Effect of Selected Mercapto Flavor Compounds on Acrylamide Elimination in a Model System

The effect of four mercapto flavor compounds (1,2-ethanedithiol, 1-butanethiol, 2-methyl-3-furanthiol, and 2-furanmethanethiol) on acrylamide elimination were investigated in model systems. The obtained results showed that mercaptans assayed were effective in elimination arylamide in a model system. Their reactivities for decreasing acrylamide content depended on mercaptan’s molecular structure and acrylamide disappearance decreased in the following order: 1,2-ethanedithiol > 2-methyl-3-furanthiol > 1-butanethiol > 2-furanmethanethiol. Mercaptans were added to acrylamide to produce the corresponding 3-(alkylthio) propionamides. This reaction was irreversible and only trace amounts of acrylamide were formed by thermal heating of 3-(alkylthio) propanamide. Although a large amount disappeared, only part of the acrylamide conversed into 3-(alkylthio) propionamides. All of these results constitute a fundamental proof of the complexity of the reactions involved in the removal of free acrylamide in foods. This implies mercapto flavor/aroma may directly or indirectly reduce the level of acrylamide in food processing. This study could be regarded as a pioneer contribution on acrylamide elimination in a model system by the addition of mercapto flavor compounds.


Introduction
Acrylamide (AA, CAS 79-06-1) as a α,β-unsaturated (conjugated) reactive molecule is widely used as a reactive monomer or intermediate in organic synthesis [1]. However, AA is a potential human carcinogen and genotoxicant and a known human neurotoxicant [2]. So it has been classified as a group 2A, "probably carcinogenic to humans", chemical by the International Agency for Research on Cancer [3]. The European Food Safety Authority (EFSA)'s Expert Panel on Contaminants in the Food Chain (CONTAM) described dietary acrylamide as potentially increasing the risk of developing cancer for consumers in all age groups [4].
Acrylamide can be formed through the Maillard reaction during food thermal processing, especially starch-rich products such as fried potato, bread, potatoes and so on [5]. Extensive research show that reaction conditions such as reaction time and temperature play important roles in the

Acrylamide Disappearance in Acrylamide/mercaptan Reaction Mixtures
Due to the inherent complexity of a food matrix, where many factors are involved that act one upon another, four mercaptans were employed to react with acrylamide under idealized conditions in a closed model system in order to evaluate the effect of mercapto flavors on the elimination of acrylamide. When heated in the presence of mercaptans, acrylamide was decreased to a certain degree. Corresponding adducts were observed and identified on the basis of their retention time and mass spectra determined by HPLC/MS using the synthesized adducts as a standard.
The reaction conditions and the amount of mercaptans added were responsible for the acrylamide disappearance. As can be seen from Figure 1, when the amount of mercaptans added was less than 5 µmol in the reaction mixture, acrylamide disappearance increased rapidly with mercaptans concentration increasing. However, acrylamide disappearance almost remained unchanged when the amount of mercaptans added was more than 10 µmol. Obviously, acrylamide disappearance increased as a function of the amount of mercaptans, and more than 80% acrylamide disappeared when 20 µmol mercaptans was added in the reaction mixture in model system. Nevertheless, the maximum allowable usage of mercapto flavors in food is more than the amount of acrylamide generally produced in food.
Molecules 2017, 22, 888 3 of 11 Nevertheless, the maximum allowable usage of mercapto flavors in food is more than the amount of acrylamide generally produced in food. In addition, the disappearance of the acrylamide depended on the reaction conditions. The heating time and temperature had a significant impact on the disappearance of the acrylamide. As shown in Figure 2, acrylamide disappearance increased rapidly and almost linearly as a function of time during the first ten minutes of the reaction. The disappearance of the acrylamide in the reaction mixture reached an apparent maximum after 20 min of heating at 180 °C, then tended to balance afterwards. The effect of the temperature as shown in Figure 3, acrylamide disappeared faster with the temperature increasing.  In addition, the disappearance of the acrylamide depended on the reaction conditions. The heating time and temperature had a significant impact on the disappearance of the acrylamide. As shown in Figure 2, acrylamide disappearance increased rapidly and almost linearly as a function of time during the first ten minutes of the reaction. The disappearance of the acrylamide in the reaction mixture reached an apparent maximum after 20 min of heating at 180 • C, then tended to balance afterwards. The effect of the temperature as shown in Figure 3, acrylamide disappeared faster with the temperature increasing. Nevertheless, the maximum allowable usage of mercapto flavors in food is more than the amount of acrylamide generally produced in food. In addition, the disappearance of the acrylamide depended on the reaction conditions. The heating time and temperature had a significant impact on the disappearance of the acrylamide. As shown in Figure 2, acrylamide disappearance increased rapidly and almost linearly as a function of time during the first ten minutes of the reaction. The disappearance of the acrylamide in the reaction mixture reached an apparent maximum after 20 min of heating at 180 °C, then tended to balance afterwards. The effect of the temperature as shown in Figure 3, acrylamide disappeared faster with the temperature increasing.  Acrylamide disappearance also depended on the pH. Acrylamide disappearance increased slowly as the pH increased from pH 2.2 to 4, while this disappearance was faster when a higher pH was assayed from pH 5 to 7 in a model system ( Figure 4).

Acrylamide Formation by Thermal Decomposition of Mercaptan-AA Adducts
When 0.2 μmol of mercaptan-acrylamide adducts were heated at 180 °C for 0-30 min, they were stable and only trace amounts of acrylamide were formed ( Figure 5). This shows that the addition reaction of the mercaptans to acrylamide were irreversible.

Acrylamide Consumption Produced to Mercaptan-Acrylamide Adducts
According to the amount of resulting adducts, it is easy to calculate the amount of acrylamide produced to mercaptan-acrylamide adducts. As can be seen from Figure 5, the consumption of Acrylamide disappearance also depended on the pH. Acrylamide disappearance increased slowly as the pH increased from pH 2.2 to 4, while this disappearance was faster when a higher pH was assayed from pH 5 to 7 in a model system ( Figure 4). Acrylamide disappearance also depended on the pH. Acrylamide disappearance increased slowly as the pH increased from pH 2.2 to 4, while this disappearance was faster when a higher pH was assayed from pH 5 to 7 in a model system ( Figure 4).

Acrylamide Formation by Thermal Decomposition of Mercaptan-AA Adducts
When 0.2 μmol of mercaptan-acrylamide adducts were heated at 180 °C for 0-30 min, they were stable and only trace amounts of acrylamide were formed ( Figure 5). This shows that the addition reaction of the mercaptans to acrylamide were irreversible.

Acrylamide Consumption Produced to Mercaptan-Acrylamide Adducts
According to the amount of resulting adducts, it is easy to calculate the amount of acrylamide produced to mercaptan-acrylamide adducts. As can be seen from Figure 5, the consumption of

Acrylamide Formation by Thermal Decomposition of Mercaptan-AA Adducts
When 0.2 µmol of mercaptan-acrylamide adducts were heated at 180 • C for 0-30 min, they were stable and only trace amounts of acrylamide were formed ( Figure 5). This shows that the addition reaction of the mercaptans to acrylamide were irreversible.

Acrylamide Consumption Produced to Mercaptan-Acrylamide Adducts
According to the amount of resulting adducts, it is easy to calculate the amount of acrylamide produced to mercaptan-acrylamide adducts. As can be seen from Figure 5, the consumption of acrylamide added to mercaptans also depended on the heating time. In the first ten minutes of the reaction, acrylamide disappearance and adducts formation increased linearly as a function of time. However, acrylamide disappearing faster than the speed of the conversion of acrylamide into adducts. In addition, the amount of acrylamide disappeared is much more than the amount of acrylamide which formed to adducts at the same reaction time. acrylamide added to mercaptans also depended on the heating time. In the first ten minutes of the reaction, acrylamide disappearance and adducts formation increased linearly as a function of time. However, acrylamide disappearing faster than the speed of the conversion of acrylamide into adducts. In addition, the amount of acrylamide disappeared is much more than the amount of acrylamide which formed to adducts at the same reaction time.  Figure 1 shows the effect of selected mercaptans on the acrylamide concentrations determined after heating acrylamide/mercaptan model system for 30 min at 180 °C. As observed in the Figure 1, acrylamide disappearance increased almost linearly as a function of the amount of the mercaptans added less than 5 μmol. By employing the disappearance rates obtained from the slopes of the lines, it was possible to concluded that the most reactive was 1,2-ethanedithiol, followed by 2-methyl-3furanthiol and 1-butanethiol, 2-furanmethanethiol was the least reactive. Similar conclusions could be drawn from Figure 2.

Comparative Reactivity of Mercaptans for Acrylamide Removal
Effect of Storage at Room Temperatures on the Acrylamide Content of Acrylamide/ Mercaptan Mixtures.
The disappearance of acrylamide in acrylamide/mercaptan reaction mixtures was also observed at room temperature to study the effect of mercaptans on acrylamide elimination under storage conditions. After all, storage conditions of family cooking food have not been rigorously isolated from the air. Table 1 shows the acrylamide disappearance at room temperature. Acrylamide disappeared as a function of the storage time. It is noteworthy that 86.89% of the initial acrylamide  Figure 1 shows the effect of selected mercaptans on the acrylamide concentrations determined after heating acrylamide/mercaptan model system for 30 min at 180 • C. As observed in the Figure 1, acrylamide disappearance increased almost linearly as a function of the amount of the mercaptans added less than 5 µmol. By employing the disappearance rates obtained from the slopes of the lines, it was possible to concluded that the most reactive was 1,2-ethanedithiol, followed by 2-methyl-3-furanthiol and 1-butanethiol, 2-furanmethanethiol was the least reactive. Similar conclusions could be drawn from Figure 2.

Comparative Reactivity of Mercaptans for Acrylamide Removal
Effect of Storage at Room Temperatures on the Acrylamide Content of Acrylamide/ Mercaptan Mixtures.
The disappearance of acrylamide in acrylamide/mercaptan reaction mixtures was also observed at room temperature to study the effect of mercaptans on acrylamide elimination under storage conditions. After all, storage conditions of family cooking food have not been rigorously isolated from the air. Table 1 shows the acrylamide disappearance at room temperature. Acrylamide disappeared as a function of the storage time. It is noteworthy that 86.89% of the initial acrylamide had disappeared in acrylamide/1,2-ethanedithiol reaction mixture after six days. The concentration of acrylamide in the other three reaction mixture were also significantly reduced after 18 days.

Discussion
In recent years, acrylamide has received worldwide attention as a consequence of its potential toxicity at the concentrations formed during thermal food processing [19]. However, as an α,β-unsaturated amide, this toxicant is not a final product. It is a good Michael acceptor and may react with a variety of nucleophiles, such as -NH 2 and -SH. The reactivity of acrylamide with various amino compounds in model reactions has been studied in recent years [20][21][22]. However, some studies had reported that cysteine was more effective to eliminate AA than other amino acids [23]. As very good Michael donors, -SH is likely to have an important effect on the concentrations of acrylamide in food. Mercaptans are often used in food as edible spices. Additionally, aroma components produced in the food thermal processing also contain a large number of mercapto compounds. These mercapto compounds may react with acrylamide during food processing. However, as a common food ingredient, this type of nucleophile is ignored in the literature and, generally, studies have focused on the effect of other compounds, such as amino acids and phenolic compounds, on the formation or the elimination of acrylamide [24]. Therefore, examining the effects of mercapto flavor has been quite poor. Under high temperatures (e.g., 180 • C), reactions in food are complex due to various food ingredients, in order to simplify the reaction, this study simulate the reaction system of food thermal processing to examine the fate of acrylamide when mercaptan spices is added. It should be noted that high concentrations of mercaptans have a very intense odor. Consequently, very small amounts of mercaptans are added in this experiment.
The present article confirms that mercapto compounds are efficient acrylamide scavengers in the model system.
However, not all of the disappearing acrylamide converted to 3-(alkylthio)propionamide. Figure 5 shows the different reactions involved. The addition of mercaptans to acrylamide may proceed via a Michael addition pathway involving an ionic process or a free radical pathway. Generally, Michael addition reaction is based on activation of mercaptan by a base or activation of the acceptor olefins with Lewis acids. In order to avoid environmental impacts, several alternative solvents, such as ionic liquids [25], supercritical fluids [26], and water [27], as viable reaction media have been developed as efficient methodology for Michael addition reaction of mercaptans to unactivated alkenes. Possibly, water has a very specific role in Michael addition pathway through hydrogen bond formation with the sulfhydryl hydrogen of the mercaptans and, thus, increases the nucleophilicity of the mercaptan ion [27].
The free-radical addition of mercaptans to acrylamide is a typical chain reaction. Mercaptan free-radicals are initiated by oxygen in the air, and subsequently add to the C=C of the acrylamide to form a carbon radical. The carbon radical then reacts with a mercaptan molecule to give the final product 3-(alkylthio)propionamide and a new thiyl radical, thus propagating the radical chain and then producing other polymers. It has been shown that the presence of oxygen in the atmosphere had a major role in the acrylamide disappearance in acrylamide/benzyl mercaptan reaction mixtures, while the presence of antioxidants prevented acrylamide losses [14]. This suggests that free radical addition is another way to eliminate acrylamide mercaptan. Figure 6 shows the different reactions involved.
However, the reactivity of the four mercaptans assayed for acrylamide removal is somewhat different. Acrylamide disappearance decreased in the following order: 1,2-ethanedithiol > 2-methyl-3-furanthiol > 1-butanethiol > 2-furanmethanethiol. Obviously, -SH plays a major role in this process. Thus, since there are two -SH groups in its molecule, 1,2-ethanedithiol is better than the other three mercaptans assayed in terms of reaction speed and elimination effect. In addition, the cleavage of an S-H bond is influenced by the structure of the mercaptans added. Thus, aromatic mercaptans are better active agents than aliphatic mercaptans, since in the former case the energy required to break the S-H bond is lowered by the conjugate stabilization of the thiyl radical or mercaptan ion formed. Therefore, 2-methyl-3-furanthiol is more reactive to react with acrylamide through Michael addition reaction or radical reaction. As for 1-butanethiol and 2-furanmethanethiol, they are all alkymercaptans with the least reactive in acrylamide elimination. However, the elimination activity of 2-furanmethanethiol is slightly weaker than 1-butanethiol, probably because there are many more side chains in the molecular of 2-furanmethanethiol. The steric effect makes it less active. the presence of antioxidants prevented acrylamide losses [14]. This suggests that free radical addition is another way to eliminate acrylamide mercaptan. Figure 6 shows the different reactions involved. However, the reactivity of the four mercaptans assayed for acrylamide removal is somewhat different. Acrylamide disappearance decreased in the following order: 1,2-ethanedithiol > 2-methyl-3-furanthiol > 1-butanethiol > 2-furanmethanethiol. Obviously, -SH plays a major role in this process. Thus, since there are two -SH groups in its molecule, 1,2-ethanedithiol is better than the other three mercaptans assayed in terms of reaction speed and elimination effect. In addition, the cleavage of an S-H bond is influenced by the structure of the mercaptans added. Thus, aromatic mercaptans are better active agents than aliphatic mercaptans, since in the former case the energy required to break the S-H bond is lowered by the conjugate stabilization of the thiyl radical or mercaptan ion formed. Therefore, 2-methyl-3-furanthiol is more reactive to react with acrylamide through Michael addition reaction or radical reaction. As for 1-butanethiol and 2-furanmethanethiol, they are all alkymercaptans with the least reactive in acrylamide elimination. However, the elimination activity of 2-furanmethanethiol is slightly weaker than 1-butanethiol, probably because there are many more side chains in the molecular of 2-furanmethanethiol. The steric effect makes it less active.
The impact of the compounds formed in these reactions on health remain to be investigated. The results obtained in this study suggest that some mercapto flavor have a good effect in eliminating acrylamide in food heat processing, and provide new evidence of the complexity of the reactions involved in the formation and elimination of acrylamide in foods.  Figure 6. Proposed pathways for the disappearance of acrylamide in the presence of mercaptans. The impact of the compounds formed in these reactions on health remain to be investigated. The results obtained in this study suggest that some mercapto flavor have a good effect in eliminating acrylamide in food heat processing, and provide new evidence of the complexity of the reactions involved in the formation and elimination of acrylamide in foods.
Mercaptan-acrylamide adducts were prepared by reaction of acrylamide with mercaptans. Briefly, acrylamide (1 mmol), mercaptans (2 mmol), Sodium hydroxide (0.4 mg) and methanol (10 mL) were added into a round-bottom flask. Then the mixture was stirred at room temperature in air, until acrylamide was completely consumed (checked by TLC). Afterwards, the pure product was obtained after purification by column chromatography or by recrystallization. The pure product was characterized by NMR spectroscopy on a 600 MHz spectrometer (Bruker, AVANCE 600, Karlsruhe, Germany). (1)

Acrylamide/Mercaptan Reactions in Aqueous Model System
Model reactions were carried out according to the methods of Zamora et al. [28] and Cai et al. [29] with slight modifications. Briefly, each 20-mL stainless-steel test tube contained 2 mL of 0.2 mol/L buffer solution (sodium citrate for pH 3-6 and sodium phosphate for pH 7-8) with 0.2 µmol acrylamide and different concentrations of mercaptans (0-20 µmol). The test tubes were capped with Teflon pad-filled stainless steel cap and the mixtures were heated at 80-180 • C in an oil bath installed with a magnetic stirrer for 0-30 min, or shaked at 180 rpm by means of a reciprocating water bath constant temperature oscillator for 0-18 day at room temperature. The assayed mercaptans were 1-butanethiol, 2-furanmethanethiol, 2-methyl-3-furanthiol, and 1,2-ethanedithiol.
After cooling (15 min at 0 • C), the test tubes were centrifuged at 2000 rpm for 10 min with a low-speed centrifuge (Jingli, Beijing, China). The reaction mixtures was decanted into 10-mL volumetric flasks. After mixing, 1 mL of the mixture were added into a 10-mL test tube, together with 10 µL of internal standard solution (1 mg/mL of labeled [1,2,3-13 C 3 ] acrylamide in methanol ), and then extracted with methanol (1 mL × 2). The extracts were studied by GC-MS for its acrylamide content. In addition, another 1 mL of the mixture was studied by LC-MS for the reaction products.

The Stability of Mercaptan-Acrylamide Adducts
Additionally, the formation of acrylamide in the thermal degradation of mercaptan-acrylamide adducts were also studied. This reaction was carried out analogously to acrylamide/mercaptan reactions. Thus, the mercaptan-acrylamide adducts (0.2 µmol) and corresponding mercaptan (20 µmol) were added into a stainless-steel test tube, together with 2 mL 0.2 mol/L sodium phosphate buffer, pH 7, and heated at 180 • C in closed test tubes for 0-30 min. After cooling, acrylamide was extracted as described above.

Preparation of Brominated Samples for the Analysis of Acrylamide
One milliliter of the extracts prepared by aforementioned pretreatment steps was treated with 100 µL sulfuric acid (10%, v/v), and then placed into refrigerating cabinet for precooling (0 • C, 30 min). Two-hundred microliters of 0.1 mol/L potassium bromate and 300 mg of potassium bromide powder, was added to the precooled solution. After shaking with a vortex blender, the reaction mixture was allowed to stand for 1 h at 4 • C. The excess of bromine was removed by addition of 1 mol/L sodium thiosulfate until the solution became colorless, and the solution was extracted with ethyl acetate (1 mL × 2), and the combined extracts were dried with sodium sulfate. One milliliter of the organic layer was evaporated until a volume of~50 µL, treated with 10 µL of triethylamine, and analyzed by GC-MS.

Analysis of Acrylamide by GC-MS
The analysis of AA content was carried out analogously to the method of Zhang et al. [30] with slight modifications. GC-MS analyses were conducted with a Hewlett-Packard 6890 GC Plus coupled with an Agilent 5975 MSD (mass selective detector-quadrupole type) operated in selected ion monitoring (SIM) mode with positive electron impact (EI) ionization. HP5-MS capillary column (polysiloxane polymers, 30 m × Ø 0.25 mm, 0.25 m, J and W Scientific, Agilent, Santa Clara, CA, USA) was used for analytical separation. One microliter of the brominated sample was injected by the autosampler into the GC-MS system using the splitless flow control mode. Helium was chosen as the carrier gas at a flow rate of 1.0 mL/min. The temperature of the column oven was programmed as follows: isothermal at 60 • C for 1 min, increasing from 60 to 200 • C at a rate of 10 • C/min, and then subjected to isothermal conditions at 300 • C for 5 min. The temperatures of injector and transfer line to MSD were set at 250 • C and 280 • C, respectively. Analysis was performed using the electric impact mode at 70 eV. The SIM ions selected for identification of 2-bromopropionamide and 2-bromo ( 13 C 3 )-propenamide were m/z 70, 149, and 152 and m/z 108, 150, and 152, respectively. Following the whole procedure described above, quantification of acrylamide was carried out by preparing standard curves. Ten different concentration levels of acrylamide (0-150 µg) were used for each curve. Acrylamide content was directly proportional to the acrylamide/internal standard area ratio (r = 0.999, p < 0.0001). Data are mean values of, at least, two experiments. The coefficients of variation at the different concentrations were lower than 10%.

Analysis of Mercaptan-Acrylamide Adducts by HPLC/MS
HPLC/MS (Waters 1525, Waters Micromass ZQ, Milford, MA, USA) analyses of the reaction mixtures were carried out to identify the reaction products. The liquid phase conditions were as follows: separations were conducted on a Symmetry C18 (5 µm, 3.9 × 150 mm) analytical column. The mobile phase was methanol-water (30:70, v/v) at a flow rate of 0.5 mL/min. The mass spectrum conditions were as follows: the capillary voltage was 3.0 kV, the cone voltage was 20 V, the ion source temperature was 100 • C and the desolution temperature was 300 • C. SIR was conducted by operating the MS in ESI+ mode. Quantification of mercaptan-acrylamide adducts was carried out by preparing standard curves of the purified product prepared above.

Statistical Analysis
Statistical analysis was performed by using Student's t-test with SPSS 19.0 software (IBM Corporation, Armonk, NY, USA). Analysis of variance (ANOVA) was tested on a significance level of p < 0.05.