which was recently proposed by

       
is interpreted as a degradation process, which tends to dominate the sorption process.

In this work, P eq C was linealy extrapolated from the last experimental point KPD, for which

P t P C P t dC d dC dt        
was monotonous (i.e. increasing or constant). The same process was applied for the concentration in the liquid. A key step in the KPD approach is calculation of the derivatives of the concentration with time when the experimental data are unevenly sampled and noisy. In this work, we use an efficient non deterministic filtering technique based on local polynomial approximants as detailed and discussed in [START_REF] Vitrac | Identification of diffusion transport properties from desorption/sorption[END_REF]. By noting with a superscript "extrap" the extrapolated quantities, the apparent partition coefficient, K app , corresponding to experimental data were calculated from:

F o r P e e r R e v i e w O n l y extrap P eq app extrap F eq C K C = (2)
It is emphasized that K app may differ from the real K value, however it is the best estimator according to the data available and our knowledge of sorption mechanisms. Confidence intervals on K app were derived by Monte Carlo sampling consisting in adding a white noise similar to the experimental error (relative error of 15 %) to kinetic data and in repeating the whole analysis.

Each confidence interval was based on the 2.5 th and 97.5 th percentiles of at least 200 Monte Carlo trials.

Statistical analysis

Sorption experiments were analyzed using a one-way ANOVA program. When the differences were significant (p<0.05), Duncan's test was used to check the differences between pairs of groups and was carried out using XLSTAT-Pro 7.0 software (Addinsoft, Paris, France).

Results and discussion

Sorption of the aroma compounds into PET and PVC

In order to monitor the sorption of aroma compounds, the strawberry syrup and the plastic material were extracted and analysed at different time points during the 330-day storage period. In the case of PVC, the presence in solvent extracts of additives and small oligomers along with absorbed volatile compounds produced more complex GC profiles than those seen with PET.

Some of these additives were tentatively identified in the extracts of the syrup. (Table 1).

[Insert Table 1 here]

These compounds originate from the degradation of additives for example plasticizer, lubricants or modifiers (Gachter and Muller, 1990). Some can migrate into the syrup such as benzaldehyde, additives were extracted and co-eluted with the aroma compounds by GC. Thus, of the 38 aroma compounds previously identified in the syrup at the initial time of storage [START_REF] Ducruet | Influence of packaging on the aroma stability of strawberry syrup during shelf life[END_REF], only 14 compounds can be quantified in both kinds of packaging and in the syrup over the 330day storage period (Table 2).

[Insert Table 2 here] The final concentration of the aroma compounds into the syrups stored in both PET and PVC bottles were compared to their initial concentrations (Figure 1). During shelf life, aroma compounds decreased by hydrolysis irrespective of the polymer. The decrease was more for short chain esters, and in particular for acetic acid esters and ethyl hexanoate (Figure 1A) compared to more stable compounds (Figure 1B). This reactivity is not related to the contact with polymer as it was previously found to occur during the storage of the syrup in a glass bottle [START_REF] Ducruet | Influence of packaging on the aroma stability of strawberry syrup during shelf life[END_REF]).

[Insert Figure 1 here] The greater reactivity was observed for short chain esters (methyl or ethyl esters) and acetic acid esters and can be explained by the low steric hindrance around their carbonyl group in contrast to linear longer chain esters (butyl, hexyl or octyl esters) and specially to branched esters (ethyl 2methylbutanoate, ethyl 3-methylbutanoate and 3-methylbutyl 3-methylbutanaote). These results demonstrated the instability of the aroma formulation for long periods of storage in acidic condition such as it was also found in the case of citrus flavoured drinks [START_REF] Sizer | Maintaining flavor and nutrient quality of aseptic orange juice[END_REF][START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF] The sorption of the aroma compounds from strawberry syrup into PET and PVC was monitored 2). The sorption into both kinds of packaging materials was found to be weak. The sorption was generally lower than 0.1 % in all cases except for octyl butanoate which reached 0.15 % in both PET samples, and 0.57 % or 0.26 % respectively in PVC1 and in PVC2 samples. After one year of storage into both samples of PET and PVC, if we consider the case of the octyl butanoate and hexyl acetate, as an example, their concentrations decreased by 30-43 % and 67-81 % respectively (Figure 1) although the sorption of these compounds into PET and PVC only represented 0.15-0.57 % and 0.013-0.023 % of the initial amount, respectively (table 1). These glassy materials showed their resistance to sorption by aroma and thus the main parameter which had an impact on the aroma formulation was the hydrolytic process into the syrup.

Few papers reported quantitative measurement of the sorption of aroma compounds into PET for real foods over a long time storage especially for thick materials such as bottles. For soft drinks flavoured with orange oil and stored in PET during 12 weeks, Nielsen (1994) showed that only 2.1 In the case of strawberry syrup, sucrose may reduce the diffusion and convection of flavour compounds in the polymer during the early stages of storage, as it increases the viscosity of the medium (Van Willige et al. 2000a). Moreover, sucrose molecules may create local hydrophobic environments in the medium which favours inclusion or interaction with hydrophobic molecules such as aroma compounds. This may lower the sorption of aroma compounds because of a competition effect between the two hydrophobic phases (polymer and food matrix) [START_REF] Roberts | Effects of sucrose, guar gum, and carboxymethylcellulose on the release of volatile flavor compounds under dynamic conditions[END_REF][START_REF] Nawar | Some variables affecting composition of headspace aroma[END_REF].

Typical kinetics of sorption into PET and PVC for the more stable compounds It is highlighted that for the set of tested aroma compounds, log P and molecular weight are not independent quantities. Log (K app ) appeared almost linearly correlated to log P values. Whatever the polymer, sorption was selective and increased with the chain length of the linear butanoic acid esters (butyl butanoate, hexyl butanoate and octyl butanoate), a behaviour quite similar to that reported by [START_REF] Shimoda | Effect of number carbon atoms of flavor compounds on diffusion, permeation and sorption with polyethylene[END_REF] with respect to polyethylene. However the correlation was poorer with the molecular mass than with the log P, due to branching into the molecules.

Branching or unsaturation in the backbone of the molecule (for an equal carbon number) caused sorption to decrease, as can be seen for 3-methylbutyl-3-methylbutanoate by comparison with hexyl butanoate in PET (Figure 3C).

Quite similar partition coefficients were obtained for both PET (PET1 and PET2) and both PVC (PVC1 and PV2). Both PET samples sorbed the esters to a lesser extent, PVC1 displayed the highest level of sorption, three to ten-fold more than both samples of PET mainly for octyl butanoate and 3-methylbutyl-3-methylbutanoate Few studies have dealt with the sorption of aroma compounds into PVC. [START_REF] Koszinowski | Food Package compatibility and migration[END_REF][START_REF] Koszinowski | Food Package compatibility and migration[END_REF][START_REF] Delassus | Sorption and diffusion of flavors in plastic packaging. Flavor-food interactions[END_REF] showed that diffusion coefficients in PVC were several orders of magnitude lower than in polyethylene and polypropylene. In contrast, their solubilities (S) were about one order of magnitude lower in polyolefins. Barrier behaviour evaluated by the permeability coefficient P (P=DxS), resulting from the kinetic effect (coefficient of diffusion, D), and the thermodynamic effect (S), only gave a small advantage for PVC over polyolefins.

With PET, the coefficients of diffusion and solubility of the volatile compounds are lower [START_REF] Pennarun | Functional barriers in PET recycled bottles. Part I. simulants[END_REF]) than those measured for polyolefins, so a low interaction can be observed with this polymer. Figure 3C and 3D shows that both PET samples were more or less equivalent and were less sorbent than PVC 2, except for cyclic esters such as ethyl cinnamate and ethyl salicylate The sorption of these compounds in PET reached the level of sorption observed in PVC2. These compounds exhibited a differential affinity for PET, probably because of the In conclusion, the variation of K app values in PET was mainly correlated to the polarity of tested compounds as assessed by their log P values. By contrast, the variations in K app values for PVC were mainly related to their chain lengths

Kinetics of sorption into PET and PVC for the unstable compounds

The sorption kinetics of some compounds, (ethyl butanoate, hexyl acetate), showed the same typical trends in both types of PET (Figure 4).

[Insert Figure 4 here] Sorption increased to a peak at 90 days and then declined until 150 days, reaching a state of equilibrium until 330 days.

Several hypotheses can be proposed to explain this behaviour.

1. [START_REF] Durning | A mathematical model for diffusion with induced crystallization (2)[END_REF] proposed a model of diffusion with induced crystallization to describe the sorption kinetics of organic liquids which might trigger PET crystallization. [START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF] showed that the absorption of aroma compounds (even at low levels) may be responsible for increasing the crystallinity of PET, over 6 months of storage. This "solvent induced crystallization" which was described in PET in contact with ethyl acetate by [START_REF] Hao | Ethyl acetate transport in poly(ethylene terephthalate)[END_REF], could lead to a structure modification of PET during 3 months of storage and then induce the release of the aroma compounds from the packaging. However this behaviour would have been observed for all the compounds specially those which are more sorbed such as octyl butanaoate, but it is not the case and this behaviour was only noticed for the more reactive compounds.

2. The sorption behaviour into PET of the more reactive aroma compounds could be explained in the following manner: during the first stage between 0 to 90 days, these compounds were slowly sorbed by PET, and hydrolysis into alcohols and acids started in the syrup. During the second period (90<t<150 days), degradation was the leading parameter, which resulted in desorption of the volatiles from PET, reaching an equilibrium between both phases (syrup and polymer). No alcohol or acid from the corresponding esters was identified in the PET extract. After 150 days, the equilibrium of aroma compounds between packaging and syrup was reached, taking account of degradation into the syrup. Thus, after 90 days, PET could play a positive role as a reserve for some of these unstable compounds.

As previously described [START_REF] Ducruet | Influence of packaging on the aroma stability of strawberry syrup during shelf life[END_REF], esterified aroma compounds were hydrolysed in glass bottles and this was not influenced by polymer contact. The hydrolysis rate of volatile compounds displayed a first order kinetic. Stronger reactivity was observed for methyl or ethyl esters of acetic acid when compared with longer linear chain esters and particularly branched esters. These compounds were rapidly hydrolysed in the syrup packaged in glass bottles during the first 150 days. Same reactivity was observed in both kinds of polymers. As oxygen transfer through amorphous PET and rigid PVC were of the same order of magnitude, respectively 4.4 10 - 11 cm 2 sec -1 Pa -1 and 3.4 10 -11 cm 2 sec -1 Pa -1 (Branrup et al. 1989), the oxidation of these compounds cannot explain the observed difference between PET and PVC.

In the case of orange juice in contact with PET, recent results had already shown that, degradation due to the acid catalysis of aroma compounds (α-pinene, limonene, linalool) is the principal parameter controlling exchanges with the glassy material [START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF]) and therefore the aromatic evolution of orange juice was not controlled by the packaging material but by reactions within the matrix itself. Polyolefins (polyethylene and polypropylene (in contact with a model medium containing highly reactive compounds such as limonene or pinene under acidic conditions may play a positive role in stabilising aroma compounds [START_REF] Lebossé | Interactions between reactive aroma compounds from model citrus juice with polypropylene packaging film[END_REF][START_REF] Feigenbaum | Polypropylene as active packaging material for aroma sorption from model orange juice[END_REF][START_REF] Reynier | Stabilization of aroma compounds through sorption-release by packaging polymers[END_REF]). Because the degradation process in the model system is of the same order of magnitude as diffusion into the polymers, the two phenomena are in competition. In the case of PET, transfer is very slow, and many studies, which considered contact times of insufficient duration, were not able to demonstrate this effect. In the case of contact between real food products during their shelf life (up to one year) and unstable aroma compounds such as short chain esters, the degradation of aroma compounds is more rapid than diffusion into PET. Release is less rapid than with polyolefins, and some stabilization effects of PET may be observed after only 150 days of storage, in line with the type trend of sorption kinetics.

In the context of food packaging interactions, the thermodynamic equilibrium of each aroma compound is expressed as the partition coefficient. Because the experimental determination of this partition coefficient may be prohibitive [START_REF] Tehrany | Partition coefficients in food packaging systems : a review[END_REF], modelling has been proposed as an alternative [START_REF] Dekker | Modelling the effect of oil/fat content in food systems on flavour absorption by LLDPE[END_REF][START_REF] Tehrany | Comparison between the different calculation methods of partition coefficient of aroma compounds with a difference chemical classes in octano/water system[END_REF]. Models are available for polyolefins but extensions to glassy polymers such as PET or PVC are still pending. The main limitations consist in the availability of reference data, which are not subjected to bias due to a competition between slow sorption kinetics (longer than 150 days) and a fast degradation process.

The production of apparent partition coefficient with minimum bias as assessed in this study using a kinetic phase diagram could be an alternative to the lack of published data. From the technological point of view, the loss of aroma for a particular food in contact with a glassy material must be envisaged as a consequence of both the sorption and degradation process. In the present case, the degradation of the aroma formulation of the syrup is mainly due to hydrolysis of esters.

Conclusion

This study shows that the interactions between aroma compounds and packaging may result in a for both PET samples and for unstable aroma compounds. It is emphasized that both processes are antagonist. Although the sorption rate is low in glassy polymers, the sorption process tends to prevent the aroma from the degradation process. By contrast, the degradation modifies the expected thermodynamic equilibrium of sorption of aroma and tends to limit the sorption of aroma. Both phenomena were simultaneously studied in this work and a general methodology was proposed to extract apparent partition coefficients, when the thermodynamic equilibrium cannot be reached due to a coupling with reactions. This methodology was used to derive the partition coefficients of 13 aroma compounds found in real fruit juices and two glassy polymers : PET and PVC. Besides, it was highlighted that the stability of the aroma profile in connection with a packaging material cannot be addressed with short contact times experiments. Additional studies and data appear desirable to help food industry to predict the behaviour of their aroma formulation during long term storage taking into account both the effects of the food matrix and the packaging.

F

  ethylhexanol. By solvent extraction of the syrup or the packaging, these

  The sorption of the 14 aroma compounds over 330 days of storage was compared to their initial concentration into the syrup (Table

%

  and 1.4 % of the initial concentrations of myrcene and of limonene were respectively sorbed by the polymer.[START_REF] Berlinet | Evolution of aroma compounds in orange juice stored in PET[END_REF] compared the initial amounts of these two aroma compounds in orange juice and the quantities adsorbed in PET after 5 months of storage. They found an adsorption of between 0.2 and 0.3% of the initial levels present. These data were in line with the findings of[START_REF] Van Willige | Influence of flavour absorption by food-packaging materials (low-density polyethylene, polycarbonate and polyethylene terephtalate) on taste perception of a model solution and orange juice[END_REF] who stated that only three flavour compounds (limonene, βmyrcene and decanal) were absorbed by PET. The percentage absorption of PET from juice containing limonene, β-myrcene, and decanal reached only 0.1%, 0.1% and 2.8% respectively, after one month of storage. When orange juice was stored in PET, the sorption of these two compounds was lower. The constituents of the juice (pulp, pectin), missing in the soft drink, may play a competitive role against the sorption into PET. The difference in the behaviour of the aroma compounds whether they are present in a simple solution or a more complex food was noticed byLeufven & Hermansson (1994). The sorption of aroma compounds into PET was lower when into a tomato juice in comparison with a model solution. These authors suggested that the aroma compounds probably remain in the tomato juice because they are retained on the natural juice constituents instead of being sorbed in the polymer.

[

  Insert Figure 2 here]In both PVC and particularly PVC1, the sorption of most aroma compounds increased with the square root of time, following a Fickian mode, as we were able to show for more stable compounds such as 3-methylbutyl-3-methylbutanoate. Their sorption kinetics and their interpretation as kinetic phase diagrams are shown in figure2. Open symbols plot experimental values as measured (duplicate experiments) whereas filled symbols plot non-deterministically filtered data as described by[START_REF] Vitrac | Identification of diffusion transport properties from desorption/sorption[END_REF] (figures 2 A-D). The continuous lines depict a cubic spline model, whose values and first derivatives fit the filtered ones. Versus the square root of time, the kinetics show a subsequent delay related to a possible external mass transfer resistance, a linear segment followed by a part with a decreasing slope. The presence of a linear segment confirmed that the sorption was controlled by the diffusion. The following sharp change in slope with a possible negative slope is related to a dynamic modification of sorption conditions.

  created by the degradation of the aroma compound in the liquid phase, which modified in return the attainable equilibrium state. The expected equilibrium concentration in the packaging material was consequently decreasing.These trends were more discernible in the kinetic phase diagram (KPD), which plots the first derivative of the concentration in the packaging material with time versus its primitive (i.e. the concentration itself)(figures 2E and 2F). Filled and open symbols plot respectively the filtered values and the 95% confidence interval as assessed by Monte Carlo sampling. A non-diffusive behaviour such as the one related to a reaction in the liquid phase is associated to a change in convexity in the diagram. Indeed, a convex shape of the KPD or a sharp decrease of the absorbed mass flux when the absorbed amount increase cannot be related anymore to diffusion (see[START_REF] Vitrac | Identification of diffusion transport properties from desorption/sorption[END_REF] for further details). This rule could not however be applied to the earlier stage of desorption kinetics since the mass flux was not estimated with enough accuracy. The KPD were used to predict the equilibrium, which would be reached if the sorption conditions as assessed during the linear part of the concentration versus the square root of times were prolonged, since in the parabolic regime of diffusion, the KPD is linear. The concentrations at equilibrium were linearly extrapolated from the convex part of the KPD to the horizontal line 0 P t d C dt = . The linear extrapolation inferred from the confidence envelope of the KPD appears as dashed lines. Affinity of aroma compounds for PVC and PET Apparent partition coefficients K app derived from the extrapolation of concentrations expected at equilibrium in absence of reaction are plotted versus logP (octanol-water partition coefficient) (figures 3A and 3B) and versus the molecular weight (figures 3C and 3D) for PET (figures 3A and 3C) and for PVS (figures 3B and 3D). [Insert Figures 3 A, B, C, D here]

  cyclic structure. This affinity could affect the typical flavour of the syrup related to the "wild strawberry note" which is associated with ethyl cinnamate, ethyl salicylate and ethyl benzoate.

  dynamic and time-dependent change in food quality during shelf life. Two combined mechanisms contribute to a loss of aroma during long-term storage: the degradation process in the food product itself and the sorption process in the packaging material. Both effects were particularly noticeable

F

Figure 1 Figure 2 Figure 3 Figure 4

 1234 Figure 1 Concentration of the aroma compounds into the strawberry syrup at t 0 day and t330 days of storage into the 4 packaging materials (n=3) 149x141mm (600 x 600 DPI)

Table 2 :

 2 Amount of aroma compounds sorbed into PET and PVC after 90 days ** and 330 days of storage at 20°C. Means of two determinations

	Volatile Compound	Code	Co*	PET1	%	PET2	%	PVC1	%	PVC2	%
			mg l -1	µg / 6dm 2	sorption	µg /6dm 2	sorption	330 days	sorption	330 days	sorption
								µg /6dm 2		µg /6dm 2
	Butyl butanoate	BB	24.6	3.7	0.015 c	3.8	0.015 c	7.8	0.032 a	4.9	0.020 b
	Hexyl butanoate	BH	9.6	2.3	0.024 c	2.6	0.027 c	11	0.115 a	5.1	0.053 b
	Octyl butanoate	BO	8.4	13.1	0.156 c	12.8	0.150 c	47.8	0.569 a	22.1	0.263 b
	Ethyl hexanoate	HE	167.5	24.5 **	0.015 c	27.8 **	0.017 b	54.3	0.032 a	29.1	0.017 b
	3-Methylbutyl 3-	3MB3MB	134.5	12	0.009 c	16.8	0.012 c	146.9	0.109 a	62.7	0.047 b
	methylbutanoate
	Ethyl 2-	2MBE	257.5	8.8	0.003 c	8.1	0.003 c	23.2	0.009 a	16.4	0.006 b
	methylbutanoate
	Ethyl 3-	3MBE	166.8	7.3	0.004 a	7.3	0.004 a	12.4	0.007 a	8.6	0.005 a
	methylbutanoate
	Hexyl acetate	AcH	152.5	23 **	0.015 b	25.8 **	0.017 b	34.6	0.023 a	19.4	0.013 c
	3(Z)-Hexenyl acetate	Ac3H	142.2	11.4 **	0.008 c	13 **	0.009 b	21.3	0.015 a	12.7	0.009 b
	Octyl acetate	AcO	10.4	9.1 **	0.087 a	8.5 **	0.082 a	9.2	0.088 a	4.3	0.041 a
	Methyl cinnamate	CIME2	530.5	149.5	0.028 b	164.9	0.031 b 283.5	0.053 a 149.7	0.028 b
	Ethyl salicylate	2OHBzE	120.4	45.5	0.038 b	40.8	0.034 b	85.7	0.071 a	42.9	0.036 b
	Ethyl benzoate	BzE	47.5	7.2	0.015 bc	6.3	0.013 c	43	0.091 a	31.9	0.067 ab
	γ-decalactone	GDL	339.2	21	0.006 c	13.6	0.005 c	132.3	0.039 a	43.2	0.013 b

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Acknowledgement

This work was supported by the Société TEISSEIRE. The authors would like to thank Mr. ROUGE and Mrs RAYNAL for their efficient partnership during study-time.