determination of acrylamide level in sangak bread … · 2015-08-26 · determination of acrylamide...

Proceedings of TheIRES 6th International Conference, Melbourne, Australia, 16th Aug. 2015, ISBN: 978-93-85465-75-8

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DETERMINATION OF ACRYLAMIDE LEVEL IN SANGAK BREAD BASED ON SPE-HPLC IN SHIRAZ, IRAN

1MOHAMMAD JAVAD TAGHIPOUR FARD ARDEKANI, 2MARYAM TAGHIPOUR FARD

ARDEKANI, 3ALI NIAZI

1,2Department of Food science and Technology, Behbahan Branch, Islamic Azad University, Behbahan, Iran

3Institute of Biotechnology, Shiraz University, Shiraz, Iran E-mail: [email protected]

Abstract- Acrylamide is an organic compound and recently presented in various food products such as baked and fried food rich in starch. It produced as a result of high temperatures during preparation and processing of the foods. The Maillard reaction, which refers to the interaction between the free amine groups of amino acids/proteins and the carbonyl group of sugars/carbohydrates, has a key role in the formation of acrylamide. The aim of this study was to evaluate the level of acrylamide in Sangak bread. In this study, 60 samples of a kind of flat bread (Sangak) were collected from Shiraz bakeries and the level of acrylamide was examined by SPE-HPLC method. Maximum and minimum amount of acrylamide in our samples were 12 ppm and 10 ppm respectively. Some factors attributed to the flour of these samples including the amounts of moisture, protein, gluten and, ashes were evaluated, but statistical analysis demonstrated no significant relation between them and their effects on acrylamide formation. Based on the result of this study further investigation are needed for reducing the level of acrylamide in these products. Keywords- Acrylamide, Sangak bread, SPE-HPLC I. INTRODUCTION Acrylamide (AA) is an odourless white crystalline organic solid and is a biodegradable substance; therefore, it does not constitute a burden on the environment. Plastic industries cause the highest environmental contamination by releasing AA (Arvanitoyannis and Dionisopoulou, 2013). In April 2002, Swedish researches found the presents of AA in some fried and backed foods, most notably potato chips and French fries, at levels of 30-2300 µg/kg. As there is no AA in unheated or boiled foods, it was concluded that it is generated due to high temperatures during preparation and processing of foods (Amany and Shaker, 2013). AA is absorbed via ingestion, inhalation or through the skin in animals and humans and was found in the thymus, liver, heart, brain, kidneys, placenta and breast milk. The oxidation of AA by cytochrome P450 leads the formation of the major metabolite AA. AA effects both human and animal health and leads to ataxia and skeletal muscle weakness as the typical symptoms (Veselá and Šucman, 2013; Fogiliano, 2011). Maillard reaction, which refers to the interaction between the free amine groups of amino acids/proteins and the carbonyl group of sugars/carbohydrates, has a key role in the formation of AA (Alper et al., 2014; Gokmen and Palazoglu, 2009). Anese et al., (2009) have shown that certain amino acids such as asparagine, especially in combination with reducing sugars during heat treatment, constitute AA (Amany and Shaker, 2013; Gokmen and Paazoglu, 2009; Anese et al., 2009). Several studies concluded that key precursor of AA formation in cereal during processing with high

temperature is asparagine (Hamlet et al., 2008; Taeymans et al., 2004). Surdyk et al., (2004) demonstrated the correlation between the amount of asparagine added to dough and AA formation in baked wheat bread while the added reducing sugar fructose did not show a similar correlation, so the limiting substrate for the AA formation was asparagine. Other wheat and rye bread model systems have also been demonstrated the clear connection between asparagine content in the dough and AA formation. (Forstova et al., 2014; Hamlet et al., 2008; Surdy et al., 2004; Brathen et al., 2005; Elmore et al., 2005). The favourite cereal breakfast food for Chinese and Asian people, fried bread sticks, has the same preparation process as Western fried foods. Foods with moderate protein and high carbohydrate level, like potatoes, have the high potential of AA formation during heat treatment (Zhang and Zhang, 2007). The aim of this study is to evaluate the level of acrylamide in Sangak bread; Also, to assess the relation between some flour factors (moisture, protein, gluten and, ashes) and AA formation in Sangak bread. II. MATERIALS AND METHODS In this study, 60 samples of Sangak bread were collected from Shiraz bakeries and the level of AA was examined by SPE-HPLC method. To check the accuracy of the developed SPE–HPLC method, 2.0 g bread samples, which were determined by HPLC to be free of AA, were spiked with 1.0 mL AA standard solution (0.25, 0.5, and 0.75 mg/L ) in a 100 mL conical flask, respectively. After being incubated for 1 hour, the spiked samples were ultrasonicated with 10 mL methanol for 30 minutes, and this step was

Determination Of Acrylamide Level In Sangak Bread Based On Spe-Hplc In Shiraz, Iran


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repeated for two more times with 10 + 10 mL of methanol. The resulting extractions were collected and centrifuged at 4,000 rpm for 30 min, and the supernatants were used for the SPE–HPLC procedure. III. PROCEDURE OF SPE COMBINED WITH HPLC The SPE cartridge was firstly rinsed with 5 mL methanol and Doubly Deionized Water (DDW), followed by loading 100 mL standard AA solution at a flow rate of 2.0 mL/ min. When the sample loading was done, the cartridge was conditioned thoroughly with 0.5 mL methanol/DDW(40:60,v/v) at 6.0 mL/min to remove the impurities, and then eluted by 0.5 mL portions of acetonitrile/acetic acid (99:1,v/v) at 6.0 mL/min to completely desorb the analytes adsorbed on the SPE microcolumn. The elution fraction volume was accurately measured. The effluent was filtered with a 0.45 lm filter membrane, and 15 lL of the filtrate was injected into HPLC for analysis. Finally, the SPE cartridge was extracted with 5 mL methanol/acetic acid (3:1,v/v), then flushed sequentially with 5 mL methanol and DDW for the next sample pre concentration. IV. INSTRUMENTATION AND HPLC ANALYSIS The HPLC system consisted of two LC-10 ATVP pumps and a Shimadzu SPD-10 AVP ultraviolet detector (Agilent Technologies). All separations were achieved on ananalytical reversed phase Zorbax eclipse C18 column (4.6 mm×150 mm) at a mobile phase flow rate of 0.5 ml/min. The mobile phase was methanol/water (15:85, v/v). AA (99.5 %) was obtained from Merk German. The HPLC grade methanol, acetonitrile and acetic acid were purchased from Merk German. DDW obtained from GFL German. The HPLC grade acetic acid was purchased from Merk German. The sample volume injected was 20 lL, and the detection was operated at 210 nm. Chemstation software was used to acquire and process spectral and chromatographic data. The actual 60 samples of Sangak bread (2.0 g) were extracted and analyzed according to the SPE-HPLC method and the results are categorized in two groups includes direct and indirect heat. In the indirect heat methods, the flame is in the steel furnace and heat transmitted via conduction, and the flame does not contact the bread directly. These devices have a thermometer and show the oven temperature but in the traditional methods the flame contact with the bread directly. Some factors attributed to the flour of these samples including the amounts of moisture, protein, gluten and, ashes were also evaluated, then statistical analysis of data is done with Chi-Square test.

IV. RESULTS Level of AA in bread The results of bread analysis show the same level of AA in both direct and indirect methods; maximum and minimum amount of AA were 12 ppm and 10 ppm respectively in our samples (tables 1 and 2), but the analysis did not show significant relationship between baking time and the amount of AA in both methods in different temperature (figure 1), as we can conclude that time changes in the range of 2 to 5.5 min (baking time of more than 90 percentages of samples are between 2 and 3.5 min) have no effect on the AA formation (figure 2). Role of the flour factors in AA formation Moisture percentage was from 12.3 to 13.4 in our samples (table 3), but according to the analysis, there is no significant relationship between the moisture content flour and the AA level that shows it does not have impact on the AA formation in this range (figure 3).

The level of measuring gluten in the sampled flour changes from 27 to 29.5 (table 3), but also the analysis did not show significant difference in AA formation and high gluten content flour (figure 4).

The maximum and minimum amounts of protein in our samples are 11.5 and 10.5 (table 3) respectively and investigation of its relationship with AA formation show that protein changes in this range have no effect on the AA level (figure 5).

Based on the results (figure 6), the percentage of ashes in the sampled flour has no relationship with AA formation and its amount changes from 1.12 to 1.3 in our samples (table 3). V. DISCUSSION AND CONCLUSION In this study, the measured amount of AA concentration in Sangak bread in Shiraz bakeries changes from 10 to 12 ppm, while the Environmental Protection Agency (EPA) and the FDA have already determined safe levels for AA in food 12 ppb in terms of the nervous system and research is ongoing in the areas of toxicology, exposure, formation, and mitigation. According to the analysis, there is no significant relationship between baking time in both direct and indirect heat methods, while generally by increasing baking time, the AA level increased, although kinds of bread and also baking time (more than 10 or 15 min) are different in other situation. Due to the small differences in numbers in baking time, we conclude that time changes in the range (2 - 5.5 min) have no effect on the AA level. Referring to table 1 and 2 baking time of more than 90 percentage of samples are between 2 -3.5 min, which is limited time changes. The high amount of AA contamination is in flat bread, since its surface area is more than other kinds of bread.



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Although, in this study various factors such as ash, protein, and gluten were measured in wheat flours, statistical analysis demonstrated no significant differences between them and their effects on AA formation. For example moisture has a key role in the formation of AA and Mailllard reaction, but due to the small difference in numbers in the moisture content, we conclude that in this range (12.3 - 13.4) it doesn't have any impact on the AA level. (The similar article has not been found). Protein and gluten show the same trend in this study, while they act as a source of amino acids in the Maillard reaction and AA formation. The ashes changes in the range (1.3 -1.12) have no effect on the AA level. However, according to previous studies, by increasing ash content of flour, the AA level increases. It is reported that by increasing the extraction of flour from 82 to 93 percent, the average amount of AA which is produce in bread is increased to 50.3 percent.

One solution which suggested for reducing AA is adding Ca salts into flour. Some authors believe that adding 0.3% of CaCl2 salts into flour decrease the amount of AA up to 30%.(Hamlet et al., 2005; Sadd et al., 2008). Also adding glycine causes AA reduction while adding high amount of amino acids increase AA amount. (Forstova, Veronika, et al., 2014). Overall, determination of extraction rate of different flours is suggested to compare the level of AA. Due to the toxicity of AA and its effect on human health, investigation of different methods for reducing the AA level in flat bread is suggested ACKNOWLEDGEMENTS This work was supported by the Behbahan branch of Islamic Azad University. We would like to thank this department and also Government Trading Company.

Table 1. The level of acrylamide determined in traditional bread with specific time and temperature.

Table 2. The level of acrylamide determined in bread in indirect device with specific time and temperature.



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Table 3. Moisture, gluten, protein and ashes value.

Figure 1. The range of baking temperature and acrylamide in

bread

Figure 2. The range of baking time and acrylamide in bread.

Figure 3. The range of moisture and acrylamide in bread.



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Figure 4. The range of gluten and acrylamide in bread.

Figure 5. The range of protein and acrylamide in bread.

Figure 6. The range of ash and acrylamide in bread.

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determination of acrylamide level in sangak bread … · 2015-08-26 · determination of acrylamide...

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