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Acrylamide formation in traditional Czech leavened wheat-rye breads and wheat rolls
Food Control 38 (2014) 221e226
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Acrylamide formation in traditional Czech leavened wheat-rye breads and wheat rolls Veronika Forstova a, Beverly Belkova a, Katerina Riddellova a, Lukas Vaclavik a, Josef Prihoda b, Jana Hajslova a, * a Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technicka 3, 166 28 Prague 6, Czech Republic b Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Department of Carbohydrates and Cereals, Technicka 3, 166 28 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2013 Received in revised form 8 October 2013 Accepted 15 October 2013
The main aim of our study was to collect the data on the acrylamide content in typical Czech leavened wheat-rye breads and wheat rolls, and to demonstrate the influence of various technological factors on its contents. We analysed several sets of industrially and laboratory baked breads to better understand the acrylamide formation. Following factors were tested: (i) the content of rye in respective bread formulae and (ii) the character of leavening procedure. We were, as well, monitoring for ten days the variability of acrylamide levels in bread baked by one bakery and, afterwards, we analysed the same type of bread baked in 12 bakeries. In the case of rolls, the influence of yeasts and vital gluten content in the dough were subjects of an assessment. The levels of acrylamide in a common type of the Czech bread baked in various bakeries were relatively low, ranging from 7 to 22 mg kg1. The content of acrylamide was rather higher in breads prepared using a commercial starter (Lactobacilli) and yeasts than in bread prepared using natural rye sourdough only. A varying content of rye flour did not seem to have any impact on acrylamide levels. The ‘size dilution effect’ was observed when comparing the same type of bread with different weight. Since acrylamide is exclusively located in the bread crust, smaller loaves contained higher acrylamide content per kg. The contents of acrylamide in standard wheat bakery goods were very low, bellow 10 mg kg1. No influence of varying content of added yeast or vital gluten was observed. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acrylamide Bread Rye flour Wheat flour Sourdough Yeasts
1. Introduction Acrylamide (prop-2-enamide) has been classified as a probable human carcinogen 2A (IARC, 1994) and is known as neurotoxin (WHO, 2002). This compound is formed during the Maillard reaction, especially in heat-processed starch-rich foods such as potato and bakery products. High acrylamide contents can be found in coffee and coffee surrogates, as well (EFSA, 2011). Currently, acrylamide is monitored in the European Union member’s states according to the European Commission Recommendation (2010/307/EU). Indicative acrylamide values based on the EFSA (European Food Safety Authority) monitoring data from 2007 to 2008 were established within the European Commission recommendation released on January 10, 2011 (“on investigations into the levels of acrylamide in food”) (EC, 2011). The indicative values are not safety thresholds, but, if exceeded, * Corresponding author. Tel.: þ420 220 443 185. E-mail address: [email protected] (J. Hajslova). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.10.022
subsequent investigation of the reasons should be conducted. Cereal products are there divided into 5 categories with relevant indicative values: Soft bread (150 mg kg1); Breakfast cereals excl. muesli and porridge (400 mg kg1); Biscuits, crackers, wafers, crisp bread and similar, excl. ginger bread (500 mg kg1); Biscuits and rusks for infants and young children (250 mg kg1); Processed cereal based foods for infants and young children, excl. biscuits and rusks (100 mg kg1) (EC, 2011). For other commodities such as potato products or coffee indicative values are available as well (EC, 2011). The latest exposure estimates in Europe evaluated by European Food Safety Authority (EFSA) were reported within the EFSA scientific report in 2011 (EFSA, 2011). Besides of potato products and coffee, bakery products, including soft bread and biscuits, have been identified as significant contributors to the overall acrylamide exposure through all groups of population (EFSA, 2011). The found mean acrylamide contents in soft breads ranged between 30 and 75 mg kg1 within the years 2007e2010 (EFSA, 2012). Despite the fact that acrylamide contents found in EU countries in soft breads are reasonably low, it is an important acrylamide source in
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European diets (EFSA, 2011). The results obtained within the EFSA report demonstrate that the contribution to the acrylamide dietary intake from soft bread in EU countries is approximately 20% among children and adolescents and up to 30% among adults (EFSA, 2011). These percentages are influenced by many factors such as age or gender of the respondents or the consumer’s habits. In the Czech Republic, the contribution of bakery products to consumers’ exposure to acrylamide is important due to their relatively high daily consumption. The contribution of soft bread to the total acrylamide dietary exposure in the Czech Republic is 13% for adults and 9e10% for children and adolescents (EFSA, 2011). The results of several studies confirmed asparagine as a key precursor of acrylamide formation in cereal based heat-treated food (Hamlet, Sadd, & Liang, 2008; Taeymans et al., 2004). According to the results of 5 years monitoring study (2006e2010), the mean asparagine content in wheat flours in the Czech Republic was found in the range from 36 to 309 mg kg1 (National project NPV II results). These values found in crop grown in the Czech Republic were comparable with those reported in other countries (69e 443 mg kg1) (Konings, Ashby, Hamlet, & Thompson, 2007). It should be noted that not only the asparagine content in flour but also the glucose/fructose ratio and other ingredients added to the dough play an important role (Hamlet et al., 2008). Several studies were concerned with a detailed monitoring of factors influencing acrylamide formation in bakery products with the aim to find the conditions enabling reduction of acrylamide. The current state of the art is summarized in the ‘Toolbox’, the document published by Food Drink Europe in 2011(FDE, 2011). For instance the addition of Ca2þ salts into flours was shown to have a potential for significant acrylamide reduction. The fortification of flour by 0.3% of Ca2þ salts is required by the U.K. law for nutritional reasons and results in up to 30% decrease according to some authors (Hamlet et al., 2005; Sadd, Hamlet, & Liang, 2008). The addition of glycine may lead to acrylamide reduction in yeast leavened bread. Nevertheless, the addition of high amounts of this amino acid may have a negative impact on yeast fermentation (Brathen, Kita, Knutsen, & Wicklund, 2005; Fink, Andersson, Rosén, & Aman, 2006). Size dilution in bread seems to be another important factor influencing the acrylamide content expressed on the whole product. The acrylamide formation is typical for the bread crust, where the temperature reaches much higher values than in the bread crumb, where the temperatures do not exceed 100 C (Surdyk, Rosén, Andersson, & Aman, 2004). Production of a larger bread loaf can reduce the average acrylamide content in a loaf due to the dilution caused by decreasing the ratio surface area to the volume ratio (Amrein, Andres, Escher, & Amado, 2007; Surdyk et al., 2004). Another possible way for reduction of the acrylamide amount is prolonged yeast fermentation during which asparagine is assimilated by yeasts (Fredriksson, Tallving, Rosén, & Aman, 2004). Some studies have shown an acrylamide reduction of about 50% after 1 h of fermentation (Hamlet et al., 2005; Sadd et al., 2008). Nevertheless, the reduction was not so successful in industrially prepared breads. During dough baking, the time and temperature are the crucial factors responsible for the extent of thermal input and, consequently, of acrylamide formation (FDE, 2011). The type of baking technology may have an impact on acrylamide contents. Infrared heating, as an alternative technology, seems to be an effective tool in acrylamide minimization, nevertheless, this approach is not fully acceptable for a production of traditional bread loaf for Czech consumers. (Ahrne, Andersson, Floberg, Rosén, & Lingnert, 2007; HEATOX Final Report, 2007). Within our study we studied several technological factors influencing the acrylamide formation during bread and rolls baking. We compared the products obtained from industrial
bakeries and the experimental products baked in the laboratory. The objective of our study was first to obtain more information about variability of acrylamide contents in commercial products (bread and rolls), prepared from locally grown cereals according to traditional recipes and baked under common industrial conditions. Second, based on the obtained data and considering current knowledge on acrylamide formation, we attempted to evaluate the factors that might contribute to the reduction of the acrylamide content in bakery products. We tested breads recipes with a varying content of rye and the use of different type of bread fermentation. 2. Experimental 2.1. Chemicals Acrylamide (CAS 79-06-1, purity 99.5%) and magnesium sulphate (p.a. purity 98%) were from SigmaeAldrich/Fluka (Switzerland). 13 C3-Acrylamide (isotopic purity 99%) was purchased from Cambridge Isotope Laboratories (USA). Sodium chloride was from Penta (Czech Republic). Aluminium oxide (basic) was from Merck (Germany). Acetonitrile and n-hexane were of HPLC grade quality and were supplied by SigmaeAldrich (Germany) and Merck (Germany), respectively. Deionized water was obtained from a Millipore apparatus (Merck Millipore, Germany). Calibration standard solutions of acrylamide were prepared in water in a concentration range of 1e 250 ng mL1 (1, 2.5, 5, 10, 50, 100, 250 ng mL1) by diluting the stock standard solutions containing 1000 or 10,000 ng mL1 with fixed amount of internal standard 13C3eacrylamide at the level of 100 ng mL1. The stock and calibration standard solutions were stored at 4 C. 2.2. Examined samples The samples analysed within the presented study differed in the production scale (industrial or laboratory), in the product type (wheat-rye fermented soft breads or wheat bakery products such as rolls) and in the recipes and technological factors which can possible influence the acrylamide content. 2.2.1. Breads 2.2.1.1. Experiment A: ten days monitoring of acrylamide contents in fermented bread - one bakery. Twenty wheat-rye bread samples from one industrial bakery were collected in ten subsequent days in two sets differing in recipes. The first set of samples represented common breads, the second set dark breads with the roasted barley malt added. The weight of bread loaves was standardized to 800 g. Within this experiment we were also determining the acrylamide content in two samples of roasted malts (black and brown), which are added in recipe when making the dark bread. Continuous belt oven was used for bread baking. 2.2.1.2. Experiment B: monitoring of acrylamide contents in fermented bread produced by various Czech bakeries. The set of 12 samples was collected from various industrial bakeries in the Czech Republic. All breads were of the same type (fermented bread) and produced, generally, using the same technology only slightly differing in a way of the dough leavening. The weight of bread loaves was standardized to 1200 g. 2.2.1.3. Experiment C: wheat-rye dough, influence of yeasts addition and size dilution (industrial bakeries, laboratory scale). Bread C1 e 26 breads from industrial bakeries made from wheat-rye flour (approx. 55e45% w/w mixture), were produced using natural rye sourdough for leavening only. The weight of bread loaves was standardized to 1300 g.
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Bread C2 e six breads were prepared in a laboratory oven, using the same formula with natural rye sourdough recipe as for bread C1. The weight of bread loaves was standardized to 450 g. Commercial standard Czech flours from industrial bakery classified as wheat flour T 1000 and rye flour T 930 were used. The oven (a laboratory oven for dough proofing, Zaklad Badawczy Przemyslu Piekarskiego, Poland) temperature was set from approx. 270e 280 C at the beginning of baking to approx. 220 C at the end of the baking process. Baking time was 30 min. Bread C3 e a group representing breads leavened with bakery yeasts included 34 samples. The bread was produced using a commercial starter (Lactobacilli) and yeasts. The weight of bread loaves was standardized to 1300 g.
was transferred into a new 50 mL centrifuge tube together with acetonitrile (10 mL), solid NaCl (0.5 g) and anhydrous MgSO4 (4 g). The mixture was vigorously shaken for 1 min and centrifuged at 10,000 rpm for 5 min. A volume of three mL of the acetonitrile layer were added to a 15 mL centrifuge tube containing 450 mg MgSO4 and 230 mg basic Al2O3. Mixture was shaken for about 1 min and centrifuged at 6000 rpm for 2 min. Supernatant was transferred into a 2 mL amber vial, solvent was removed by a gentle stream of nitrogen and water was added before the instrumental analysis. An aliquot (20 mL) of the water layer was injected into HPLC-MS/MS. A liquid chromatograph Alliance 2695 (Waters, USA) coupled to a mass spectrometer Quattro Premier XE (Waters/Micromass, USA/ UK) were used for measurements.
2.2.1.4. Experiment D: wheat-rye dough, dried rye sourdough, influence of rye flour content (laboratory scale). Within this experiment, we examined eight laboratory produced bread samples varying in the content of rye and wheat flours (15e85%). Because it is not technologically possible to use natural sourdough with the rye flour content lower than 42%, otherwise required texture is not obtained, dried rye sourdough was used. For that reason, it was necessary to add yeast as the leavening agent. Recipe: 300 g of flours; wheat flour (T1000) 15e85%; rye flour (T930) 85e15%, yeast 4%, dry sourdough 3%; salt solution 8%, water (addition to the final consistence of 400e450 Farinograph units). The oven temperature and baking profile was the same as in the experiment C2.
2.3.2. Instrumental analysis 2.3.2.1. High-performance liquid chromatographic separation. Chromatographic separation was carried out using an AtlantisÒ T3 analytical column (150 mm 3 mm; 3 mm). The column temperature was maintained at 35 C, with a column heater. Isocratic elution of acrylamide was achieved with mobile phase composed of acetonitrile and water (3:97, v/v). The flow rate was set to 0.3 mL min1 and injection volume was 20 mL. Under these HPLC condition the retention time of acrylamide was 4.3 min.
2.2.2. Rolls 2.2.2.1. Wheat rolls, industrial production. Several types of roll samples from industrial bakeries with typical weight of 60 g were collected. The continual production line with the continuous belt oven or a discontinuous box oven was used for baking. 2.2.2.2. Wheat bakery goods, the influence of the yeasts content (laboratory scale). Laboratory baked products corresponding to standard products from industrial bakeries were prepared according to the following recipe: wheat flour (T530) 100%, salt 1.5%, sunflower oil 3%, yeast 2.0e 4.0%, water (addition to the final consistence of 500 Farinograph units). The oven temperature was set from 240 C at the beginning of baking to approximately 210 C at the end of the baking process. The baking time was approx. 13e15 min. 2.2.2.3. Wheat bakery goods, the influence of vital gluten content (laboratory scale). Laboratory baked products corresponding to standard products from industrial bakeries were prepared according to the following recipe: wheat flour (T530) 100%, salt 1.5%, sunflower oil 3%, yeast 3%, vital gluten 0.5e2.5%, water (addition to the final consistence of 500 Farinograph units). The oven temperature and time profile was the same as in the previous experiment. 2.3. Acrylamide analysis 2.3.1. Sample preparation Before extraction, each analysed sample was divided into the crust and crumb. Samples were homogenized in a laboratory blender. An amount of 2 g of a representative sample were shaken for about 1 min with 10 mL of n-hexane in a 50 mL centrifuge tube. Afterwards, 20 mL of water and 200 mL (10 mg mL1) of an internal standard 13C3-acrylamide (an added amount corresponding to the concentration of 1000 mg kg1 matrix) were added and the mixture was shaken once again for approx. 1 min. Centrifugation at 10,000 rpm for 5 min followed. Ten mL aliquot of aqueous extract
2.3.2.2. Mass spectrometric detection. The mass spectrometer equipped with an electrospray interface (ESI) was operated in the positive ionization mode with a voltage of 4.5 kV on the capillary needle. Typical settings of the instrument were as follows: nitrogen desolvation gas flow: 700 L h1 at 450 C, nitrogen cone gas flow: 100 L h1, source temperature: 120 C, cone voltage: 21 V. The argon was used as a collision gas and its pressure was 9 103 bar. The two monitored transitions at unit resolution were for: acrylamide: m/z 72 / 55 (collision 10 V) and m/z 72 / 54 (collision 12 V); and one transition for 13C3eacrylamide (internal standard): m/z 75 / 58 (collision 10 V). The dwell time for each of MRM (multiple reaction monitoring) transitions was 0.1 s and inter channel delay was 0.02 s. MassLynx 4.1 equipped with an application manager QuanLynx was used for the data acquisition and processing. Each sample was measured in two replicates, the limit of detection (LOD) was 5 mg kg1. Due to supposed acrylamide low content in breads, to achieve reliable analytical data, acrylamide content was measured in bread crust and then recalculated to the whole loaf of bread. 3. Results and discussion 3.1. Experiment A: ten days monitoring of acrylamide contents in fermented bread from one Czech bakery As outlined in the Introduction, there are many factors that may influence acrylamide contents at producer’s site. Not only the changes in raw materials composition as regards acrylamide precursors content, but also (even small) fluctuation of technological process parameters are related to acrylamide formation (FDE, 2011). To learn more about the variability of acrylamide content in typical Czech fermented soft breads, we organized, in cooperation with one of high capacity bakeries, a small monitoring survey. The results of analysis of day after day collected crusts from freshly baked breads are shown in Fig. 1. In spite of a low detection limit of employed LCeMS/MS method (LOD ¼ 5 mg kg1), no acrylamide was found in any of bread crumbs. Commonly, in internal parts of loaf, the temperature during baking does not exceed 100 C and moisture content is around 40%, thus Maillard reaction originating acrylamide does not occur (Surdyk et al., 2004). On the other hand, in line with these studies reporting on distribution of acrylamide in
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fluctuating in a very narrow range. The explanation of a sudden increase after the day 5, become evident, when the temperature setting was controlled back: following the repair of baking oven thermostat, the new calibration setting was by 10 C higher. To understand more about possible contribution to the acrylamide content we analysed black and brown roasted barley malts which are used as colouring additives in a dough (0.3 and 0.6% w/w into the dough for black and brown malt, respectively) for production of ‘dark’ breads. While the ‘brown’ malt contained 502 mg kg1 of acrylamide, in the ‘black’ malt (prepared at prolonged roasting time) its content was by one order of magnitude lower e 37 mg kg1, probably due to the acrylamide reduction which can be observed as well during prolonged coffee roasting (Lantz et al., 2006). The hypothetical contribution of roasted malts to the total acrylamide content in bread loaf was calculated to be around 3 mg per 800 g loaf. Taking into account generally low contents of acrylamide in examined soft breads (2e20 mg kg1), the contribution of added malts can be, in some cases, relatively significant. Nevertheless, even in dark breads analysed within our study, the acrylamide contents were still very low compared to crisp breads in which the average contents above 200 mg kg1 were reported (EFSA, 2011). 3.2. Experiment B monitoring of acrylamide contents in bread produced in various Czech bakeries
Fig. 1. Ten days monitoring of acrylamide content in crust of typical Czech fermented dark (A) and common (B) breads produced in one of bakeries (Error bars indicate the analytical uncertainty).
similar breads (Surdyk et al., 2004), this compound was only occurred in the bread crusts. Nevertheless, thanks to specific baking technology of Czech breads, acrylamide formation is not too extensive. According to traditional process, the dough loaf surface is moistened before it is put into the oven which is then steamed. This set-up results in an additional wetting and cooling of the loaf surface by condensed steam. On this account, the temperature at the bread surface at the end of baking is not higher than 160 C. Generally, temperature increase of bread surface during baking is relatively slow, although in the oven, the highest temperature is typically at the beginning of the baking process e 260 C (then the temperature continuously decreases to 190 C). The delay of bread crust formation in spite of high oven temperature explains a limited extent of acrylamide formation in this, otherwise starch rich bakery product. The mean content of acrylamide in crusts was not influenced by the addition of malt (dark bread) that can represent a potential source of acrylamide. Considering that the separated crust represented approximately 16% of the loaf weight in this type of breads, the acrylamide contents recalculated to an 800 g loaf ranged, as documented in Fig. 1, from 2 to 20 mg kg1 of bread. Unexpectedly, the mean content found for the days 1e5 was significantly lower (2 and 4 mg kg1 for dark and common breads, respectively) compared to that in the second part of the survey (5 and 13 mg kg1 for dark and common breads, respectively). When tracing back the reason for this difference, all the relevant factors were considered. The same batches of wheat and rye flours, sourdoughs and other ingredients were used over the entire monitored period (their amount used for dough preparation was sampled automatically), also the pH value of the dough recorded by the producer was
The next phase of our study was focused on monitoring of acrylamide contents in traditional soft breads from various Czech bakeries that employ practically the same dough recipes and similar bread making technology, possibly only slightly differing in the way of dough leavening. The results of crust analyses shown in Fig. 2 illustrate relatively small variations of acrylamide contents among the crusts of traditional soft breads available at the Czech market. The acrylamide content in 12 tested samples ranged from 7 to 22 mg kg1 with a mean of 16 mg kg1. The calculated relative standard deviation for the data set was 30%. While in most cases, sourdough for leavening was prepared using a commercial mixture of Lactobacilli as a starter together with commercial bakery yeast, the breads from bakeries no. 5 and 9, that contained the lowest acrylamide contents, were leavened using a natural rye (home-made) sourdough obtained by spontaneous fermentation of rye flour. It contains naturally originated microorganisms as Saccharomyces cerevisiae. We assumed that pH dough value of the breads no. 5 and 9 might be lower due to the higher acidity of the used sourdough, therefore responsible for reduction of acrylamide formation. This trend would be in an agreement with the results of several other studies demonstrating reduced extent of contamination in fermented bakery products (Claus, Mongili, Weisz, Schreiber, & Carle, 2008; Fredriksson et al.,
Fig. 2. The acrylamide content in crust of breads baked in different Czech bakeries (Error bars indicate the analytical uncertainty).
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2004). To learn more about the effect of the way of leavening, we conducted additional complementary experiments. 3.3. Experiment C e wheat-rye breads, the effect of the type of leavening Within this set of experiments, we compared two basic types of mixed wheat-rye bread leavening procedures. The first type of bread was leavened using a natural rye sourdough, the baking was conducted either in industrial bakery or under laboratory conditions in a small oven. (Experiments C1 and C2, resp.). For the second type of breads, a sourdough was prepared from a commercial mixture of Lactobacilli and bakery yeasts. These breads were baked in an industrial bakery (Experiment C3). As illustrated in Fig. 3, the highest mean acrylamide content in bread crusts, 150 mg kg1, was found in the Experiment C3 (1300 g loaf). In the C2 (450 g loaf) and C1 (1300 g loaf) experiments the contamination was lower, 95 and 73 mg kg1, respectively). After recalculating the acryalamide content from the crust to the whole loaf, the mean content in the experimental breads C3, C2 and C1 was 25, 15 and 10 mg kg1, resp. The C2/C1 acrylamide content ratio (3/2) reflects the crust/crumb ratio which is higher in smaller C2 loaves. 3.4. Experiment D wheat-rye dough, dried rye sourdough, influence of rye/wheat flour ratio (laboratory scale) As mentioned above, the composition of raw materials for making particular type of bread leavened using natural rye sourdough may slightly differ among bakeries. The weight ratios of wheat and rye flours in traditional Czech soft fermented bread recipes are typically around 1:1, the actual value often reflects the availability of particular commodity at the market (and its cost as well). Nevertheless the ratio should not exceed 2:3 to ensure optimal fermentation without yeast addition. To enable the use of even higher ratios of rye/wheat flour and assess the impact of this raw material on the extent of acrylamide formation in bread, dry rye sourdough had to be used. The initiation of leavening was achieved by bakery yeasts addition. The acrylamide content in wheat rye breads ranged from 30 to 47 mg kg1 of bread loaf. As shown in Fig. 4, no relationship between the portion of rye flour in dough and the final acrylamide content in bread was found. 3.5. Acrylamide content in standard wheat bakery goods (industrial and laboratory scale) The acrylamide content in commercially baked wheat rolls available at the Czech market was generally very low with no
Fig. 3. The average values (including the variability of the data sets with the lowest and the highest contents) of the acrylamide contents in bread crusts depending on the type of leavening.
Fig. 4. The acrylamide contents in bread loaves (450 g) depending on the portion of rye flour in recipe.
impact of the type of baking oven. When calculated to the whole roll (typically 60 g), the acrylamide content was lower than 10 mg kg1 of roll. The same result was obtained for laboratory scale baking, the acrylamide contents were lower than 10 mg kg1 of roll. Varying amount of yeast or vital gluten did not seem to have any systemic impact on the acrylamide contents, as can be seen in Figs. 5 and 6. 4. Conclusions The current study concerned both with the monitoring of the acrylamide occurrence in several types of commercial breads available at the Czech market and prepared by experimental baking aimed at the assessment of various factors related to acrylamide formation. The main outcomes can be summarized as follows: The contents of acrylamide found in traditional Czech soft breads available at the market (16 mg kg1 mean/17 mg kg1 median) were lower compared to values reported by EFSA (EFSA, 2012) for years 2007e2010 (75/25, 53/24, 46/20, 30/ 18 mg kg1, resp.); in none of them the target analyte exceeded 21 mg kg1 that is more than one order of magnitude less than maximum contents reported in EU database. It might be assumed that delayed crust formation (in spite of high oven temperature) during specific regime in baking oven (temperature/humidity) is responsible for a rather limited extent of the Maillard reaction. The maximum content of acrylamide in bread crust (approx. 15% w/w for 1200 g loaf) from commercial breads was 155 mg kg1. Considering the growing popularity of small soft bread loafs with higher crust/crumb ratio, the per portion acrylamide intake might become higher, since acrylamide is contained exclusively in a bread crust.
Fig. 5. The acrylamide contents in laboratory baked rolls depending on the portion of added yeast in recipe.
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Fig. 6. The acrylamide contents in laboratory baked rolls depending on the portion of added vital gluten in recipe.
Lower acrylamide contents were found in breads leavened by natural rye sourdough, probably due to lower pH values of dough achieved during fermentation. No relationship was found between the rye/wheat ratio in the laboratory baked breads and the amount of acrylamide in crust. Use of brown/black barley malts for breads colouring influenced acrylamide contents insignificantly; although contamination of these additives was relatively high compared to bread. The changes in formulation of wheat rolls such as addition of various amounts of vital gluten or bakery yeasts did not show any systemic impact on acrylamide contents which were generally low, not exceeding 10 mg kg1 in 60 g product. Finally it can be conclude, that the contents of acrylamide in the Czech breads are relatively low, which is in agreement with the recent EFSA report (EFSA, 2011), which identified soft breads, contrary to crisp breads, as a food category with a low acrylamide content. In spite of that, the contribution to the acrylamide dietary intake cannot be overlooked since soft bread consumption in the Czech Republic is relatively high. As mentioned in the Introduction, the contribution of soft bread to the total acrylamide dietary exposure in the Czech Republic is 13% for adults and 9e10% for children and adolescents (EFSA, 2011). Acknowledgement This study was carried out with the support from the following projects financed by the Ministry of Education, Youth and Sports of the Czech Republic: (i) the NPV II. project 2B06168; (ii) project MEB 080882 and (iii) Specific University Research (MSMT No. 20/2013). References Ahrne, L., Andersson, C. G., Floberg, P., Rosén, J., & Lingnert, H. (2007). Effect of crust temperature and water content on acrylamide formation during baking of
white bread: steam and falling temperature baking. LWT - Food Science and Technology, 40, 1708e1715. Amrein, T., Andres, L., Escher, F., & Amado, R. (2007). Occurrence of acrylamide in selected foods and mitigation options. Food Additives and Contaminants, 24(S1), 13e25. Brathen, E., Kita, A., Knutsen, S. H., & Wicklund, T. (2005). Addition of glycine reduces the content of acrylamide in cereal and potato products. Journal of Agricultural and Food Chemistry, 53, 3259e3262. Claus, A., Mongili, M., Weisz, G., Schreiber, A., & Carle, R. (2008). Impact of formulation and technological factors on the acrylamide content of wheat bread and bread rolls. Journal of Cereal Science, 47, 546e554. EC. (2011). Commission recommendation on investigations into the levels of acrylamide in food Document C(2010) 9681 final of 10.1.2011. EFSA. (2011). European Food Safety Authority. Results on acrylamide levels in food from monitoring years 2007e2009 and exposure assessment, Scientific report of EFSA, Parma, Italy. EFSA Journal, 9(4), 2133. EFSA. (2012). European Food Safety Authority. Update on acrylamide levels in food from monitoring years 2007 to 2010, Scientific report of EFSA, Parma, Italy. EFSA Journal, 10(10), 2938. European Commission Recommendation from the 2nd of June 2010 (on the monitoring of acrylamide levels in food; 2010/307/EU). FDE. (2011). FoodDrinkEurope acrylamide Toolbox. http://www.fooddrinkeurope.eu/ uploads/publications_documents/Toolboxfinal260911.pdf Accessed 19.07.13. Fink, M., Andersson, R., Rosén, J., & Aman, P. (2006). Effect of added asparagine and glycine on acrylamide content in yeast-leavened bread. Cereal Chemistry, 83, 218e222. Fredriksson, H., Tallving, J., Rosén, J., & Aman, P. (2004). Fermentation reduces free asparagine in dough and acrylamide content in bread. Cereal Chemistry, 81, 650e653. Hamlet, C. G., Baxter, D. E., Sadd, P. A., Slaiding, I., Liang, L., Muller, R., et al. (2005). Exploiting process factors to reduce acrylamide in cereal-based foods. High Wycombe: RHM Technology Ltd. C03032 and C03026. Report C014 prepared on behalf of the UK Food Standards Agency. Hamlet, C. G., Sadd, P. A., & Liang, L. (2008). Correlations between the amounts of free asparagine and saccharides present in commercial cereal flours in the United Kingdom and the generation of acrylamide during cooking. Journal of Agricultural and Food Chemistry, 56, 6145e6153. HEATOX. (2007). HEATOX: Heat-generated food toxicants: Identification, characterisation and risk minimisation. Final report. Lund, Sweden: Lund University http:// www.slv.se/upload/heatox/documents/Heatox_Final%20_report.pdf Accessed 19.06.13. IARC. (1994). Acrylamide. In IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans (Vol. 60; pp. 389e433). Lyon, France: International Agency for Research on Cancer. Konings, E. J. M., Ashby, P., Hamlet, C. G., & Thompson, G. A. K. (2007). Acrylamide in cereal and cereal products: a review on progress in level reduction. Food Additives and Contaminants, 24(S1), 47e59. Lantz, I., Ternite, R., Wilkens, J., Hoenicke, K., Guenther, H., & van der Stegen, G. H. D. (2006). Studies on acrylamide levels in roasting, storage and brewing of coffee. Molecular Nutrition & Food Research, 50, 1039e1046. project n. 2B06168National project NPV II results2006)2011). Ministry of Education, Youth and Sports of the Czech Republic. Sadd, P. A., Hamlet, C. G., & Liang, L. (2008). Effectiveness of methods for reducing acrylamide in bakery products. Journal of Agricultural and Food Chemistry, 56, 6154e6161. Surdyk, N., Rosén, J., Andersson, R., & Aman, P. (2004). Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread. Journal of Agricultural and Food Chemistry, 52, 2047e2051. Taeymans, D., Ashby, P., Blank, I., Gondé, P., van Eijck, P., Lalljie, S., et al. (2004). A review of acrylamide: an industry perspective on research, analysis, formation and control. Critical Reviews in Food Science and Nutrition, 44, 323e347. WHO. (2002). FAO/WHO consultation on the health implications of acrylamide in food. Summary report of a meeting held in Geneva, 25e27 June 2002. Geneva, Switzerland: World Health Organization. http://www.who.int/foodsafety/ publications/chem/acrylamide_june2002/en/ Accessed 19.06.13.
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Acrylamide formation in traditional Czech leavened wheat-rye breads and wheat rolls Veronika Forstova a, Beverly Belkova a, Katerina Riddellova a, Lukas Vaclavik a, Josef Prihoda b, Jana Hajslova a, * a Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technicka 3, 166 28 Prague 6, Czech Republic b Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Department of Carbohydrates and Cereals, Technicka 3, 166 28 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2013 Received in revised form 8 October 2013 Accepted 15 October 2013
The main aim of our study was to collect the data on the acrylamide content in typical Czech leavened wheat-rye breads and wheat rolls, and to demonstrate the influence of various technological factors on its contents. We analysed several sets of industrially and laboratory baked breads to better understand the acrylamide formation. Following factors were tested: (i) the content of rye in respective bread formulae and (ii) the character of leavening procedure. We were, as well, monitoring for ten days the variability of acrylamide levels in bread baked by one bakery and, afterwards, we analysed the same type of bread baked in 12 bakeries. In the case of rolls, the influence of yeasts and vital gluten content in the dough were subjects of an assessment. The levels of acrylamide in a common type of the Czech bread baked in various bakeries were relatively low, ranging from 7 to 22 mg kg1. The content of acrylamide was rather higher in breads prepared using a commercial starter (Lactobacilli) and yeasts than in bread prepared using natural rye sourdough only. A varying content of rye flour did not seem to have any impact on acrylamide levels. The ‘size dilution effect’ was observed when comparing the same type of bread with different weight. Since acrylamide is exclusively located in the bread crust, smaller loaves contained higher acrylamide content per kg. The contents of acrylamide in standard wheat bakery goods were very low, bellow 10 mg kg1. No influence of varying content of added yeast or vital gluten was observed. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Acrylamide Bread Rye flour Wheat flour Sourdough Yeasts
1. Introduction Acrylamide (prop-2-enamide) has been classified as a probable human carcinogen 2A (IARC, 1994) and is known as neurotoxin (WHO, 2002). This compound is formed during the Maillard reaction, especially in heat-processed starch-rich foods such as potato and bakery products. High acrylamide contents can be found in coffee and coffee surrogates, as well (EFSA, 2011). Currently, acrylamide is monitored in the European Union member’s states according to the European Commission Recommendation (2010/307/EU). Indicative acrylamide values based on the EFSA (European Food Safety Authority) monitoring data from 2007 to 2008 were established within the European Commission recommendation released on January 10, 2011 (“on investigations into the levels of acrylamide in food”) (EC, 2011). The indicative values are not safety thresholds, but, if exceeded, * Corresponding author. Tel.: þ420 220 443 185. E-mail address: [email protected] (J. Hajslova). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.10.022
subsequent investigation of the reasons should be conducted. Cereal products are there divided into 5 categories with relevant indicative values: Soft bread (150 mg kg1); Breakfast cereals excl. muesli and porridge (400 mg kg1); Biscuits, crackers, wafers, crisp bread and similar, excl. ginger bread (500 mg kg1); Biscuits and rusks for infants and young children (250 mg kg1); Processed cereal based foods for infants and young children, excl. biscuits and rusks (100 mg kg1) (EC, 2011). For other commodities such as potato products or coffee indicative values are available as well (EC, 2011). The latest exposure estimates in Europe evaluated by European Food Safety Authority (EFSA) were reported within the EFSA scientific report in 2011 (EFSA, 2011). Besides of potato products and coffee, bakery products, including soft bread and biscuits, have been identified as significant contributors to the overall acrylamide exposure through all groups of population (EFSA, 2011). The found mean acrylamide contents in soft breads ranged between 30 and 75 mg kg1 within the years 2007e2010 (EFSA, 2012). Despite the fact that acrylamide contents found in EU countries in soft breads are reasonably low, it is an important acrylamide source in
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European diets (EFSA, 2011). The results obtained within the EFSA report demonstrate that the contribution to the acrylamide dietary intake from soft bread in EU countries is approximately 20% among children and adolescents and up to 30% among adults (EFSA, 2011). These percentages are influenced by many factors such as age or gender of the respondents or the consumer’s habits. In the Czech Republic, the contribution of bakery products to consumers’ exposure to acrylamide is important due to their relatively high daily consumption. The contribution of soft bread to the total acrylamide dietary exposure in the Czech Republic is 13% for adults and 9e10% for children and adolescents (EFSA, 2011). The results of several studies confirmed asparagine as a key precursor of acrylamide formation in cereal based heat-treated food (Hamlet, Sadd, & Liang, 2008; Taeymans et al., 2004). According to the results of 5 years monitoring study (2006e2010), the mean asparagine content in wheat flours in the Czech Republic was found in the range from 36 to 309 mg kg1 (National project NPV II results). These values found in crop grown in the Czech Republic were comparable with those reported in other countries (69e 443 mg kg1) (Konings, Ashby, Hamlet, & Thompson, 2007). It should be noted that not only the asparagine content in flour but also the glucose/fructose ratio and other ingredients added to the dough play an important role (Hamlet et al., 2008). Several studies were concerned with a detailed monitoring of factors influencing acrylamide formation in bakery products with the aim to find the conditions enabling reduction of acrylamide. The current state of the art is summarized in the ‘Toolbox’, the document published by Food Drink Europe in 2011(FDE, 2011). For instance the addition of Ca2þ salts into flours was shown to have a potential for significant acrylamide reduction. The fortification of flour by 0.3% of Ca2þ salts is required by the U.K. law for nutritional reasons and results in up to 30% decrease according to some authors (Hamlet et al., 2005; Sadd, Hamlet, & Liang, 2008). The addition of glycine may lead to acrylamide reduction in yeast leavened bread. Nevertheless, the addition of high amounts of this amino acid may have a negative impact on yeast fermentation (Brathen, Kita, Knutsen, & Wicklund, 2005; Fink, Andersson, Rosén, & Aman, 2006). Size dilution in bread seems to be another important factor influencing the acrylamide content expressed on the whole product. The acrylamide formation is typical for the bread crust, where the temperature reaches much higher values than in the bread crumb, where the temperatures do not exceed 100 C (Surdyk, Rosén, Andersson, & Aman, 2004). Production of a larger bread loaf can reduce the average acrylamide content in a loaf due to the dilution caused by decreasing the ratio surface area to the volume ratio (Amrein, Andres, Escher, & Amado, 2007; Surdyk et al., 2004). Another possible way for reduction of the acrylamide amount is prolonged yeast fermentation during which asparagine is assimilated by yeasts (Fredriksson, Tallving, Rosén, & Aman, 2004). Some studies have shown an acrylamide reduction of about 50% after 1 h of fermentation (Hamlet et al., 2005; Sadd et al., 2008). Nevertheless, the reduction was not so successful in industrially prepared breads. During dough baking, the time and temperature are the crucial factors responsible for the extent of thermal input and, consequently, of acrylamide formation (FDE, 2011). The type of baking technology may have an impact on acrylamide contents. Infrared heating, as an alternative technology, seems to be an effective tool in acrylamide minimization, nevertheless, this approach is not fully acceptable for a production of traditional bread loaf for Czech consumers. (Ahrne, Andersson, Floberg, Rosén, & Lingnert, 2007; HEATOX Final Report, 2007). Within our study we studied several technological factors influencing the acrylamide formation during bread and rolls baking. We compared the products obtained from industrial
bakeries and the experimental products baked in the laboratory. The objective of our study was first to obtain more information about variability of acrylamide contents in commercial products (bread and rolls), prepared from locally grown cereals according to traditional recipes and baked under common industrial conditions. Second, based on the obtained data and considering current knowledge on acrylamide formation, we attempted to evaluate the factors that might contribute to the reduction of the acrylamide content in bakery products. We tested breads recipes with a varying content of rye and the use of different type of bread fermentation. 2. Experimental 2.1. Chemicals Acrylamide (CAS 79-06-1, purity 99.5%) and magnesium sulphate (p.a. purity 98%) were from SigmaeAldrich/Fluka (Switzerland). 13 C3-Acrylamide (isotopic purity 99%) was purchased from Cambridge Isotope Laboratories (USA). Sodium chloride was from Penta (Czech Republic). Aluminium oxide (basic) was from Merck (Germany). Acetonitrile and n-hexane were of HPLC grade quality and were supplied by SigmaeAldrich (Germany) and Merck (Germany), respectively. Deionized water was obtained from a Millipore apparatus (Merck Millipore, Germany). Calibration standard solutions of acrylamide were prepared in water in a concentration range of 1e 250 ng mL1 (1, 2.5, 5, 10, 50, 100, 250 ng mL1) by diluting the stock standard solutions containing 1000 or 10,000 ng mL1 with fixed amount of internal standard 13C3eacrylamide at the level of 100 ng mL1. The stock and calibration standard solutions were stored at 4 C. 2.2. Examined samples The samples analysed within the presented study differed in the production scale (industrial or laboratory), in the product type (wheat-rye fermented soft breads or wheat bakery products such as rolls) and in the recipes and technological factors which can possible influence the acrylamide content. 2.2.1. Breads 2.2.1.1. Experiment A: ten days monitoring of acrylamide contents in fermented bread - one bakery. Twenty wheat-rye bread samples from one industrial bakery were collected in ten subsequent days in two sets differing in recipes. The first set of samples represented common breads, the second set dark breads with the roasted barley malt added. The weight of bread loaves was standardized to 800 g. Within this experiment we were also determining the acrylamide content in two samples of roasted malts (black and brown), which are added in recipe when making the dark bread. Continuous belt oven was used for bread baking. 2.2.1.2. Experiment B: monitoring of acrylamide contents in fermented bread produced by various Czech bakeries. The set of 12 samples was collected from various industrial bakeries in the Czech Republic. All breads were of the same type (fermented bread) and produced, generally, using the same technology only slightly differing in a way of the dough leavening. The weight of bread loaves was standardized to 1200 g. 2.2.1.3. Experiment C: wheat-rye dough, influence of yeasts addition and size dilution (industrial bakeries, laboratory scale). Bread C1 e 26 breads from industrial bakeries made from wheat-rye flour (approx. 55e45% w/w mixture), were produced using natural rye sourdough for leavening only. The weight of bread loaves was standardized to 1300 g.
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Bread C2 e six breads were prepared in a laboratory oven, using the same formula with natural rye sourdough recipe as for bread C1. The weight of bread loaves was standardized to 450 g. Commercial standard Czech flours from industrial bakery classified as wheat flour T 1000 and rye flour T 930 were used. The oven (a laboratory oven for dough proofing, Zaklad Badawczy Przemyslu Piekarskiego, Poland) temperature was set from approx. 270e 280 C at the beginning of baking to approx. 220 C at the end of the baking process. Baking time was 30 min. Bread C3 e a group representing breads leavened with bakery yeasts included 34 samples. The bread was produced using a commercial starter (Lactobacilli) and yeasts. The weight of bread loaves was standardized to 1300 g.
was transferred into a new 50 mL centrifuge tube together with acetonitrile (10 mL), solid NaCl (0.5 g) and anhydrous MgSO4 (4 g). The mixture was vigorously shaken for 1 min and centrifuged at 10,000 rpm for 5 min. A volume of three mL of the acetonitrile layer were added to a 15 mL centrifuge tube containing 450 mg MgSO4 and 230 mg basic Al2O3. Mixture was shaken for about 1 min and centrifuged at 6000 rpm for 2 min. Supernatant was transferred into a 2 mL amber vial, solvent was removed by a gentle stream of nitrogen and water was added before the instrumental analysis. An aliquot (20 mL) of the water layer was injected into HPLC-MS/MS. A liquid chromatograph Alliance 2695 (Waters, USA) coupled to a mass spectrometer Quattro Premier XE (Waters/Micromass, USA/ UK) were used for measurements.
2.2.1.4. Experiment D: wheat-rye dough, dried rye sourdough, influence of rye flour content (laboratory scale). Within this experiment, we examined eight laboratory produced bread samples varying in the content of rye and wheat flours (15e85%). Because it is not technologically possible to use natural sourdough with the rye flour content lower than 42%, otherwise required texture is not obtained, dried rye sourdough was used. For that reason, it was necessary to add yeast as the leavening agent. Recipe: 300 g of flours; wheat flour (T1000) 15e85%; rye flour (T930) 85e15%, yeast 4%, dry sourdough 3%; salt solution 8%, water (addition to the final consistence of 400e450 Farinograph units). The oven temperature and baking profile was the same as in the experiment C2.
2.3.2. Instrumental analysis 2.3.2.1. High-performance liquid chromatographic separation. Chromatographic separation was carried out using an AtlantisÒ T3 analytical column (150 mm 3 mm; 3 mm). The column temperature was maintained at 35 C, with a column heater. Isocratic elution of acrylamide was achieved with mobile phase composed of acetonitrile and water (3:97, v/v). The flow rate was set to 0.3 mL min1 and injection volume was 20 mL. Under these HPLC condition the retention time of acrylamide was 4.3 min.
2.2.2. Rolls 2.2.2.1. Wheat rolls, industrial production. Several types of roll samples from industrial bakeries with typical weight of 60 g were collected. The continual production line with the continuous belt oven or a discontinuous box oven was used for baking. 2.2.2.2. Wheat bakery goods, the influence of the yeasts content (laboratory scale). Laboratory baked products corresponding to standard products from industrial bakeries were prepared according to the following recipe: wheat flour (T530) 100%, salt 1.5%, sunflower oil 3%, yeast 2.0e 4.0%, water (addition to the final consistence of 500 Farinograph units). The oven temperature was set from 240 C at the beginning of baking to approximately 210 C at the end of the baking process. The baking time was approx. 13e15 min. 2.2.2.3. Wheat bakery goods, the influence of vital gluten content (laboratory scale). Laboratory baked products corresponding to standard products from industrial bakeries were prepared according to the following recipe: wheat flour (T530) 100%, salt 1.5%, sunflower oil 3%, yeast 3%, vital gluten 0.5e2.5%, water (addition to the final consistence of 500 Farinograph units). The oven temperature and time profile was the same as in the previous experiment. 2.3. Acrylamide analysis 2.3.1. Sample preparation Before extraction, each analysed sample was divided into the crust and crumb. Samples were homogenized in a laboratory blender. An amount of 2 g of a representative sample were shaken for about 1 min with 10 mL of n-hexane in a 50 mL centrifuge tube. Afterwards, 20 mL of water and 200 mL (10 mg mL1) of an internal standard 13C3-acrylamide (an added amount corresponding to the concentration of 1000 mg kg1 matrix) were added and the mixture was shaken once again for approx. 1 min. Centrifugation at 10,000 rpm for 5 min followed. Ten mL aliquot of aqueous extract
2.3.2.2. Mass spectrometric detection. The mass spectrometer equipped with an electrospray interface (ESI) was operated in the positive ionization mode with a voltage of 4.5 kV on the capillary needle. Typical settings of the instrument were as follows: nitrogen desolvation gas flow: 700 L h1 at 450 C, nitrogen cone gas flow: 100 L h1, source temperature: 120 C, cone voltage: 21 V. The argon was used as a collision gas and its pressure was 9 103 bar. The two monitored transitions at unit resolution were for: acrylamide: m/z 72 / 55 (collision 10 V) and m/z 72 / 54 (collision 12 V); and one transition for 13C3eacrylamide (internal standard): m/z 75 / 58 (collision 10 V). The dwell time for each of MRM (multiple reaction monitoring) transitions was 0.1 s and inter channel delay was 0.02 s. MassLynx 4.1 equipped with an application manager QuanLynx was used for the data acquisition and processing. Each sample was measured in two replicates, the limit of detection (LOD) was 5 mg kg1. Due to supposed acrylamide low content in breads, to achieve reliable analytical data, acrylamide content was measured in bread crust and then recalculated to the whole loaf of bread. 3. Results and discussion 3.1. Experiment A: ten days monitoring of acrylamide contents in fermented bread from one Czech bakery As outlined in the Introduction, there are many factors that may influence acrylamide contents at producer’s site. Not only the changes in raw materials composition as regards acrylamide precursors content, but also (even small) fluctuation of technological process parameters are related to acrylamide formation (FDE, 2011). To learn more about the variability of acrylamide content in typical Czech fermented soft breads, we organized, in cooperation with one of high capacity bakeries, a small monitoring survey. The results of analysis of day after day collected crusts from freshly baked breads are shown in Fig. 1. In spite of a low detection limit of employed LCeMS/MS method (LOD ¼ 5 mg kg1), no acrylamide was found in any of bread crumbs. Commonly, in internal parts of loaf, the temperature during baking does not exceed 100 C and moisture content is around 40%, thus Maillard reaction originating acrylamide does not occur (Surdyk et al., 2004). On the other hand, in line with these studies reporting on distribution of acrylamide in
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fluctuating in a very narrow range. The explanation of a sudden increase after the day 5, become evident, when the temperature setting was controlled back: following the repair of baking oven thermostat, the new calibration setting was by 10 C higher. To understand more about possible contribution to the acrylamide content we analysed black and brown roasted barley malts which are used as colouring additives in a dough (0.3 and 0.6% w/w into the dough for black and brown malt, respectively) for production of ‘dark’ breads. While the ‘brown’ malt contained 502 mg kg1 of acrylamide, in the ‘black’ malt (prepared at prolonged roasting time) its content was by one order of magnitude lower e 37 mg kg1, probably due to the acrylamide reduction which can be observed as well during prolonged coffee roasting (Lantz et al., 2006). The hypothetical contribution of roasted malts to the total acrylamide content in bread loaf was calculated to be around 3 mg per 800 g loaf. Taking into account generally low contents of acrylamide in examined soft breads (2e20 mg kg1), the contribution of added malts can be, in some cases, relatively significant. Nevertheless, even in dark breads analysed within our study, the acrylamide contents were still very low compared to crisp breads in which the average contents above 200 mg kg1 were reported (EFSA, 2011). 3.2. Experiment B monitoring of acrylamide contents in bread produced in various Czech bakeries
Fig. 1. Ten days monitoring of acrylamide content in crust of typical Czech fermented dark (A) and common (B) breads produced in one of bakeries (Error bars indicate the analytical uncertainty).
similar breads (Surdyk et al., 2004), this compound was only occurred in the bread crusts. Nevertheless, thanks to specific baking technology of Czech breads, acrylamide formation is not too extensive. According to traditional process, the dough loaf surface is moistened before it is put into the oven which is then steamed. This set-up results in an additional wetting and cooling of the loaf surface by condensed steam. On this account, the temperature at the bread surface at the end of baking is not higher than 160 C. Generally, temperature increase of bread surface during baking is relatively slow, although in the oven, the highest temperature is typically at the beginning of the baking process e 260 C (then the temperature continuously decreases to 190 C). The delay of bread crust formation in spite of high oven temperature explains a limited extent of acrylamide formation in this, otherwise starch rich bakery product. The mean content of acrylamide in crusts was not influenced by the addition of malt (dark bread) that can represent a potential source of acrylamide. Considering that the separated crust represented approximately 16% of the loaf weight in this type of breads, the acrylamide contents recalculated to an 800 g loaf ranged, as documented in Fig. 1, from 2 to 20 mg kg1 of bread. Unexpectedly, the mean content found for the days 1e5 was significantly lower (2 and 4 mg kg1 for dark and common breads, respectively) compared to that in the second part of the survey (5 and 13 mg kg1 for dark and common breads, respectively). When tracing back the reason for this difference, all the relevant factors were considered. The same batches of wheat and rye flours, sourdoughs and other ingredients were used over the entire monitored period (their amount used for dough preparation was sampled automatically), also the pH value of the dough recorded by the producer was
The next phase of our study was focused on monitoring of acrylamide contents in traditional soft breads from various Czech bakeries that employ practically the same dough recipes and similar bread making technology, possibly only slightly differing in the way of dough leavening. The results of crust analyses shown in Fig. 2 illustrate relatively small variations of acrylamide contents among the crusts of traditional soft breads available at the Czech market. The acrylamide content in 12 tested samples ranged from 7 to 22 mg kg1 with a mean of 16 mg kg1. The calculated relative standard deviation for the data set was 30%. While in most cases, sourdough for leavening was prepared using a commercial mixture of Lactobacilli as a starter together with commercial bakery yeast, the breads from bakeries no. 5 and 9, that contained the lowest acrylamide contents, were leavened using a natural rye (home-made) sourdough obtained by spontaneous fermentation of rye flour. It contains naturally originated microorganisms as Saccharomyces cerevisiae. We assumed that pH dough value of the breads no. 5 and 9 might be lower due to the higher acidity of the used sourdough, therefore responsible for reduction of acrylamide formation. This trend would be in an agreement with the results of several other studies demonstrating reduced extent of contamination in fermented bakery products (Claus, Mongili, Weisz, Schreiber, & Carle, 2008; Fredriksson et al.,
Fig. 2. The acrylamide content in crust of breads baked in different Czech bakeries (Error bars indicate the analytical uncertainty).
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2004). To learn more about the effect of the way of leavening, we conducted additional complementary experiments. 3.3. Experiment C e wheat-rye breads, the effect of the type of leavening Within this set of experiments, we compared two basic types of mixed wheat-rye bread leavening procedures. The first type of bread was leavened using a natural rye sourdough, the baking was conducted either in industrial bakery or under laboratory conditions in a small oven. (Experiments C1 and C2, resp.). For the second type of breads, a sourdough was prepared from a commercial mixture of Lactobacilli and bakery yeasts. These breads were baked in an industrial bakery (Experiment C3). As illustrated in Fig. 3, the highest mean acrylamide content in bread crusts, 150 mg kg1, was found in the Experiment C3 (1300 g loaf). In the C2 (450 g loaf) and C1 (1300 g loaf) experiments the contamination was lower, 95 and 73 mg kg1, respectively). After recalculating the acryalamide content from the crust to the whole loaf, the mean content in the experimental breads C3, C2 and C1 was 25, 15 and 10 mg kg1, resp. The C2/C1 acrylamide content ratio (3/2) reflects the crust/crumb ratio which is higher in smaller C2 loaves. 3.4. Experiment D wheat-rye dough, dried rye sourdough, influence of rye/wheat flour ratio (laboratory scale) As mentioned above, the composition of raw materials for making particular type of bread leavened using natural rye sourdough may slightly differ among bakeries. The weight ratios of wheat and rye flours in traditional Czech soft fermented bread recipes are typically around 1:1, the actual value often reflects the availability of particular commodity at the market (and its cost as well). Nevertheless the ratio should not exceed 2:3 to ensure optimal fermentation without yeast addition. To enable the use of even higher ratios of rye/wheat flour and assess the impact of this raw material on the extent of acrylamide formation in bread, dry rye sourdough had to be used. The initiation of leavening was achieved by bakery yeasts addition. The acrylamide content in wheat rye breads ranged from 30 to 47 mg kg1 of bread loaf. As shown in Fig. 4, no relationship between the portion of rye flour in dough and the final acrylamide content in bread was found. 3.5. Acrylamide content in standard wheat bakery goods (industrial and laboratory scale) The acrylamide content in commercially baked wheat rolls available at the Czech market was generally very low with no
Fig. 3. The average values (including the variability of the data sets with the lowest and the highest contents) of the acrylamide contents in bread crusts depending on the type of leavening.
Fig. 4. The acrylamide contents in bread loaves (450 g) depending on the portion of rye flour in recipe.
impact of the type of baking oven. When calculated to the whole roll (typically 60 g), the acrylamide content was lower than 10 mg kg1 of roll. The same result was obtained for laboratory scale baking, the acrylamide contents were lower than 10 mg kg1 of roll. Varying amount of yeast or vital gluten did not seem to have any systemic impact on the acrylamide contents, as can be seen in Figs. 5 and 6. 4. Conclusions The current study concerned both with the monitoring of the acrylamide occurrence in several types of commercial breads available at the Czech market and prepared by experimental baking aimed at the assessment of various factors related to acrylamide formation. The main outcomes can be summarized as follows: The contents of acrylamide found in traditional Czech soft breads available at the market (16 mg kg1 mean/17 mg kg1 median) were lower compared to values reported by EFSA (EFSA, 2012) for years 2007e2010 (75/25, 53/24, 46/20, 30/ 18 mg kg1, resp.); in none of them the target analyte exceeded 21 mg kg1 that is more than one order of magnitude less than maximum contents reported in EU database. It might be assumed that delayed crust formation (in spite of high oven temperature) during specific regime in baking oven (temperature/humidity) is responsible for a rather limited extent of the Maillard reaction. The maximum content of acrylamide in bread crust (approx. 15% w/w for 1200 g loaf) from commercial breads was 155 mg kg1. Considering the growing popularity of small soft bread loafs with higher crust/crumb ratio, the per portion acrylamide intake might become higher, since acrylamide is contained exclusively in a bread crust.
Fig. 5. The acrylamide contents in laboratory baked rolls depending on the portion of added yeast in recipe.
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Fig. 6. The acrylamide contents in laboratory baked rolls depending on the portion of added vital gluten in recipe.
Lower acrylamide contents were found in breads leavened by natural rye sourdough, probably due to lower pH values of dough achieved during fermentation. No relationship was found between the rye/wheat ratio in the laboratory baked breads and the amount of acrylamide in crust. Use of brown/black barley malts for breads colouring influenced acrylamide contents insignificantly; although contamination of these additives was relatively high compared to bread. The changes in formulation of wheat rolls such as addition of various amounts of vital gluten or bakery yeasts did not show any systemic impact on acrylamide contents which were generally low, not exceeding 10 mg kg1 in 60 g product. Finally it can be conclude, that the contents of acrylamide in the Czech breads are relatively low, which is in agreement with the recent EFSA report (EFSA, 2011), which identified soft breads, contrary to crisp breads, as a food category with a low acrylamide content. In spite of that, the contribution to the acrylamide dietary intake cannot be overlooked since soft bread consumption in the Czech Republic is relatively high. As mentioned in the Introduction, the contribution of soft bread to the total acrylamide dietary exposure in the Czech Republic is 13% for adults and 9e10% for children and adolescents (EFSA, 2011). Acknowledgement This study was carried out with the support from the following projects financed by the Ministry of Education, Youth and Sports of the Czech Republic: (i) the NPV II. project 2B06168; (ii) project MEB 080882 and (iii) Specific University Research (MSMT No. 20/2013). References Ahrne, L., Andersson, C. G., Floberg, P., Rosén, J., & Lingnert, H. (2007). Effect of crust temperature and water content on acrylamide formation during baking of
white bread: steam and falling temperature baking. LWT - Food Science and Technology, 40, 1708e1715. Amrein, T., Andres, L., Escher, F., & Amado, R. (2007). Occurrence of acrylamide in selected foods and mitigation options. Food Additives and Contaminants, 24(S1), 13e25. Brathen, E., Kita, A., Knutsen, S. H., & Wicklund, T. (2005). Addition of glycine reduces the content of acrylamide in cereal and potato products. Journal of Agricultural and Food Chemistry, 53, 3259e3262. Claus, A., Mongili, M., Weisz, G., Schreiber, A., & Carle, R. (2008). Impact of formulation and technological factors on the acrylamide content of wheat bread and bread rolls. Journal of Cereal Science, 47, 546e554. EC. (2011). Commission recommendation on investigations into the levels of acrylamide in food Document C(2010) 9681 final of 10.1.2011. EFSA. (2011). European Food Safety Authority. Results on acrylamide levels in food from monitoring years 2007e2009 and exposure assessment, Scientific report of EFSA, Parma, Italy. EFSA Journal, 9(4), 2133. EFSA. (2012). European Food Safety Authority. Update on acrylamide levels in food from monitoring years 2007 to 2010, Scientific report of EFSA, Parma, Italy. EFSA Journal, 10(10), 2938. European Commission Recommendation from the 2nd of June 2010 (on the monitoring of acrylamide levels in food; 2010/307/EU). FDE. (2011). FoodDrinkEurope acrylamide Toolbox. http://www.fooddrinkeurope.eu/ uploads/publications_documents/Toolboxfinal260911.pdf Accessed 19.07.13. Fink, M., Andersson, R., Rosén, J., & Aman, P. (2006). Effect of added asparagine and glycine on acrylamide content in yeast-leavened bread. Cereal Chemistry, 83, 218e222. Fredriksson, H., Tallving, J., Rosén, J., & Aman, P. (2004). Fermentation reduces free asparagine in dough and acrylamide content in bread. Cereal Chemistry, 81, 650e653. Hamlet, C. G., Baxter, D. E., Sadd, P. A., Slaiding, I., Liang, L., Muller, R., et al. (2005). Exploiting process factors to reduce acrylamide in cereal-based foods. High Wycombe: RHM Technology Ltd. C03032 and C03026. Report C014 prepared on behalf of the UK Food Standards Agency. Hamlet, C. G., Sadd, P. A., & Liang, L. (2008). Correlations between the amounts of free asparagine and saccharides present in commercial cereal flours in the United Kingdom and the generation of acrylamide during cooking. Journal of Agricultural and Food Chemistry, 56, 6145e6153. HEATOX. (2007). HEATOX: Heat-generated food toxicants: Identification, characterisation and risk minimisation. Final report. Lund, Sweden: Lund University http:// www.slv.se/upload/heatox/documents/Heatox_Final%20_report.pdf Accessed 19.06.13. IARC. (1994). Acrylamide. In IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans (Vol. 60; pp. 389e433). Lyon, France: International Agency for Research on Cancer. Konings, E. J. M., Ashby, P., Hamlet, C. G., & Thompson, G. A. K. (2007). Acrylamide in cereal and cereal products: a review on progress in level reduction. Food Additives and Contaminants, 24(S1), 47e59. Lantz, I., Ternite, R., Wilkens, J., Hoenicke, K., Guenther, H., & van der Stegen, G. H. D. (2006). Studies on acrylamide levels in roasting, storage and brewing of coffee. Molecular Nutrition & Food Research, 50, 1039e1046. project n. 2B06168National project NPV II results2006)2011). Ministry of Education, Youth and Sports of the Czech Republic. Sadd, P. A., Hamlet, C. G., & Liang, L. (2008). Effectiveness of methods for reducing acrylamide in bakery products. Journal of Agricultural and Food Chemistry, 56, 6154e6161. Surdyk, N., Rosén, J., Andersson, R., & Aman, P. (2004). Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread. Journal of Agricultural and Food Chemistry, 52, 2047e2051. Taeymans, D., Ashby, P., Blank, I., Gondé, P., van Eijck, P., Lalljie, S., et al. (2004). A review of acrylamide: an industry perspective on research, analysis, formation and control. Critical Reviews in Food Science and Nutrition, 44, 323e347. WHO. (2002). FAO/WHO consultation on the health implications of acrylamide in food. Summary report of a meeting held in Geneva, 25e27 June 2002. Geneva, Switzerland: World Health Organization. http://www.who.int/foodsafety/ publications/chem/acrylamide_june2002/en/ Accessed 19.06.13.