Cysteine alone or in combination with glycine simultaneously reduced the contents of acrylamide and hydroxymethylfurfural

LWT - Food Science and Technology 63 (2015) 275e280

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Cysteine alone or in combination with glycine simultaneously reduced the contents of acrylamide and hydroxymethylfurfural Yueyu Zou, Caihuan Huang, Kehan Pei, Yun Cai, Guangwen Zhang, Changying Hu, Shiyi Ou* Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 October 2014 Received in revised form 12 February 2015 Accepted 28 March 2015 Available online 8 April 2015

Inhibition of acrylamide formation in food has been extensively reported, but some mitigation methods result in a concomitant increase in hydroxymethylfurfural (HMF), a food contaminant mainly produced through Maillard reaction. Mitigation strategies to reduce HMF are not yet available. This study showed that cysteine alone or in combination with glycine could simultaneously and significantly reduce the content of acrylamide and HMF in asparagine/glucose model as well as in biscuits. Mixing 0.36 g/100 g of cysteine and 0.2 g/100 g (w/w) of glycine into the dough reduced 97.8% and 93.2% of acrylamide and HMF, respectively, in biscuits. Cysteine reduced HMF content possibly by reacting with formed HMF through Michael adduction and Maillard reaction. © 2015 Published by Elsevier Ltd.

Keywords: Acrylamide Hydroxymethylfurfural Cysteine Glycine

1. Introduction Acrylamide and hydroxymethylfurfural (HMF) are two food contaminants produced during high-temperature food processing. Acrylamide is mainly formed from the reaction between asparagines and carbonyl compounds (reducing sugars). This reaction firstly formed a Schiff base and then decarboxylate to form an azomethine ylide. Afterward, acrylamide may be formed directly from azomethine ylide, through b-elimination of the decarboxylated Amadori compound, or through deamination of 3aminopropionamide (Jin, Wu, & Zhang, 2013). Acrylamide induces tumours in several organs in mice and rats and has been designated by the International Agency for Research on Cancer as a “probable human carcinogen” (Capuano & Fogliano, 2011). It is rapidly absorbed from the gastrointestinal tract and can be metabolised to glycidamide, a compound more reactive with DNA and proteins than acrylamide (Pedreschi, Mariotti, & Granby, 2014). Acrylamide is a neurotoxin that inhibits kinesin-based fast axonal

Abbreviations: Cys, cysteine; Gly, glycine; HMF, hydroxymethylfurfural; LC-MS, liquid chromatographyemass spectroscopy; ORP, oxidationereduction potential; PBS, phosphate buffer solution; SMF, 5-sulfooxymethylfurfural; SULTs, sulfotransferases. * Corresponding author. Tel.: þ86 20 85224235; fax: þ86 20 85226630. E-mail address: [email protected] (S. Ou). http://dx.doi.org/10.1016/j.lwt.2015.03.104 0023-6438/© 2015 Published by Elsevier Ltd.

transport and decreases neurotransmitter levels, thereby inhibiting neurotransmission (Erkekoglu & Baydar, 2014). The neurotoxic effects of acrylamide can be observed at low dose with long exposures (Erkekoglu & Baydar, 2014), suggesting that dietary acrylamide is harmful to humans, especially children. The presence of acrylamide in food remains a health risk. According to WHO, the mean margin of exposure (MOE) value based on the carcinogenic effect of acrylamide in mammary glands is 300e310 (Pedreschi et al., 2014), which is lower than 10,000, a criterion regarded as low health concern. Moreover, the detected concentrations of acrylamide and glycidamide haemoglobin adducts in Canadian teenagers indicate the need to reduce acrylamide exposure in the population (Brisson et al., 2014). 5-Hydroxymethylfurfural (HMF), a heterocyclic compound, is a thermal process contaminant in food. It is formed after 1,2enolization, dehydration and cyclisation reactions from hexose sugars and Amadori product degradation during Maillard reaction, or from direct dehydration of sugars under acidic conditions (Capuano & Fogliano, 2011; Goncuoglu & Gokmen, 2013). HMF content ranges from 1.9 mg/kg to 20 mg/kg in baking products to several grams/kg in coffee, toasted chicory and dried fruits (Capuano & Fogliano, 2011; Goncuoglu & Gokmen, 2013; Petisca, Henriques, Perez-Palacios, Pinho, & Ferreira, 2014). HMF content in food is related to the heat load applied during processing, which is a common index to evaluate thermal

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damage, ageing, and sensory properties in food products (Anese, Bot, & Suman, 2014; Goncuoglu & Gokmen, 2013; Petisca et al., 2014). HMF causes death in honey bees (Zirbes et al., 2013), induces genotoxic and mutagenic effects in bacterial and human cells (Svendsen et al., 2012), and promotes colon cancer in rats (Svendsen et al., 2012), although conflicting views exist with regard to the effect of this substance on human health (Abraham et al., 2011; Severin, Dumont, Jondeau-Cabaton, Graillot, & Chagnon, 2010). HMF initiates colonic aberrant crypt foci in rats and skin papillomas and hepatocellular adenomas in mice. HMF is inactive in in vitro genotoxicity tests using standard activating systems but is activated to a mutagen, 5-sulfooxymethylfurfural (SMF), by sulfotransferases (SULTs) (Monien, Engst, Barknowitz, Seidel, & Glattt, 2012; Monien, Frank, Seidel, & Glatt, 2009; Monien & Glatt, 2012). Monien et al. (2009) intravenously injected HMF (793 mmol/kg) into mice and detected a maximum SMF plasma level 2.5 min after HMF administration; 452e551 ppm of the initial HMF dose was converted to SMF and reached the circulation. In contrast to rodents, which mainly express sulfotransferases in liver, humans express sulfotransferases in many tissues, including the intestine, implying that humans could be more susceptible to HMF than conventional rodent models (Svendsen et al., 2012). Moreover, HMF can be converted to acrylamide and furan during € kmen, Kocadagli, Go €ncüoglu, Maillard reaction, (Cai et al., 2014; Go & Mogol, 2012; Mesias-Garcia, Guerra-Hernandez, & GarciaVillanova, 2010) which are classified as “possibly carcinogenic to humans”. Numerous studies focus on HMF content and toxicology as well as the influence of composition and process variables on HMF formation, but mitigation strategies specifically addressed to reduce HMF content in foods are not yet available (Capuano & Fogliano, 2011). Preventive strategies are based on changes in formulation, reduction of thermal impact, and removal of HMF formed in the product by vacuum treatments (Anese & Suman, 2013). However, HMF cannot be removed by vacuum treatments in biscuits (Anese et al., 2014). A number of food additives, such as CaCl2, cysteine, and glycine, have been proven to effectively inhibit acrylamide formation. Mixing these inhibitors with raw materials or immersing of fresh food in solutions containing inhibitors does not significantly affect the processing technology, making this method practical in the food industry (Friedman & Levin, 2008; Ou et al., 2008; Pedreschi et al., 2014). In our previous study, cysteine and glycine significantly influence HMF formation during Maillard reaction; reaction of glucose with cysteine and glycine produces less HMF than reaction with lysine and glutamate (Jiang et al., 2013). Whether adding these additives alone or in combination can simultaneously inhibit the formation of acrylamide and HMF is unknown. Therefore, this study we used CaCl2, cysteine and glycine alone or their mixtures to investigate their inhibition effect on acrylamide and HMF formation. 2. Materials and methods 2.1. Chemicals Cysteine, glycine, asparagine, glucose, 2-mercaptoethanol, ethylamine, and calcium chloride were purchased from Aladdin Reagents Database Inc. (Shanghai, China). HMF and acrylamide standard (>99.8%) were obtained from SigmaeAldrich Company (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade methanol and polyphenol oxidase (845 U/mg) were

obtained from J. T. Baker (USA) and Worthington Biochemical Corporation (Lakewood, NJ, USA), respectively. 2.2. Preparation of cysteine, glycine and/or CaCl2 containing asparagine/glucose model reaction systems An equimolar asparagine/glucose Maillard reaction system was used to investigate the effects of additives on the formation of acrylamide and HMF. Each 20-ml stainless steel test tube contained 4 mL of 0.1 mol/L phosphate buffer solution (pH ¼ 5.7 and 7.0) with 1 mmol asparagine and 1 mmol glucose, as well as different concentrations of cysteine, glycine and CaCl2 alone (0.05, 0.1, 0.25, 0.375 and 0.5 mol/L), As described in our previous research (Cai et al., 2014), the test tubes were capped with Teflon pad-filled stainless steel cap and the mixtures were heated at 160  C for 15 min in an oil bath installed with a magnetic stirrer (DF-101S, Yuhua Instrument Co. Ltd., Gongyi, Henan Province, China). After cooling in an ice bath, the reaction mixtures were decanted into 14ml centrifuge tubes and deionized water was added to make a total volume of 10 mL in each tube. The mixtures were then centrifuged at 3000 g for 20 min on an Allegra 21 R centrifuge (Beckman Coulter Inc., Miami, USA). The concentrations of acrylamide and HMF in the supernatant were then determined. 2.3. Preparation of biscuits added with additive mixtures Biscuits were prepared according to the recipe of Van Der FelsKlerx et al. (2014) with slight modification. The dough contained 80.0 g wheat flour (passing through a 0.074 mm-sieve), refined palm oil (20.0 g), sucrose (35.0 g), NaHCO3 (0.8 g), water (16.0 g), NH4HCO3 (0.4 g) and NaCl (1.0 g). Oil, sucrose, NaHCO3, NH4HCO3, NaCl and the additives were initially mixed in water and then mixed with flour to prepare the dough. Based on the optimal concentration obtained from the results of the asparagine/glucose model reaction system, six kinds of additive mixtures (on the basis of dough; their concentrations in added water were similar to that in the model reaction system) were tested: A. 0.08 g/100 g CaCl2 þ 0.4 g/100 g cysteine; B. 0.06 g/100 g CaCl2 þ 0.65 g/100 g cysteine; C. 0.16 g/100 g CaCl2 þ 0.54 g/100 g cysteine; D. 0.18 g/ 100 g cysteine þ0.3 g/100 g glycine; E. 0.36 g/100 g cysteine þ0.2 g/ 100 g glycine; and F. 0.06 g/100 g CaCl2 þ 0.29 g/100 g cysteine þ0.2 g/100 g glycine The dough (3  5  0.3 cm) were baked in a convention oven at 190  C for 15 min, then cooled. 5.0 g of biscuits was ground and ultrasound extracted three times using 20 mL of 800 mL/L methanol according to Michalak, Gujska, and Kuncewicz (2013). Methanol and water in the extracts were removed by a rotary evaporator (RE-52AAA, Shanghai JiaPeng Technology co., LTD, Shanghai, China) under vacuum at 60  C, the residues were dissolved in 2.0 mL deionized water for the determination of acrylamide and HMF. 2.4. Effect of amino acids, 2-mercaptoethanol and ethylamine on HMF elimination The mixture (4 mL) in the test tubes, containing 0.1 mmol of HMF and 1.0 mmol of amino acids (Cys or Gly) or 2mercaptoethanol (SH containing compounds) or ethylamine (NH2 containing compounds), was allowed to react in an oil bath at 160  C for 15 min. The residual HMF was detected by HPLC. 2.5. Acrylamide and HMF analysis Acrylamide and HMF were determined by LCeMS and HPLC, respectively, as we previously described (Cai et al., 2014).

Y. Zou et al. / LWT - Food Science and Technology 63 (2015) 275e280

2.5.1. Acrylamide analysis The samples were diluted to 10 mL using deionized water, and 4.8 mL of diluted sample was mixed with 0.2 mL of 13C3labelled acrylamide internal standard (2 mg/mL). Liquid chromatographyemass spectrometry/mass spectrometry (LC-MS/ MS) analysis of the samples was conducted on a Shimadzu LC20AT system (Shimadzu, Kyoto, Japan) consisting of an LC10ATvp pump, a SIL-HTa autosampler, and a CTO-10Asvp temperature-controlled column oven, coupled to an API3000 MS detector equipped with an atmospheric pressure chemical ionization interface. A total volume of 20 mL of the samples was eluted on a YMC-Pack ODS-AQ C18 column (50 mm  4.6 mm, 5 mm) at 40  C using an isocratic mixture of 0.5 mL/100 mL methanol/0.1 mL/100 mL acetic acid in deionized water at a flow rate of 0.5 mL/min. MS/MS was performed in positive ESI mode. The transitions m/z 72 / 55 and 75 / 58 were used in the identification and quantification of acrylamide and 13C3-labelled acrylamide, respectively. 2.5.2. HMF analysis Samples were filtered through a 0.45 mm membrane. HMF quantification was performed on a Shimadzu LC-20AT system (Shimadzu, Kyoto, Japan) equipped with a diode array detector and LC-solution software. A Zorbax SB-Aq column (4.6 mm  250 mm, 5 mm) (Agilent Technologies Co., Ltd.) was selected for HMF analysis. The injection volume was 5 mL. Elution was conducted at a flow rate of 0.5 mL/min under isocratic conditions at 40  C, using 5 mL/100 mL of aqueous methanol solution as the mobile phase. HMF was detected at 284 nm and quantified using the standard curve. The range of standards used was 0, 1.0, 2.0, 4.0, 6.0, 8, and 10.0 mg/mL for low concentration of HMF, and 0, 50,100, 200, 400, 600, 800 mg/mL for high concentration of HMF. 2.6. Determination of oxidationereduction potential in the Maillard reaction system Oxidationereduction potential (ORP) in the supernatant of the Maillard reaction system was determined using an ORP-422 model ORP detector (Beijing Zhongxi Yuanda Scientific Instrument Co., Ltd., Beijing, China). 2.7. Determination of adducts of amino acids with HMF Samples (10 mL) were analysed on an LCeMS system composed of a 4000Q-TRAP mass spectrometer (Applied BiosystemSciex) and a 1100 HPLC system equipped with a Venusil MP C18 column (2.1 mm  100 mm, 3 mm) (Agela Technologies Inc, Newark, USA) maintained at 35  C. Samples were eluted with 0.5 mL/100 mL formic acid in water (eluent A) and acetonitrile (eluent B) at a flow rate of 0.4 mL/min and the total run time was 20 min. The gradient elution program was as follows: 100% eluent A isocratically (8 min), from 100% A to 85% A and 15% B (12 min). Full scan MS (MS2SCAN) was conducted using electrospray ionisation (ESI) with selected ion recording. Mass spectra of the precursor and product ions were obtained to determine the molecular weight of product components and identify amino acid and HMF adducts.

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3. Results and discussion 3.1. Effect of additives on acrylamide and HMF formation in asparagine/glucose model reaction system The effect of cysteine, glycine and CaCl2 on acrylamide and HMF formation in asparagine/glucose model reaction system was tested at pH 7.0 and 5.7 (the internal pH of potato tuber reported by Rydberg et al., 2003) respectively. The results in Table 1 showed that adding cysteine, glycine and CaCl2 significantly reduced acrylamide formation on both pH values, and the reduction was dose dependent. At the addition levels from 0.05 to 0.25 mol/L, cysteine was the most effective, followed by CaCl2 and glycine (except for that at addition level of 0.375 and 0.5 mol/L); when the concentration of the additives further increased, glycine was more effective than CaCl2. Cysteine and glycine were less effective at lower pH (5.7) than at higher pH (Table 1), possibly because that higher pH favours Michael addition reaction and Maillard reaction (competition with asparagine for reaction with reducing sugar). Cysteine and glycine significantly inhibited the heat-induced decrease in the ORP (increased the negative voltages) of the Maillard reaction system, whereas CaCl2 increased ORP (Table 2). Thus, adding cysteine and glycine may not promote acrylamide elimination through free radicals produced during the Maillard reaction (Friedman & Levin, 2008); They may inhibit acrylamide formation by competing with asparagine (Rydberg et al., 2003), or eliminating the formed acrylamide by Michael adduction (Koutsidis et al., 2009; Liu, Chen, Man, Dong, & Hu, 2011; Yu, Ou, Deng, Huang, & Zhang, 2013; Zamora, Delgado, & Hidalgo, 2010). Cysteine reduced HMF formation at both pH levels and all addition levels; Lower pH levels seemed to favour its inhibition effect (Table 2), by an unknown mechanism. Adding glycine at lower concentration levels increased HMF formation (Table 3); however, higher addition level of glycine (0.2 mol/L) decreased HMF content, especially at pH 7.0. We proposed that glycine increased HMF formation through Maillard reaction and eliminated the formed HMF through Michael addition (which will be discussed later); the addition level of glycine may determine the balance between the formation and elimination of HMF. By contrast, adding CaCl2 significantly increased HMF formation (Table 3). Gokmen and Senyuva (2007) proposed that cations effectively prevent the formation of Schiff's base, thus inhibiting acrylamide formation and changing the reaction path toward the dehydration of glucose to produce HMF. Since excessive addition of cysteine produces unpleasant flavour (Ou et al., 2008) and Michael adduction between amino acids and acrylamide is a reversible reaction (Koutsidis et al., 2009; Zamora et al., 2010); this research evaluated the mixture of the two amino acids and CaCl2 on the inhibitory effect on acrylamide and HMF formation. Table 4 shows that the combination of cysteine and CaCl2 or the mixtures of the three additives reduced acrylamide and HMF formation in asparagine/glucose model reaction system. Adding 0.125 mol/L CaCl2 combined with 0.375 mol/L cysteine inhibited acrylamide formation by 97.3%, and completely inhibited HMF production at pH 5.7. Moreover, 98.2% and 86.4% of acrylamide and HMF were reduced respectively at pH 7.0 after addition of 0.05 mol/L CaCl2 in combination with 0.25 mol/L cysteine, and 0.2 mol/L glycine (Table 4).

2.8. Statistical analysis 3.2. Effect of additives on acrylamide and HMF formation in biscuits Each treatment was conducted in triplicates. Data were expressed as means ± standard error. Statistics were determined with SPSS 13.0 for Windows procedure. Duncan's multiple-range test (p < 0.05) was used to determine significance of differences between means.

Thin biscuits (with 3 mm thickness) were oven baked (190  C for 15 min) under conditions found to produce a high amount of acrylamide and HMF. These biscuits were used to evaluate the effect of additives on the formation of acrylamide and HMF. All

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Table 1 Effect of cysteine, glycine and calcium chloride on acrylamide (mg/ml) formation in asparagine/glucose model reaction system at pH 5.7 and 7.0. Concentration (mol/L)

Cysteine

Glycine

pH ¼ 5.7 0 0.05 0.1 0.2 0.25 0.375 0.5 a b

60.3 34.9 14.1 3.4 3.3 4.4 3.7

± ± ± ± ± ± ±

pH ¼ 7.0

1.1d 1.9c 3.8b 0.7a 0.7a 2.0a 1.6a

a

68.0 33.0 14.4 6.7 2.5 NDb ND

± ± ± ± ±

CaCl2

pH ¼ 5.7

3.3e 8.3d 1.2c 2.4b 0.9a

60.3 38.7 27.3 15.9 6.4 13.1 ND

± ± ± ± ± ±

pH ¼ 7.0

1.1e 4.5d 2.1c 2.5b 2.1a 3.4b

68.0 45.9 30.0 19.6 17.3 5.1 2.3

± ± ± ± ± ± ±

pH ¼ 5.7

3.3f 3.7e 0.5d 1.8c 1.8c 0.1b 0.3a

60.3 34.1 18.6 15.8 9.3 10.4 10.2

± ± ± ± ± ± ±

pH ¼ 7.0

1.1g 5.6f 0.7e 0.8d 0.3a 1.2bc 0.4b

68.0 22.5 13.1 11.6 9.9 5.1 5.0

± ± ± ± ± ± ±

3.3e 1.5d 1.7c 0.7bc 4.7b 1.3a 0.4a

Values (means ± SD, n ¼ 3) with different letters within a column are significantly different at 5% level. Not detected.

Table 2 Effect of cysteine, glycine and calcium chloride on oxidationereduction potential (mv) in asparagine/glucose model reaction system at pH 5.7 and 7.0. Concentration (mol/L)

Cysteine

Glycine

pH ¼ 5.7 0 0.05 0.1 0.2 0.25 0.375 0.5 a

20 11 81 129 140 176 181

± ± ± ± ± ± ±

pH ¼ 7.0

7fa 5e 3d 8c 4b 21a 12a

22 13 50 102 140 153 170

± ± ± ± ± ± ±

CaCl2

pH ¼ 5.7

9f 4e 6d 5c 11b 5b 4a

20 19 17 15 13 18 29

± ± ± ± ± ± ±

pH ¼ 7.0

7c 2c 10bc 4b 6b 7a 4a

22 22 22 20 17 14 28

± ± ± ± ± ± ±

pH ¼ 5.7

9d 2d 7d 3cd 5cv 4b 9a

20 78 72 70 79 79 85

± ± ± ± ± ± ±

7a 5bc 1b 6b 2bc 7bc 4cd

pH ¼ 7.0 22 46 70 81 66 73 81

± ± ± ± ± ± ±

9a 6b 8cd 2e 5c 4cd 1e

Values (means ± SD, n ¼ 3) with different letters within a column are significantly different at 5% level.

Table 3 Effect of cysteine, glycine and calcium chloride on HMF (mg/ml) formation in asparagine/glucose model reaction system at pH 5.7 and 7.0. Concentration (mol/L)

Cysteine

Glycine

pH ¼ 5.7 0 0.05 0.1 0.2 0.25 0.375 0.5 a b

46.5 43.7 16.5 3.4 0.9 NDb ND

± ± ± ± ±

pH ¼ 7.0

1.5ea 3.4d 1.8c 0.9b 0.2a

35.4 33.7 21.1 3.1 3.2 1.1 ND

± ± ± ± ± ±

2.9e 0.2d 3.5c 0.3b 0.5b 0.2a

CaCl2

pH ¼ 5.7 46.5 53.6 56.7 52.0 41.9 34.8 32.6

± ± ± ± ± ± ±

pH ¼ 7.0

1.5c 0.5d 3.3d 2.4d 3.4b 1.8a 1.7a

35.4 40.5 44.2 28.2 16.6 12.5 7.6

± ± ± ± ± ± ±

pH ¼ 5.7

2.9e 0.9f 2.6g 4.4d 2.2c 2.7b 0.0a

46.5 162.9 265.8 300.6 336.9 388.5 433.8

± ± ± ± ± ± ±

1.5a 8.5b 2.3c 11.8d 8.4e 10.3f 4.0g

pH ¼ 7.0 35.4 82.6 119.7 169.6 306.9 371.9 430.4

± ± ± ± ± ± ±

2.9a 2.1b 11.6c 27.1d 8.7e 16.3f 4.6g

Values (means ± SD, n ¼ 3) with different letters within a column are significantly different at 5% level. Not detected.

additive mixtures reduced the contents of acrylamide and HMF (Table 5). The mixture of CaCl2 along with amino acids decreased the content of acrylamide but showed no effect on HMF reduction (Table 5). The optimal formulation was 0.36 g/100 g cysteine and

0.2 g/100 g glycine (approximately 0.25 mol/L cysteine or glycine in added water of the dough). In this treatment, the contents of acrylamide and HMF were reduced by 97.8% and 93.2%, respectively, and no unpleasant favour was produced from cysteine.

Table 4 Effect of the mixture of cysteine, glycine and calcium chloride on the formation of acrylamide and HMF in asparagine/glucose model reaction system at pH 5.7 and 7.0. Con. In the mixture (mol/L)a

Acrylamide (mg/ml)

Control CaCl20.05 þ Gly0.45 CaCl20.125 þ Gly0.375 CaCl20.2 þ Gly0.3 CaCl20.05 þ Cys0.45 CaCl20.125 þ Cys0.375 CaCl20.2 þ Cys0.3 Gly0.375 þ Cys0.125 Gly0.25 þ Cys0.25 Gly0.125 þ Cys0.375 CaCl20.05 þ Gly0.2 þ Cys0.25 CaCl20.05 þ Gly0.25 þ Cys0.2

60.3 7.6 6.4 14.6 2.7 1.9 2.0 4.3 2.6 2.4 3.0 3.5

pH ¼ 5.7

a b

± ± ± ± ± ± ± ± ± ± ± ±

1.1ja 0.6h 0.3g 0.7i 0.1c 0.1a 0.1a 0.2f 0.0c 0.0b 0.0d 0.1e

HMF (mg/ml) pH ¼ 7.0 68.0 5.8 1.5 2.0 0.7 0.8 0.2 0.3 0.3 1.9 1.2 1.3

± ± ± ± ± ± ± ± ± ± ± ±

13.3h 0.3g 0.1e 0.1f 0.0c 0.0c 0.0a 0.0b 0.0b 0.0f 0.0d 0.1d

Values (means ± SD, n ¼ 3) with different letters within a column are significantly different at 5% level. Not detected.

pH ¼ 5.7 46.5 147.5 248.3 496.7 631.7 NDb ND 19.8 4.1 1.8 6.0 10.6

± ± ± ± ±

1.5f 8.2g 13.9h 23.0i 32.6j

± ± ± ± ±

0.6e 0.8b 0.2a 0.5c 0.1d

pH ¼ 7.0 35.4 173.1 414.1 382.6 ND ND 13.5 16.5 4.1 ND 4.8 9.6

± ± ± ±

6.9e 3.3h 34.6j 23.9i

± 4.7d ± 0.8d ± 0.0a ± 0.0b ± 0.8c

Y. Zou et al. / LWT - Food Science and Technology 63 (2015) 275e280

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Table 5 Effect of the mixture of cysteine, glycine and calcium chloride on the formation of acrylamide and HMF in biscuits. Formulation

Control

Aa

B

C

D

E

F

Acrylamide (mg/kg) HMF(mg/kg)

6849 ± 35gb 2450 ± 57

3755 ± 27f (45.2)c 1528 ± 123d (37.6)

3653 ± 31e (46.7) 2276 ± 28e (7.1)

3196 ± 35d (53.3) 1424 ± 14d (41.9)

2739 ± 21c (60.0) 371 ± 9b (84.8)

149 ± 7b (97.8) 167 ± 4a (93.2)

137 ± 6a (98.0) 1206 ± 21c (50.7)

a A. 0.08 g/100 g CaCl2 þ 0.4 g/100 g cysteine; B. 0.06 g/100 g CaCl2 þ 0.65 g/100 g cysteine; C. 0.16 g/100 g CaCl2 þ 0.54 g/100 g cysteine; D. 0.18 g/100 g cysteine þ 0.3 g/ 100 g glycine; E. 0.36 g/100 g cysteine þ 0.2 g/100 g glycine; and F. 0.06 g/100 g CaCl2 þ 0.29 g/100 g cysteine þ 0.2 g/100 g glycine. b Values (means ± SD, n ¼ 3) with different letters within a line are significantly different at 5% level. c Data in the brackets were inhibition percentage of acrylamide or HMF by food additives.

3.3. Possible inhibition mechanism of cysteine and glycine for HMF Cysteine and glycine reduce acrylamide content in heat-treated foods by competing with asparagine for the carbonyl group of the sugar moiety and by forming adducts with acrylamide after its formation (Friedman & Levin, 2008). The mechanism of HMF reduction by the two amino acids remains unknown. HMF may react with NH2 group of amino acids through Maillard reaction, or with NH2 or SH group through Michael adduction because HMF contains a carbonyl group and two double bonds (a,b-unsaturated carbonyl compounds). In this study, 1 mmol of cysteine (which contains a-NH2 and eSH groups), glycine (which only contains a-NH2), 2-mercaptoethanol (which only contains eSH group), and ethylamine (which only contains eNH2) were separately reacted with 0.1 mmol of HMF (12.61 mg) in a 4 mL solution at 160  C for 15 min, and the residual HMF were 162.1 ± 3.6, 1433.8 ± 46.0, 729.4 ± 6.3, and 877.3 ± 4.7 mg/mL, respectively. HMF was depleted by 94.9%, 54.5%, 76.9%, and 72.2% respectively after the addition of cysteine, glycine, 2mercaptoethanol and ethylamine. Cysteine depleted 18% more HMF than 2-mercaptoethanol, which only contains eSH group and can only react with HMF through Michael reaction. This finding suggests that cysteine reduced the formed HMF through two pathways: by reacting with the formed MF through Michael adduction (dominant one), and by reaction with HMF possibly through the Maillard reaction. Because eSH group in 2mercaptoethanol showed higher reaction activity than eNH2 in ethylamine, the Maillard reaction between amino acids and HMF may make less contribution to eliminate the produced HMF than Michael addition. HPLC-MS showed that a high peak-area compound (MW ¼ 350) was produced, which has fragment ions (m/z) as: 315, 228, 166, 150, and 136. We speculated that it is an adduct of one molecule of HMF with two molecules of cysteine as shown in the supplementary materials. The accurate structure of this adduct need to be further investigated. Cysteine also reduced HMF formation by inhibiting Maillard reaction. Adding 0.2 mmol of cysteine to asparagine/glucose model reaction system decreased the A420 value (an index for assessment of browning of Maillard reaction) from 0.648 to 0.152. In summary, cysteine simultaneously reduced the contents of acrylamide and HMF through inhibiting the Maillard reaction and formed adduct with the formed acrylamide and HMF. On the contrary, glycine can react with glucose to form HMF; however, it also reacts with HMF and thus reduces HMF content at high addition level. 4. Conclusion Cysteine significantly decreased the formation of acrylamide and HMF in asparagine/glucose model reaction system; it completely inhibited their formation at the concentration of 0.375e0.5 mol/L. Glycine can reduce the usage amount of cysteine, addition of 0.36 g/100 g cysteine and 0.2 g/100 g glycine into the dough decreased the contents of acrylamide and HMF by 97.8% and

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