Acrylamide Contents of Commonly Consumed Bread Types in Turkey

Abstract

In this study, levels of acrylamide in different bread samples obtained from a Turkish market were investigated. Acrylamide contents of the samples were determined by a gas chromatography-mass spectrometry method. The samples (n = 43) were commonly consumed, different types of breads, including white wheat bread, stone oven wheat bread, wheat bran bread, rye bread, whole grain bread, and whole wheat bread. Results revealed that the acrylamide contents of the samples show a considerable variation among different types of breads as well as within the same type. Crumb-crust ratio of the samples was also considered during the experiments. The contents of acrylamide in breads ranged from below the limit of quantification to 695 μg kg⁻¹ and the mean acrylamide content was 225 μg kg⁻¹. The highest mean level of acrylamide was detected in whole wheat bread.

Keywords:

• Acrylamide
• Asparagine
• Bread
• GC-MS
• Rye
• Wheat

INTRODUCTION

Acrylamide is known to be neurotoxic in both animals and humans, and has been classified as “probably carcinogenic to humans” by the International Agency for Research on Cancer.^[1] Moreover, acrylamide is converted to its epoxide metabolite glycidamide by cytochrome P450 2E1 (CYP2E1) in the body and both acrylamide and glycidamide are genotoxic compounds by causing clastogenic effects, such as chromosome aberrations, micronuclei, sister chromatid exchanges, and forming DNA adducts that leads to point mutations.^[2−4] Presence of acrylamide in foods has attracted considerable attention worldwide because of these negative health effects of this contaminant.

Numerous mechanisms have been reported for the formation of acrylamide in foods, but the Maillard reaction is proposed as the most probable mechanism.^[5−7] According to the reaction, free asparagine in combination with reducing sugars (mainly glucose, fructose, and maltose) generate significant amounts of acrylamide when pyrolyzed at temperatures above 120°C.^[5,6] High levels of acrylamide have been detected in foods processed at high temperatures, especially in potato products, such as French fries and potato crisps; cereal products including bread, breakfast cereals, cakes, and biscuits; and roasted coffee.^[8−11] Among these foods, bread is an important source of dietary acrylamide exposure due to its relatively high consumption rate.^[10,12−23] According to the recent monitoring reports about exposure levels of acrylamide, bread was one of the major contributors to acrylamide exposure ranging from 10 to 30% in the adult population in the European countries.^[10]

The content of acrylamide in breads has been found to be mainly controlled by the effects of time and temperature of baking.^[24] In addition, there have been some precursors that affect acrylamide formation in breads. The contents of acrylamide precursors, namely, free asparagine and reducing sugars, are influenced by both plant variety and cultivars. For example, a wide range of asparagine contents has been reported in different wheat and spelt cultivars, but no great variation has been observed in rye.^[25,26] Addition of the enzyme asparagines has been reported as a measure to reduce the formation of asparagine since it results in the hydrolysis of asparagine to aspartic acid and ammonia.^[7] The baking agent NH₄HCO₃ has been found to enhance the formation of acrylamide in bakery products in both model systems and practical production.^[27] Studies of the effect of yeast fermentation on acrylamide reduction in yeast-leavened wheat bread suggest that prolongation of the fermentation time results in a substantial reduction in acrylamide content in bread and bread rolls.^[28] Acrylamide is intensively found in the outer part of the breads. In soft wheat bread, more than 99% of the acrylamide content is determined in the crust and the content of acrylamide linearly increases with time and baking temperature, an effect that can be attributed to a high rate of water loss from the surface of the bread.^[24]

Bread occupies an important part of a Turkish diet. It is a staple food for Turkish people. According to the Guinness book of world records, as of 2000, the country with the largest per capita consumption of bread is Turkey with 199.6 kg per person. Turkish people consume bread more than three times their own body weight annually. Turkey is followed in bread consumption by Serbia and Montenegro with 135 kg and Bulgaria with 133.1 kg. According to FAO Food Balance Sheets, the major percentage of energy in Turkish people comes from bread (44%) and bread with other cereals (58%).^[29] As a result of high consumption rate of bread in Turkey, monitoring of acrylamide in different bread types has become an essential necessity for consumers, producers, and institutions concerned with standards and quality control management. Therefore, the aim of this study was to determine the acrylamide levels in different types of breads commonly consumed in Turkey.

MATERIALS AND METHODS

Chemicals and Standards

Acrylamide standard (>99%) was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). ¹³C₃-acrylamide (1,2,3-¹³C₃-acrylamide, >99%), which was used as the acrylamide internal standard (IS), and the acrylamide derivative (2,3 dibromopropionamide >99.5%) were obtained from Cambridge Isotope Laboratories Inc. (Andover, MA, USA) and Chem Service Inc. (West Chester, PA, USA), respectively. Ethyl acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium bromide, hydrobromic acid (47%), bromine, triethylamine, sodium thiosulfate, potassium hexacyanoferrate (II) trihydrate, and zinc sulphate heptahydrate were all obtained from Merck KGaA (Darmstadt, Germany). Water was produced by an ultrapure (18.2 MΩ cm at 25°C) purification system from Millipore (Bedford, MA, USA). A series of different types of bread were purchased from five different bakeries and markets between February 2012 and April 2012 in Turkey.

Sampling

Widely consumed bread types were selected for the study. For this purpose, 43 samples of different bread types, including white wheat bread (n = 6), stone oven wheat bread, which is a traditional white wheat bread that is baked in the stone ovens (n = 13), wheat bran bread (n = 8), rye bread (n = 4), whole grain bread (n = 4), whole wheat bread (n = 4), and other breads that are used for special foods, such as hamburger or toast (n = 4), were collected from local bakeries. The bread samples were analyzed within 12 h of collection. Typical ingredients of tested bread types are illustrated in Table 1. The information about the ingredients is supplied from the Turkish Food Codex.^[30]

TABLE 1 Ingredients of different bread types

Bread type	Ingredients
White wheat bread	Wheat flour, water, yeast, salt, bread improver
Stone oven wheat bread	Wheat flour, water, yeast, salt, bread improver
Wheat bran bread	Wheat flour, wheat bran (10–30%), water, yeast, salt, bread improver
Rye bread	Wheat flour, rye flour (min 30%), water, yeast, salt, bread improver
Whole wheat bread	Wheat flour, whole wheat flour (min 60%), water, yeast, salt, bread improver
Whole grain bread	Wheat flour, whole wheat flour, some other cereals, such as corn, barley, rye, etc. (min 5% for each), water, yeast, salt, bread improver
Other breads	Wheat flour, water, yeast, salt, bread improver

Special ingredients for each type of bread appear in boldface.

There are a few basic steps for the production of these types of breads. They can be listed as follows: (i) mixing of wheat flour and water, together with yeast and salt, and other specified ingredients in appropriate ratios, (ii) subdivision of the dough mass into unit pieces, (iii) fermentation and expansion of the shaped dough pieces during proof (typical proof conditions are 40–45°C and 85% relative humidity), (iv) baking of the dough after proofing conditions (typical oven temperatures lie in the range of 220–250°C.), and (v) cooling and storage of the final product before consumption.

Reagents

Bromine solution (15.2 g of potassium bromide, 0.8 ml of hydrobromic acid, 5 ml of 1.6% saturated bromine water, and 60 ml of distilled water) that is used for derivatization was stored in a dark bottle. Carrez I solution was prepared by dissolving 1.44 g of potassium hexacyanoferrate (II) trihydrate in 5 ml of water, and Carrez II solution by dissolving 2.88 g of zinc sulfate heptahydrate in 5 ml of water. All of the solutions were stored at 4°C.

Sample Extraction

The bread samples were manually divided into crumb (inner portion) and crust (outer portion) and dried. Each portion was weighed and crumb/crust ratio of the samples was calculated as the mass of crust divided by the mass of crumb. Because the particle sizes of crumb and crust are different, this difference may be problematic during the sieving step. These divided parts of bread samples were separately pulverized and sieved through 212 meshes. After the sieving step, crumb and crust parts of each bread type were combined as previously calculated. From the sieved sample, one gram of subsample was suspended in 8.2 ml of water at 60°C in a beaker. The suspension was spiked with 200 μl of ¹³C₃ IS at a concentration of 15 mg L⁻¹ and stirred at 60°C on a magnetic heater stirrer (Heidolph MR 3001 K, Heidolph Instruments, Germany) for 20 min. Then, the mixtures were transferred to centrifuge tubes. In order to remove the proteins, 300 μl of Carrez I and 300 μl of Carrez II clearing agents were added to the tubes. Then, the mixture was centrifuged at 8000 rpm for 30 min. Finally, 3 ml of clarified aqueous layer was filtered through a 0.45-μm syringe filter into another tube prior to derivatization.

Bromination

Bromination of the extracts was carried out according to a procedure reported previously.^[13,31] Briefly, the clarified solutions were treated with 300 μL of bromine solution and covered with aluminium foil. The reaction mixture was transferred into an ice bath where reaction was allowed to occur for 1 h in the dark. After reaction was completed, the excess bromine was decomposed by adding 10 μl of 1 M sodium thiosulfate solution until the yellow color disappeared. Then the mixture was extracted with 2 ml of ethyl acetate using a vortex for 30 s. After the extraction of acrylamide derivative, 2,3 dibromopropionamide (2,3-DBPA), the tubes were centrifuged at 5000 rpm for 10 min. The upper phase (1.8 ml) was transferred to a gas chromatography-mass spectrometry (GC-MS) vial and 10% of triethylamine (180 μl) was added to convert the 2,3-DBPA to a more stable derivative, 2-bromopropionamide (2-BPA), prior to analyses.

GC-MS Conditions

A Thermo Scientific ISQ GC-MS system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a fused capillary column (TR-WAX, 30 m × 0.25 mm × 0.25 μm) was used for the determination of acrylamide in analyzed samples. The oven temperature program was as follows: A 50°C initial temperature was held for 1 min, then increased to 180°C at 20°C min⁻¹, then to 260°C by a rate of 10°C min⁻¹, and held for 10 min at this temperature. The injection block, detector, and ion source temperatures were 240, 250, and 230°C, respectively. Carrier gas (helium) flowing through the column was 1 ml min⁻¹. Injection volume was 2 μl and identification was determined using a Selective Ion Monitoring (SIM) mode.

RESULTS AND DISCUSSION

Quality Control

The calibration curve was linear (R² = 0.999) in the range of 10–1000 μg L⁻¹. A representative chromatogram for a bread sample that contains acrylamide is shown in Fig. 1. The chromatogram in the figure showed that acrylamide derivative (2-BPA) was well separated with no interfering peaks and the noise was similar regardless of the matrices, suggesting excellent selectivity of this method. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be 7.46 and 24.88 μg kg⁻¹, respectively. For a recovery test, the acrylamide standard was added to a mixed crumb and crust sample at a final concentration of 1 mg kg⁻¹. The absence of acrylamide in the sample was confirmed by chromatographic analysis prior to the recovery studies. The same extraction procedures and GC-MS conditions of those applied for the sample analyses were used for recovery studies. In this way, the obtained recovery was 83% ± 3.5 (n = 6). This result agrees with those obtained in previous studies.^[32−34]

TABLE 2 Levels of acrylamide in different types of bread

		Acrylamide levels (μg kg^−1)
Food groups	Number of samples	Mean ± SD	Minimum–Maximum
White wheat bread	6	121 ± 103	<LOQ -254
Stone oven wheat bread	13	171 ± 184	<LOQ -468
Wheat bran bread	8	307 ± 258	<LOQ -695
Rye bread	4	432 ± 214	209–624
Whole wheat bread	4	479 ± 325	<LOQ -692
Whole grain bread	4	151 ± 211	<LOQ -448
Other breads	4	<LOQ	<LOQ
Bread types	43	225 ± 235	<LOQ -695

LOQ: limit of quantification.

FIGURE 1 A sample chromatogram of a bread sample.

Acrylamide Content of Different Types of Breads

Acrylamide contents of the samples ranged from <LOQ to 695 μg kg⁻¹ depending on the bread types (Table 2). The mean value of acrylamide levels was found to be 225 ± 235 μg kg⁻¹. The acrylamide contents in all bread types were aligned from high to low as whole wheat bread (479 ± 325 μg kg⁻¹) > rye bread (432 ± 214 μg kg⁻¹) > wheat bran bread (307 ± 258 μg kg⁻¹) > stone oven wheat bread (171 ± 184 μg kg⁻¹) > whole grain bread (151 ± 211 μg kg⁻¹) > white wheat bread (121 ± 103 μg kg⁻¹) > other breads (<LOQ). Moreover, Fig. 2 shows how many samples in the ranges of <LOQ, LOQ–200, 200–400, 400–600, and 600 < μg acrylamide kg⁻¹ sample.

FIGURE 2 Numbers of bread samples in various ranges of acrylamide (μg kg⁻¹).

The results revealed that acrylamide content of different bread types shows a wide variation as indicated from the high levels of standard deviation. It can be assumed that differences in types and quality of raw materials, formulations, processing methods, and processing parameters may effect the formation of acrylamide. The highest level of acrylamide was found in whole wheat breads. This result can be attributed to the fact that this bread may have various levels of some fractions of wheat, such as germ and bran containing significant amounts of asparagine, the main precursor of acrylamide in cereal products. Free asparagine in combination with reducing sugars generates significant amounts of acrylamide when pyrolyzed at temperatures >120°C.^[5,6] Fredriksson et al.^[28] reported that the free asparagine in wheat was found to be 55.5, 1.48, and 0.17 g kg⁻¹ in the fraction of germ, bran, and flour, respectively. In addition, Mustafa^[24] reported that the highest and lowest concentrations of free asparagine in both wheat samples and rye samples were found in their bran and sifted fraction, respectively.

The second high level of acrylamide was found to be 432 ± 214 μg kg⁻¹ in rye bread samples. The mean acrylamide content of rye bread samples was found to be much higher than that of the breads, which are made using wheat flour. This result may be explained by the fact that the rye flour contains higher free asparagine than that of wheat flour. Fredriksson et al.^[28] reported that free asparagine content of rye flour was 0.68 g kg⁻¹ but wheat was 0.17 g kg⁻¹. Amrein et al.^[35] also reported that rye flour contained more free asparagine and free amino acids than that of other flours.

The levels of acrylamide may also be affected by crust thickness of the breads. Şenyuva and Gökmen^[36] reported that the acrylamide was not found in any of the bread crumb. They also reported that the crusts of Trabzon, rye, and oat bread contained 420, 526, and 718 ng g⁻¹ acrylamide, respectively. Surdyk et al.^[37] reported that acrylamide is predominantly generated in the outer crust layer where more than 99% can be found. This could be explained by the heat transfer mechanism during the baking process. The heat is transported from the oven air to the bread, and since bread is a poor heat conductor, a temperature and water profile arises in the bread. At the end of the baking process, loaf surface heat rises to 230–250°C, and the interior of the loaf may reach 100°C or slightly higher. It was reported that acrylamide formation was found to occur at temperatures above 120°C.^[5,38] Since the total baking time is not long enough to increase the crumb temperature of bread above 120°C, at which point acrylamide begins to form, acrylamide may not be formed.

The determined mean acrylamide level in breads (225 μg kg⁻¹) tested in this study was higher than those obtained by Ölmez et al.^[39] In that study, 22 bread samples sold in Turkey were analyzed for acrylamide residues and the mean and highest acrylamide levels in the breads were reported as 38 and 85 μg kg⁻¹, respectively. Higher results were reported by Şenyuva and Gökmen,^[36] who found that the mean acrylamide content of 16 Turkish breads was 108 μg kg⁻¹ (maximum 287 μg kg⁻¹). They also reported that the highest acrylamide content was found in rye bread. The difference between our results and Ölmez et al.^[39] could be explained by considering that the crumb/crust ratio may result in a higher level of acrylamide since almost all of the acrylamide is found in the crust.

A wide range of acrylamide levels, which may be achieved using different raw materials, ingredients, and cooking methods, were reported all over the world. According to the report of the Food and Drug Administration (FDA), acrylamide levels in breads were different depending on the bread types, such as 59 μg kg⁻¹ for grain bread, 25 and 45 μg kg⁻¹ for different brands of whole wheat bread, and 31 μg kg⁻¹ for rye bread.^[40] The mean and highest acrylamide levels in breads were reported as 30 and 425 μg kg⁻¹, respectively, in the European Food Safety Authority’s (EFSA) monitoring report about acrylamide levels in food from 2007 to 2010 in Europe.^[10] Arisseto et al.^[21] determined the acrylamide levels in breads in Brazil in the range from <20 to 71 μg kg⁻¹. Similar results obtained the mean and highest acrylamide levels in breads as 25 and 70 μg kg⁻¹, respectively, and were reported from the Netherlands.^[41] Svensson et al.^[18] determined the mean and highest acrylamide levels in breads in Sweden as 50 and 160 μg kg⁻¹, respectively. Mean acrylamide levels determined in breads in Egypt and Belgium were 64 and 30 μg kg⁻¹, respectively.^[15,20] In another study, Chinese foods were analyzed for their acrylamide contents, and mean acrylamide level in breads was 38 μg kg⁻¹ and the range was 10–133 μg kg^−1.[42]

CONCLUSIONS

This study provides information on acrylamide levels in different bread types sold in Turkey. The mean acrylamide level of breads was 225 μg kg⁻¹. Acrylamide levels in different bread types changed depending on the ingredients and processing conditions. The concentration of precursors in raw material and process temperature are important factors for acrylamide formation in food. Acrylamide is probably a carcinogenic compound to humans and for a compound that is carcinogenic and, presumably, acts via a genotoxic mechanism, no safe levels of intake can be estimated, meaning that the level in foods should be as low as reasonably achievable. Since bread is an important acrylamide source due to high consumption rate, effective mitigation strategies should be researched and applied in the food industry to decrease acrylamide in breads, and thus the acrylamide exposure. The results obtained in this study will be able to shed light on the fate of acrylamide during the baking process and may also prove to be valuable knowledge on estimating potential risks from dietary exposure.

FUNDING

The authors are thankful to the Research Fund of Akdeniz University for a grant (Project 2012.02.0121.001) supporting this study.