Acrylamide formation in biscuits made of different wholegrain flours depending on their free asparagine content and baking conditions

Journal Pre-proofs Acrylamide formation in biscuits made of different wholegrain flours depending on their free asparagine content and baking conditions Slađana Žilić, Iş ıl Gürsul Aktağ, Dejan Dodig, Milomir Filipović, Vural Gökmen PII: DOI: Reference:

S0963-9969(20)30134-4 https://doi.org/10.1016/j.foodres.2020.109109 FRIN 109109

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Food Research International

Received Date: Revised Date: Accepted Date:

20 September 2019 10 February 2020 18 February 2020

Please cite this article as: Žilić, S., Gürsul Aktağ, I., Dodig, D., Filipović, M., Gökmen, V., Acrylamide formation in biscuits made of different wholegrain flours depending on their free asparagine content and baking conditions, Food Research International (2020), doi: https://doi.org/10.1016/j.foodres.2020.109109

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Acrylamide formation in biscuits made of different wholegrain flours depending on their free asparagine content and baking conditions Slađana Žilića1, Işıl Gürsul Aktağb, Dejan Dodiga2, Milomir Filipovića2, Vural Gökmenb* a

Maize Research Institute, 1Laboratory of Food Technology and Biochemistry and 2Breeding

Department, Slobodana Bajića 1, 11185 Belgrad- Zemun, Serbia b

Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering,

Hacettepe University, 06800 Beytepe, Ankara, Turkey

*Corresponding author: Vural Gökmen Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, 06800 Beytepe, Ankara, Turkey. E-mail address: [email protected] (V. Gökmen)

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ABSTRACT Due to a high content of bioactive compounds with beneficial health effects, wholegrain flours of different cereals have been extensively used in the confectionery industry. However, according to our study, cereal species and their varieties have different potential for the formation of acrylamide in biscuits. In this study, wholegrain flours of eight genotypes of small grain cereals (bread wheat, durum wheat, soft wheat, hard wheat, triticale, rye, hulless barley and hulless oat) and four genotypes of maize (white-, yellow- and red-coloured standard seeded maize, and blue-coloured popping maize) were used to prepare biscuits. The biscuits were baked at 180oC for 7, 10 and 13 min. At 180oC, acrylamide was detected at all baking times, reaching a final concentration of 72.3 up to 861.7 μg/kg after 13 min of baking in refined bread wheat-based biscuits and hulless oatbased biscuits, respectively. Data indicated that acrylamide in biscuits could not exactly correspond to free asparagine in flour. However, hulless oat, durum wheat and rye flour with the highest content of free asparagine of 859.8, 603.2 and 530.3 mg/kg, respectively, generated most acrylamide in biscuits baked for 13 min. The lowest content of acrylamide was found in biscuits prepared from refined bread wheat flour and wholegrain red maize flour that also contained the lowest content of free asparagine. After baking for 7, 10 and 13 min, the content of acrylamide in these samples was 17.9 and 24.4 μg/kg, 51.9 and 28.7 μg/kg and 72.3 and 95.2 μg/kg, respectively. The results suggest that the use of cereal flours low in free asparagine can be an effective strategy for acrylamide mitigation in biscuits, together with the use of lower thermal load during baking. Keywords: Whole grains, cereal flours, biscuits, free asparagine, acrylamide

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1. Introduction Cereals play an important role in the food production today. Over 73% of the total world harvested area is cultivated with cereals, which also make more than 60% of the world food production (Das, Raychaudhuri, & Chakraborty, 2012). Cereals are basic ingredients of bread, other bakery products, pastas, as well as many confectionery products, and thus they present the main source of energy and nutrients to the most of the world human population. Numerous epidemiological and intervention studies performed during the last 15 years confirm the contribution of whole grains to health and chronic disease prevention. A higher intake of whole grains, i.e. unrefined cereal products is associated with a lower risk of cancer, as well as cardiovascular disease (Aune et al. 2016), and at the same time, with the beneficial effect on glucose-insulin homeostasis, blood lipids, and gastrointestinal health (Ye, Chacko, Chou, Kugizaki, & Liu, 2012). In most cases, health beneficial compounds such as dietary fibers, minerals, vitamins and phenolic compounds are concentrated in outer layers, i.e. bran and aleurone of the grain (Žilić, Serpen, Akıllıoğlu, Janković, & Gökmen, 2012). Aleurone is also a protein-rich tissue with a good amino acid profile. However, as such, it is the main source of precursors in acrylamide formation in whole grain cereal processed food. According to results of Capuano et al. (2009), under the same baking conditions wholegrain wheat flour in bread crisps produced more acrylamide than refined flour free of aleurone by the sieving. As already known, acrylamide is one of the food-borne toxicants that has acquired much attention since its discovery in heated food. On the basis of its carcinogenic action in rodents, the International Agency for Research on Cancer has classified acrylamide as probably carcinogenic to humans (Group 2a) (IARC, 1994). At high doses acrylamide has been also indicated as genotoxic and neurotoxic to animals (Exon, 2006). 3

In addition to processed potatoes and coffee, fried, roasted or baked cereal products are groceries with the highest level of acrylamide. The contribution of cereal products to dietary intake of acrylamide varies from country to country due to dietary preferences. In the United States and Sweden, acrylamide from cereal products make up 33% and 22%, respectively, of the total daily intake (Mucci & Wilson, 2008). However, based on data presented by the European Food Safety Authority (2011), the contribution of cereal products to dietary acrylamide intake for adults in France, Germany, Sweden and United Kingdom was 40.9, 45.4, 41.2 and 31.9%, respectively. Except in Sweden, in mentioned countries the contribution of acrylamide from cereal products is highest in bread with 25.7, 32.0 and 15%, respectively, followed by biscuits and breakfast cereals. Muesli was a main cereal contributor of acrylamide (13%) to total dietary intake in Sweden (Curtis & Halford, 2016). The precursors of acrylamide in potatoes, coffee and cereal products are the same, i.e. free asparagine and reducing sugars. However, the relative importance of these two, differs substantially among the three species. Reducing sugars abound in cereal grains, so the concentration of free asparagine is the most important or the rate limiting factor in affecting acrylamide formation in baked products (Seal et al., 2008). The content of asparagine varies among, as well as within cereal species. The average content of free asparagine in widely used cereal species was found to be between 426 ± 144 and 1179 ± 359 mg/kg (Žilić et al., 2017). According to the same study, the species had a mean free asparagine content in the following descending order: rye > hulless oat > dent maize > hulless barley > durum wheat > bread wheat. The study of Curtis, Powers, Wanga and Halford (2018) confirmed that the asparagine content in cereals, and in this regard their potential for acrylamide formation, is affected by common factors such as the genetic basis, growing conditions, time of harvest and post-harvest storage conditions. Taking all of these into account, one of the food 4

industry approaches toward reducing levels of acrylamide in its products include selecting cereal species or their varieties which contain low levels of acrylamide precursors (primarily asparagine). Hence, in order to determine a genetic resource with reduced potential for acrylamide formation, the aim of this study was to investigate the effect of the different whole grain cereal flours (12 genotypes belonging to seven species) on the acrylamide content in biscuits. The interrelationship between the initial content of proteins and free asparagine in cereal flours and acrylamide in the biscuits, as well as the correlation between contents of acrylamide and free asparagine in biscuits baked at 180oC for 7, 10 and 13 min was analyzed for this purpose. Using cereal with low potential for acrylamide formation in biscuits would be important especially for children and adolescents who tend to eat acrylamide-rich foods, such as confectionery. 2. Material and methods 2.1. Flour samples The experimental material used to make flour consisted of eight and four genotypes of small grain cereals and maize, respectively. One genotype each of bread wheat (Triticum aestivum var. lutescens), durum wheat (Triticum durum Desf.), soft wheat (Triticum aestivum var. alba), hard wheat (Triticum aestivum var. compactum), triticale (Triticosecale var. hexaploid), rye (Secale cereale), hulless oat (Avena sativa var. nudum) and hulless barley (Hordeum vulgare L. var. nudum Hook. f.) was used. Maize genotypes were distinguished by the kernel colour. Three maize genotypes of standardised white-, yellow- and red-colour kernel type (Zea mays L. var. indentata), as well as one popping maize (Zea mays L. var. everta) genotype with kernels of blue colour were used. All genotypes were grown in the field at the Maize 5

Research Institute, Belgrade, Serbia, in the 2017/2018 growing season. Standard cropping practices were applied to provide adequate nutrition and to keep the disease- and weedfree plots. One sample of refined wheat flour type 400 was purchased from a local supermarket and used. Wholegrain flours were used to prepare biscuits. Whole grains were milled in the Perten 120 lab mill (Perten Instruments, Hägersten, Sweden) (particle size < 500 μm). 2.2. Preparation of biscuits The dough was prepared according to the recipe described in the AACC Methods 10-54 with some modifications (AACC, 2000). The recipe contained the following ingredients: 40.0 g of flour, 16.8 g of sucrose, 16.0 g of shortening (refined palm oil), 0.5 g of sodium chloride, 0.6 g of sodium bicarbonate, 0.6 g high-fructose maize syrup, 0.4 g of nonfat dry milk and 8.8 ml of water. All ingredients were mixed thoroughly in accordance with the AACC Method 10-54 procedure using the Kenwood CHEF dough mixer. The dough was rolled out to disks with a diameter of 5.5 cm and a height of 3 mm, and baked in convection oven (Memmert, UNE 400) at 180°C for 7, 10 and 13 min. The fan and flap were set to 10%. All baking experiments were performed in triplicate. The biscuits were placed in the same position inside the oven. Dough and biscuits are shown in Fig. 1. 2.3. Analytical procedures 2.3.1. Analysis of proteins and non-protein nitrogen The standard AOAC (1995) chemical methods were applied to determine the content of ash, total proteins, as well as non-protein nitrogen in cereal flours. Non-protein nitrogen was determined from the supernatant after extraction of salts of soluble proteins with 0.5M NaCl and their precipitation with 10% of trichloroacetic acid (TCA). The total fibre content was 6

determined according to the AOAC 985.29 method (AOAC, 1985). All results are expressed in the percentages per dry matter (d.m.). 2.3.2. Analysis of free asparagine (Asn) Cereal flours and biscuits were evaluated for free asparagine according to the method described previously with some modifications (Kocadağlı, Özdemir, & Gökmen, 2013). One gram of sample was extracted with 20 mL of 10 mM formic acid in water at triple stage (10, 5, and 5 mL) by vortexing for 3 min. The combined extract was clarified by adding Carrez I and Carrez II solutions. The mixture was centrifuged at 10000 g for 3 min. The supernatants were kept at -70°C until analysis. All extractions were performed in duplicate per each sample. The clear supernatant was mixed with an equal volume of acetonitrile and centrifuged at 12000 g for 3 min. The supernatant was passed through a 0.45-µm nylon filter and collected in a vial. The samples were analyzed by a Waters Acquity UPLC system coupled to a triple quadrupole detector operated in positive electrospray ionization mode. Chromatographic separations were performed on an Thermo Scientific Syncronis HILIC column (100 × 2.1 mm i.d., 1.7 µm) by using a gradient mixture of 5 mM ammonium formate in water with 0.5% formic acid (A) and 5 mM ammonium formate in water : acetonitrile (v/v 1:9) with 0.5% formic acid (B). A flow rate was 0.7 mL/min. The eluent composition starting with 0% A linearly increased to 80% in 7.10 min and held for 2.9 min. Then, it was linearly decreased to the initial conditions (0% A) in 1 min and held for 4 min. The column was at 40°C and the Waters ACQUITY FTN autosampler was at 10°C during the analysis. The electrospray source had the following settings: capillary voltage of 3.5 kV; cone voltage of 20 V; extractor voltage of 3 V; source temperature of 120°C; desolvation temperature of 370°C; and desolvation gas

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(nitrogen) flow of 900 L/h. Quantifications were performed by means of external calibration curves built for asparagine in a range between 0.05 and 2.0 mg/L. The accuracy of asparagine analysis was checked by adding 0.5 mg/L of theanine as internal standard to both working standards and extracts (Yılmaz, Göncüoğlu Taş, Kocadağlı, & Gökmen, 2019). The molecular ion of asparagine was m/z 133 and its fragment ions were m/z 87 and 116. The molecular ion of internal standard, theanine, was m/z 175 and its fragment ions were m/z 46, 129 and 158. The quantifier ions were 158 and 116 for asparagine and theanine, respectively. The results were expressed as mg per kg of d.m. 2.3.3. Analysis of acrylamide Cereal flours and biscuits were evaluated for acrylamide according to the method described previously with some modifications (Gökmen, Morales, Ataç, Serpen, & Arribas-Lorenzo, 2009). Employing the multiple extraction strategy during the analysis of acrylamide in cereal products was found to increase the analytical accuracy. One gram of ground sample was extracted with 20 mL of 10 mM formic acid in water at triple stage (10, 5, and 5 mL) by vortexing for 3 min. The combined extract was clarified by adding Carrez I and Carrez II solutions. The mixture was centrifuged at 10000 g for 3 min. The supernatants were kept at 70°C until analysis. All extractions were performed in duplicate per each sample. The clear supernatant was passed through a preconditioned Oasis MCX solid phase extraction cartridge and the pure extract was analyzed using the LC-MS/MS. A Waters Acquity H Class UPLC system (Millford, MA) coupled to a TQ detector with electrospray ionization operated in a positive mode was used to analyze the extracts for acrylamide. The chromatographic separations were performed on a Thermo Scientific Hypercarb column (100 × 2.1 mm i.d., 3 μm) using 0.1% formic acid in water as the mobile phase at a flow rate

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of 0.2 mL/min. The column equilibrated at 50°C and Waters Acquity FTN autosampler was held at 10 °C during the analysis. The electrospray source had the following settings: capillary voltage of 2.00 kV; cone voltage of 22 V; extractor voltage of 4 V; source temperature of 120°C; desolvation temperature of 400°C; and desolvation gas (nitrogen) flow of 900 L/h. The flow rate of the collision gas (argon) was set to 100 L/h. Acrylamide was identified by multiple reaction monitoring (MRM) of two channels. The precursor ion [M + H]+ 72 was fragmented and the product ions at m/z 55 (collision energy of 9 V) and m/z 44 (collision energy of 12 V) were monitored. The dwell time was 0.2 s for all MRM transitions. The stock solution of acrylamide was prepared in water to a concentration of 1 mg/mL. Working solutions were prepared by diluting the stock solution with water. The concentration of acrylamide in samples was calculated by means of a calibration curve built in the range between 1 and 20 ng/mL (1, 2, 5, 10, 20 ng/mL). The results were expressed as μg per kg of d.m. The limit of detection and limit of quantitation of acrylamide were 3 and 10 ng/g, respectively. 2.4. Statistical analysis The analytical data were reported as mean ± standard deviation of at least duplicate independent extractions. Significance of differences among species means were analyzed by Tukey’s (HSD) test. Differences at p < 0.05 were considered significant. The genotype-by-trait biplot was used to observe relationships amongst asparagine, total proteins, non-protein nitrogen and acrylamide across flour samples and baking treatment. These inter-relations were analyzed using the Statistica software Minitab® (Minitab, LLC, Pennsylvania, USA). Correlations between parameters were examined using a linear regression model in Excel ® (Microsoft).

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3. Results and discussion 3.1. Content of ash, total fibers, proteins and free asparagine in cereal flours In this study, the content of free asparagine, as a limiting factor affecting acrylamide formation, was analyzed in wholegrain flour of 12 genotypes of the most cultivated cereal species and in one sample of refined wheat flour. At the same time, the content of ash, total fibers, total proteins, as well as non-protein nitrogen as an indicator of the total free amino acids, was evaluated with the purpose of a better characterization of cereal flour types, as well as of further determining their relationship with the content of acrylamide in cookies (Table 1). Studies show that reformulation of traditional wheat recipes with innovative plant species, including different cereals, could increase the acrylamide content in the biscuits despite the mitigation strategies applied in past ten years in the confectionery industry (Mesías, Holgado, Márquez-Ruiz, & Morales, 2016). By the content of total proteins, wholegrain flour of hulless oat that stood out with 16.80% was followed by wholegrain flour of hard wheat and blue popping maize with 14.80 and 13.11%, respectively. In other cereal flour samples the content ranged from 9.56 to 12.22%. Hulless oat flour had also the highest content of free asparagine, while this was not the case with hard wheat and blue popping maize flours. Proteins and free asparagine accumulation in grains is affected by different genetic and growing conditions. Therefore, these compounds do not have to be positively correlated. According to the study of Gao et al. (2016) the highest free asparagine accumulation in cereal grain occurs when the plant has a plentiful supply of nitrogen but is unable to maintain a normal level of protein synthesis because of deficiencies in other nutrients. Rye and durum wheat whole grain flours, with free asparagine content of 603.2 and 530.3 mg/kg, respectively, followed hulless oat flour.

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The content of free asparagine in wholegrain flour of hard wheat, blue popping maize, as well as yellow and white standard maize and triticale ranged from 387.9 to 480.6 mg/kg. The third group, with the content of free asparagine varying from 189.7 to 297.0 mg/kg, consisted of wholegrain flour of red standard maize, hulless barley, soft and bread wheat. Similarly, the free asparagine content varied among cereal species and their varieties (Žilić et al., 2017). In these studies, the lowest content of free asparagine was measured in refined bread wheat flour released from bran and aleurone by the sieving. Compared to wholegrain flour of bread wheat, in this sample the content of free asparagine was 4.2 times lower (Table 1). Previous studies showed that the ash and fiber content of wholegrain flour influenced the acrylamide content in bakery products (Claus, Carle, & Schieber, 2006). The content of ash and fibre in wholegrain flour is directly related to the content of free amino acids in it, since all these compounds are localized in the outer layer of the cereal grain. According to our research, a significantly high positive correlation (r = 0.82) between the content of ash and the content of free asparagine in cereal flours was established. The lowest (0.47%) and the highest (2.44%) content of ash was found in refined bread wheat flour and wholegrain flour of hulless oat, respectively, which coincides with the content of free asparagine in them. Similarly, the total fiber content in the examined cereal flour samples varied from 7.1 to 15.2%. The correlation between the content of non-protein nitrogen and free asparagine in cereal flours has not been established. In general, maize flour samples had a lower content of non-protein nitrogen compared to flour of small grain cereals, while the highest content of total free amino acids, 45.6% of total proteins, was found in rye flour.

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3.2. Content of free asparagine and acrylamide in biscuits Previous studies showed that baking of biscuits promote the formation of compounds with antioxidant activity (Serpen & Gökmen, 2009; Žilić, Kocadağlı, Vančetović, & Gökmen, 2016), as well as with an important role in flavour and the development of browning (Palazoğlu, Coşkun, Tuta, Mogol, & Gökmen, 2015; Kocadağlı et al., 2016) but also potentially harmful ones such as acrylamide (Morales, Martin, Açar, Arribas-Lorenzo, & Gökmen, 2009; van der Fels-Klerx et al., 2014). In order to prove the effect of the initial content of free asparagine in the flour, as well as the type of cereal flour on the acrylamide formation, the content of acrylamide and free asparagine was analyzed in different biscuits after baking at 180oC for 7, 10 and 13 min (Fig. 2). Generally, the reduction in the asparagine content, and the increase in the acrylamide content was apparently caused by the thermal treatment. Compared to the initial content in flours, the relative asparagine reduction in biscuits baked for 7 min (on average 69±16%) was lower than in biscuits baked for 13 min (on average 87±8%). In these biscuits samples the residual content of free asparagine ranged from 0 to 265.6 mg/kg and from 0 to 91.4 mg/kg, respectively. The complete reduction of asparagine was observed in hulless oat biscuits. On the other hand, the lowest degree of asparagine reduction was found in hulless barley biscuits baked for 7, 10 and 13 min amounting to 58, 50 and 69%, respectively. Despite the significant degree of reduction, a relatively high content of free asparagine was found in durum wheat biscuits (265.5, 166.6 and 85.0 mg/kg), then in hulless barley biscuits, hard wheat biscuits, white maize and yellow maize biscuits. In addition to hulless oat biscuits, the lowest asparagine content was measured in refined bread wheat biscuits (24.6, 19.1 and 8.8 mg/kg), as well as in red maize biscuits (38.1, 30.0 and 25.9 mg/kg). The results show that

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the heating time appeared to have a different effect on the asparagine content reduction in biscuits. In the majority of samples (biscuits made from the refined bread wheat flour, as well as from wholegrain flour of durum wheat, soft wheat, rye, yellow and blue popping maize), a gradual decrease in the asparagine content was observed with the extension of the baking time. However, in biscuits made from the wholegrain flour of bread wheat and triticale the content was rapidly reduced to an apparent minimum at 180° C for 10 min of baking (10.0 and 80.2 mg/kg, respectively), followed by a statistically insignificant increase in biscuits baked for 13 min. As a consequence of the thermal protein hydrolysis, in biscuits baked for 10 min and made from the proteins-rich wholegrain flour of hard wheat, hulless oat and hulless barley, the content of free asparagine was a higher (128.8, 55,6 and 151.3 mg/kg, respectively) than in the biscuits baked for 7 min (114.4, 0, 124.5 mg/kg, respectively). The effect of the heating time and the temperature on asparagine stability and its content in food has been investigated by several researchers (Hamlet, Sadd, & Liang, 2008; Curtis, Postles, & Halford, 2014; Weiss, Muth, Drumm, & Kirchner, 2018). A total 49 biscuits prepared from different cereal flours were analyzed for their acrylamide content (Fig. 2). Acrylamide content of cereal flours was found to be lower than LOQ. At 180oC, acrylamide was detected at all baking times, reaching a final content from 72.3 to 861.7 μg/kg after 13 min of baking in refined bread wheat-based biscuits and hulless oatbased biscuits, respectively. The content of acrylamide in biscuits baked for 7 min ranged from 17.9 μg/kg 167.6 μg/kg. Higher variations were found in biscuits baked for 10 min. The content varied from 28.7 μg/kg in red maize-based biscuits to 341.6 μg/kg in yellow maizebased biscuits. Contrary to asparagine, a gradual increase in acrylamide content was observed with the extension of the baking time in most samples. However, the acrylamide content was decreased after 10 min of baking in biscuits made from the bread wheat, hard 13

wheat and yellow maize. Many authors reported a partial degradation of acrylamide at high temperatures and a prolonged heating time (Bråthen & Knutsen, 2005; Gökmen & Senyuva, 2006) as a result of the reaction with other compounds or polymerization (Hidalgo, Delgado, & Zamora, 2011). The results reported by these authors may help to better understand the processes responsible for acrylamide disappearance as a consequence of the Michael addition of sulfuric amino acids, highly represented in cereals, to acrylamide. Deviation from the gradual increase of acrylamide content with extending the baking time was also established in hulless oat-based biscuits. Namely, compared to shorter treatment times, a rapid increase in the acrylamide content of 72% was observed in the biscuits after 13 minutes of baking. In addition to the temperature and the time of baking, cereal ingredients were the basic factor in the formation of acrylamide in biscuits. The lowest content of acrylamide was found in refined bread wheat-based biscuits and red maize-based biscuits. After baking for 7, 10 and 13 min, the content of acrylamide in these samples amounted to 17.9 and 24.4 μg/kg, 51.9 and 28.7 μg/kg and 72.3 and 95.2 μg/kg, respectively. These samples were followed by biscuits prepared from soft wheat flour with less than 300 mg/kg of the initial free asparagine content in it. Our investigation in biscuits baked for 7, 10 and 13 min at 180oC indicates that the use of wholegrain bread wheat flour increases the acrylamide content by about 89, 76 and 65%, respectively, compared with refined wheat flour. Fig. 2 shows that the acrylamide content after 13-minute baking was higher in biscuits made of hulless oat, durum wheat and rye than those made of other cereals. However, compared to durum wheat-based biscuits and rye-based biscuits, hulless oat-based biscuits baked for 7 and 13 min had the acrylamide content higher by 35% on average. For these categories of biscuits, the acrylamide content was lower than that reported by Mesías, Morales and Delgado-Andrade (2019). According to research of Mesías, Morales and 14

Delgado-Andrade (2019), among the 80 commercial biscuits marketed in Spain, the highest acrylamide contents were exhibited by rye-based biscuits (2144 μg/kg) followed by those formulated with oat (on average 1424 μg/kg). According to research of Capei, Pettini, Lo Nostro and Pesavento (2015), among the 44 commercial biscuits marketed in Italy, the highest acrylamide contents were exhibited by biscuits based on mix of ingredients such as wheat, oat, barley, rice, maize and rye (940 μg/kg). Our data for the acrylamide content in biscuits made of white, yellow and red maize flour are close to the range of values in Syrian maize-based biscuits (57 to 325 μg/kg, n = 77) (Alyousef, Wang, Al-Hajj, & Koko, 2016). A benchmark value settled by the European Commission for acrylamide in biscuits and wafers is 350 μg/kg (European Commission, 2017). According to this value, all cereal-based biscuits samples baked at 180oC for 7 and 10 min were below the benchmark level established by the EU Regulation. A total of 30% of the samples baked for 13 min, including durum wheat-, triticale-, rye- and hulless oat-based biscuits, exceeded the reference level. Our data are close to those of Mesías, Morales and Delgado-Andrade (2019) for the commercial cereal-based biscuits marketed in Spain. Considering the benchmark level for acrylamide of 150 μg/kg for biscuits

and rusks for infants and young children, only 35% of the total cookies baked for 7, 10 and 13 min are acceptable for eating. If only the acrylamide content is taken into account, all refined wheat-based biscuits and red maize-based biscuits were health-safe. 3.3. Relationship between the initial content of proteins and free asparagine in cereal flours and acrylamide in biscuits The content of acrylamide in the biscuits samples baked for 7, 10 and 13 min was correlated with the initial content of free asparagine, total proteins and non-protein nitrogen in the flours using principle component (PC) and the regression analysis. Both interrelationship (Fig. 3a) and correlation (Fig. 3b) analyses showed that the initial content of free asparagine 15

in cereal flour had the highest effect on acrylamide, particularly in biscuits baked for 13 min. Non-protein nitrogen had a low influence on acrylamide in biscuits regardless of baking time, while correlations between proteins in the flour and acrylamide in biscuits were moderate (except for biscuits baked for 10 min). The correlation data (Fig. 3a), according to the results published by Capuano et al. (2009), indicate that in biscuits acrylamide could not exactly correspond to the free asparagine in flour. In fact, the height and significance of the correlation depended on the duration of dough exposure to the elevated temperature. The regression coefficient between free asparagine in the flour and acrylamide in the biscuits baked for 13 min was 0.81. Correlating free asparagine with acrylamide in biscuits baked for 7 and 10 min indicated that the correlation coefficient was lower by 1.7 times. According to results of Granby et al. (2008), there is a high relationship between asparagine in rye and wheat flour and acrylamide in bread after medium (r2 = 0.86) or hard (r2 = 0.88) toasting. A strong positive correlation was between the free asparagine concentration of rye grain (n = 5) and he acrylamide formed upon heating at 160oC for 20 min (Postles, Powers, Elmore, Mottram, & Halford, 2013). 3.4. Correlation between contents of free asparagine and acrylamide in cereal flours and biscuits The interdependence of free asparagine and acrylamide under the influence of high temperature during 7, 10 and 13 min of biscuits baking was determined by the application of statistical methods (Fig. 4). A negative correlation between the content of free asparagine and acrylamide after baking for different times indicates that the asparagine content reduction was accompanied by an increase in the acrylamide content. In seven of 13 sample groups, the analysis shows a medium (r2 = 0.6 to 0.7), high (r2 = 0.7 to 0.9) and very high (r2 =

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over 0.9) dependence on the change in asparagine and acrylamide contents after baking of biscuits. The highest correlation between these parameters was found in wholegrain bread wheat- and white maize-based biscuits. However, the correlations between contents of free asparagine and acrylamide in triticale, hulless oat, hulless barley, yellow maize, red maize and blue maize flours and biscuits baked at 180oC for 7, 10 and 13 min were as low as r2 = 0.56. Initial values for asparagine and acrylamide in flour significantly influenced the correlation between changes of mentioned compounds during baking of biscuits. This impact was directed towards both increasing and decreasing the correlation coefficient. Even so, our results suggest that changes in the acrylamide content in biscuits under the influence of the prolonged baking time do not exactly match changes in free asparagine. The correlations between the acrylamide content in biscuits and the differences in the free asparagine content before and after baking showed the same trend. These correlations were positive and ranged from r2 = 0.0001 in triticale-based cookies to r2 = 1 in white maize-based biscuits. It indicates that the formation of acrylamide in the biscuits was also influenced by other factors and properties of flour, or that intermediate compounds were formed at shorter baking times. The drastic decrease in the free asparagine content after baking at a lower temperature, not accompanied by a significant increase of acrylamide in biscuits, could be due to the formation of 3-aminopropionamide (3-APA) as one of the key transient intermediates in acrylamide formation during thermal degradation of asparagine initiated by reducing carbohydrates or aldehydes. According to the results of Gökmen, Kocadağlı, Göncüoğlu and Ataç Mogol (2012), quantification of 3-APA indicated clearly its accumulation in asparagine-glucose and asparagine-5-hydroxymethyl-2-furfural (HMF) model systems during heating at 180oC. It should be emphasized that numerous authors have studied the impact of various factors, such as water content, the physical state of the food, phenolic 17

profile, lipid oxidation products, type of reducing sugar, free amino acid profile, chemical leavening agents, yeast amount and yeast types, fermentation time and fermentation temperature etc. on acceleration or mitigation of the acrylamide formation in a complex food system such as bread and biscuits (Ciesarova, Kiss, & Kolek, 2016; Arribas-Lorenzo, Fogliano, & Morales, 2009; Koutsidis et al., 2009; Masatcioglu, Gökmen, Ng, & Köksel, 2014; Katsaiti, & Granby, 2016). 4. Conclusion Despite the mitigation strategies applied, the modification of standard recipes with different cereal species or innovative genotypes can increase the acrylamide content in biscuits. According to obtained results, it can be concluded that genotypes of small grain cereals and maize had different potential for the acrylamide formation. Our results confirm that free asparagine in cereal flour is one of the main factors in the formation of acrylamide in biscuits during baking. Although the correlation data indicated that acrylamide in biscuits could not exactly correspond to the free asparagine in flour, it is evident that the biscuits prepared from flour with the highest content of free asparagine had the highest content of acrylamide. Given that asparagine is one of the nonessential amino acids in the diet, industrial approaches to reducing the acrylamide in biscuits may include the use of existing cereal species and their varieties with a low free asparagine content in the grain. Data show that hulless oat, durum wheat and rye produced most acrylamide in biscuits baked for 13 min. The lowest content of acrylamide was found in refined bread wheat-based biscuits and red maize-based biscuits. Our investigation also confirmed that the use of wholegrain bread wheat flour increased the acrylamide content in biscuits by 65 to 89% compared with refined wheat flour. Acrylamide levels in cereal biscuits baked for 13 min a greatly varied,

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ranging from lower than 72.3 μg/kg to 861.7 μg/kg. According to the benchmark level established by the European Regulation for biscuits and wafers, 30% of the biscuits baked at 180oC for 13 min exceeded the reference level. Biscuits baked for a shorter time were below the benchmark level. Therefore, it can be concluded that, in addition to the low content of free asparagine contained by cereal flour, the use of adequate thermal treatments in the biscuits production is an effective acrylamide mitigation strategy. More varieties within species grown in different environments should be tested to further validate the findings of this study on the effect of different cereals on acrylamide formation in bakery products. Acknowledgement This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia [grants number TR31069]. Declaration of Competing Interest All the authors declare no conflict of interest.

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Highlights 

Higher the content of free asparagine in flours resulted in higher amounts of acrylamide formed in biscuits.



The highest amount of acrylamide occurred in biscuits made of flour with the highest content of free asparagine.



The lowest amount of acrylamide was found in refined bread wheat-based biscuits.



Hull-less oat, durum wheat and rye flours generated higher amounts of acrylamide.

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GA

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Fig. 1. Appearance of dough and cookies made of different whole grain cereals baked at 180oC for 7, 10 and 13 min Dough Wheat flour T 400

180oC / 7 min

180oC / 10min

180oC / 13 min

Bread wheat – Wholegrain

Durum wheat - Wholegrain

Soft wheat - Wholegrain

Hard wheat - Wholegrain

29

Fig. 1. (continued) Dough Triticale - Wholegrain

180oC / 7 min

180oC / 10min

180oC / 13 min

Rye - Wholegrain

Hull-less oat - Wholegrain

Hull-less barley - Wholegrain

White maize - Wholegrain

30

Fig. 1. (continued) Dough Yellow maize- Wholegrain

180oC / 7 min

180oC / 10min

180oC / 13 min

Red maize - Wholegrain

Blue popping maize - Wholegrain

31

Fig. 2. Contents of free asparagine and acrylamide in cereal flour and cookies made of different whole grain cereals baked at 180oC for 7, 10 and 13 min

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Fig. 3. The interrelationship A) and the correlation B) between the initial content of proteins, non-protein nitrogen and free asparagine in cereal flours and acrylamide in cereal cookies A) flour/cookies 7 min

flour/cookies 10 min 0.75

0.50

flour/cookies 13 min 0.50

Proteins

Proteins

Proteins

FAsn

-0.25 -0.50

Non-protein nitrogen

0.25

FAsn

0.00

0.1

0.2

0.3

0.4

0.5

PC1 (63.2%)

0.6

0.7

0.8

0.9

-0.25

ACR

Non-protein nitrogen

-0.75

Non-protein nitrogen

-0.50 0.0

FAsn ACR

0.00

-0.50

-0.25

-0.75

PC2 (19.9%)

0.00

0.25

0.50

ACR PC2 (21.2%)

PC2 (20.2%)

0.25

0.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.2

0.4

0.6

0.9 PC1 (66.6%)

PC1 (58.5%)

B)

33

0.8

1.0

Fig. 4. Correlation between contents of free asparagine and acrylamide in cereal flours and cookies baked at 180oC for 7, 10 and 13 min

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Acrylamide formation in biscuits made of different whole grain flours depending on their free asparagine content and baking conditions Slađana Žilića1, Işıl Gürsul Aktağb, Dejan Dodiga2, Milomir Filipovića2, Vural Gökmenb* a Maize

Research Institute, 1Department of Food Technology and Biochemistry and 2Breeding

Department, Slobodana Bajića 1, 11080 Zemun-Belgrade, Serbia b

Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering,

Hacettepe University, 06800 Beytepe, Ankara, Turkey

Conflict of Interest File Declarations of interest: none

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Table 1. Contents of proteins, non-protein nitrogen and free asparagine in different cereal flours Proteins

Non-protein

Free asparagine

Ash

Total fiber

(%)

nitrogen (%)

(mg/kg)

(%)

(%)

Wheat flour T 400

10.42±0.02h

2.91±0.07c

61.6±5.4i

0.47±0.01e

7.1±0.11g

Bread wheat

11.81±0.02e

2.32±0.11d

260.0±21.6gh

1.60±0.08cd

7.6±0.18fg

Durum wheat

10.83±0.08e

2.86±0.10c

530.3±19.9cd

1.68±0.08bcd

10.0±0.21d

Soft wheat

11.64±0.12e

1.79±0.01e

275.8±22.4g

1.63±0.07cd

9.0±0.30e

Hard wheat

14.80±0.12b

3.39±0.08b

480.6±48.4de

2.09±0.06ab

9.8±0.26d

Triticale

12.22±0.06d

1.96±0.01de

449.4±21.2def

1.75±0.01bc

13.1±0.52a

Rye

11.45±0.05f

5.22±0.32a

603.2±24.4bc

2.36±0.08a

15.2±0.20a

Hull-less oat

16.80±0.11a

2.91±0.02c

859.8±72.8a

2.44±0.11a

11.9±0.30c

Hull-less barley

11.74±0.01e

2.14±0.12d

297.0±33.0g

1.81±0.11bc

12.0±0.31c

White maize

10.67±0.06g

1.76±0.07e

420.2±7.4ef

1.34±0.06d

8.0±0.25f

Yellow maize

10.31±0.06h

1.78±0.02e

470.5±35.1de

1.63±0.03cd

8.5±0.48ef

9.56±0.07i

1.57±0.03e

189.7±12.1h

1.49±0.12cd

9.2±0.31de

13.11±0.11c

1.76±0.05e

387.9±24.1f

1.48±0.09cd

11.9±0.41c

Red maize Blue popping maize

Means followed by the same letter within the same row are not significantly different, according to Tukey’s test (α=0.05%).

36