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Chapter 4
Formation of Acrylamide in Thermally Processed Foods and Its Reactions during in Vitro Digestion A. Hamzalıoğlu and V. Gökmen* Food Quality and Safety (FoQuS) Research Group, Department of Food Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey *E-mail:
[email protected] Thermal process is applied to foods for processing or preservation purposes, and several chemical reactions proceed during thermal process, resulting in formation of chemical compounds. While these compounds provide many desired characteristics in foods, they may also cause the formation of undesired compounds. Acrylamide, a probable human carcinogen, is formed in foods as a result of heating. It is derived from free asparagine and formed through the Maillard reaction. Coffee, bakery, and potato products are some of the most important foods containing high levels of acrylamide. Acrylamide has an unsaturated carbonyl, which makes it highly electrophilic; owing to this, it reacts with nucleophilic groups present and/or released in foods during digestion.
© 2019 American Chemical Society Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Introduction Acrylamide, 2-propenamid, is a reactive amide and has a chemical formula C3H5NO (Figure 1).
Figure 1. Chemical structure of acrylamide.
As it is also an α, β-unsaturated carbonyl, it was reported to be highly reactive in covalently interacting with nucleophilic groups in the body. In addition, glycidamide, formed as a result of its conversion, was reported to react with DNA (1). It has been classified as a “probable human carcinogen” in Group 2A by the International Agency for Research on Cancer in 1994. However, acrylamide as a food contaminant was first discovered in 2000 (2) by Swedish researchers and in 2002, its presence was observed in foods that are exposed to thermal treatment such as fried potato products and bakery products (3).
Formation Mechanisms Major Pathways After the discovery of acrylamide in certain heated foods, researchers undertook an extensive effort to evaluate the mechanism of its formation in these foods. It was reported that the Maillard reaction, the reaction between an amine and a carbonyl group, was the key responsible mechanism for the formation of acrylamide in heated foods (3–5). Further experiments in order to evaluate the key precursors of acrylamide formation were then conducted, and through data provided by one of these studies, it was confirmed that acrylamide was mainly derived from asparagine by using carbon and nitrogen labeled asparagine (6). Despite the fact that asparagine was concluded as the main amino and carbon source for acrylamide formation, it was found that asparagine was rapidly converted to acrylamide in the presence of carbonyl compounds (7). The acrylamide formation pathway is given in Figure 2. In this mechanism, the first step is the carbonylation of α-amino group of asparagine by carbonyl source, resulting in carbinolamine, N-glycosyl-asparagine, formation. Under dry conditions, a water molecule is cleaved from N-glycosyl-asparagine, forming Schiff base upon heating at elevated temperatures. Schiff base might also rearrange to form Amadori compounds under aqueous conditions, but these rearrangement products are highly stable and do not lead to acrylamide formation. They are mainly responsible for color and flavor development (9).
46 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 2. Formation pathway of acrylamide. Adapted with permission from ref (8). Copyright 2004 American Chemical Society.
The second step in acrylamide formation is the decarboxylation step, which yields an azomethine ylide from Schiff base. Azomethine ylide might form imine I through Schiff base betaine; it might also proceed to imine II formation through oxazolidin-5-one (6, 7). Acrylamide formation from imine II could proceed in two ways: (1) the formation of acrylamide directly through β-elimination or (2) the hydrolysis of imine II to 3-aminopropionamide (3-APA) followed by deamination to form acrylamide (6). On the other hand, imine I is hydrolyzed to Strecker aldehyde, which is also assumed as one of the direct precursors of acrylamide (4). 47 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Minor Pathways There are also minor pathways for the formation of acrylamide other than the Maillard reaction. Acrylamide is mainly formed in oils through the formation of acrolein as a result of decomposition of oils after heating at high temperatures. Acrolein is then converted to acrylic acid that reacts with ammonia to form acrylamide (10). Additionally, acrylamide might also be formed by conversion of asparagine itself through a non-Maillard pathway under certain conditions. Conversion of asparagine to acrylamide mainly proceeds through 3-APA. As was previously mentioned in this chapter, 3-APA formation is followed by deamination, resulting in acrylamide (11, 12). Even though acrylamide is mostly formed in dry foods that are exposed to excess heating conditions, it is also possible to form under aqueous conditions and at relatively low temperatures. 3-APA might also be formed as a result of enzymatic conversion of acrylamide, and, rapidly converted to acrylamide (11, 13). Factors Affecting Acrylamide Formation in Thermally Processed Foods The studies on formation kinetics of acrylamide carried out to date provided information about the factors affecting its formation in detail (14, 15). Food processing conditions and the presence and/or amount of key precursors are the most important ones for acrylamide formation.
Asparagine As stated previously in this chapter, asparagine forms the backbone of acrylamide; accordingly, acrylamide content in a heated food is affected by free asparagine concentration. Generally, asparagine is mostly concentrated in plant-based foods such as potatoes and cereals (16). The ratio of free asparagine to reducing sugars is comparably higher in potatoes; thus, sugar concentration is the limiting factor for acrylamide formation in potato products (17). On the other hand, in cereals, reducing sugar is the limiting reactant due to higher concentrations than free asparagine (18). Hence, in order to control the acrylamide levels, use of asparagine-low variety cereals might be one of the mitigation strategies. In addition to this, despite some limitations in its use, application of asparaginase to degrade some of the reactive asparagine is still an important mitigation strategy for acrylamide.
Carbonyl Compounds In principle, any carbonyl compound could contribute to conversion of asparagine into acrylamide, but in different yields. Reaction efficiency mostly depends on the decarboxylation step, which highly affects the conversion rate of asparagine into acrylamide (19) Most of the studies showed that hydroxycarbonyl 48 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
compounds such as reducing sugars were highly effective in converting free asparagine into acrylamide (20). As sucrose is one of the main ingredients of some heated foodstuffs, effect of sucrose on acrylamide formation was also studied, and its hydrolysis to reducing sugars during heating was found to be mainly responsible for the acrylamide formation (21). In addition to hydroxycarbonyl compounds, dicarbonyl compounds such as methylglyoxal and glyoxal were also found to promote acrylamide formation as a result of heating (22, 23). Accordingly, in the study of Gökmen et al. (24), 5-hydroxymethylfurfural (HMF), a sugar dehydration product, was found to be a potent carbonyl compound promoting acrylamide formation. HMF is simultaneously formed together with acrylamide through the Maillard reaction during heating of foods. In the study, acrylamide levels in a model system composed of HMF and asparagine were monitored in order to understand the contribution of HMF to acrylamide formation during heating. Other sugar dehydration products bearing carbonyl moiety were also shown as precursors in acrylamide formation, and 3-APA was concluded as one of the key intermediates. Moreover, lipid oxidation products might also participate in acrylamide formation. As Hidalgo and his research group have reported, aldehydes and ketones produced as a result of lipid oxidation could take role as carbonyl compounds in acrylamide formation (25–28). It was also reported that short chain carbonyl compounds were found to be more reactive in converting asparagine into acrylamide; however, aldehydes were reported to be more effective compared to ketones. Additionally, curcumin, as a bioactive carbonyl, was expected to be a potent precursor of acrylamide formation. The role of curcumin on acrylamide formation was investigated in the study of Hamzalioğlu et al. (29), and the results indicated that curcumin was effective in reacting with asparagine, resulting in acrylamide formation as a result of heating. In the study conducted by Hamzalioğlu and Gökmen (30), vanillin, dehydroascorbic acid, ascorbic acid, and silymarin were also tested as bioactive carbonyl compounds for their reactivity in acrylamide formation in the case of heating together with asparagine. All the bioactive carbonyls tested efficiently reacted with asparagine to form acrylamide in different extents. Compared to fructose, vanillin was found to be more reactive; however, other bioactive carbonyl compounds such as curcumin, silymarin, ascorbic acid, and dehydroascorbic acid were not as effective as fructose in reacting with asparagine. Formation mechanism was also proposed for the reaction between asparagine and bioactive carbonyl compounds by high-resolution mass spectrometry analysis (Figure 3). According to the proposed mechanism, Schiff base is formed as a result of carbonylation of the α-amino group of asparagine by curcumin in the asparagine–curcumin model system. It then decarboxylates to form decarboxylated Schiff base that might directly result in acrylamide formation. On the other hand, formation of 3-APA was also confirmed in the asparagine–curcumin model system during heating. Reactivity of carbonyl compounds depends on its resonance, presence or absence of leaving group, magnitude of δ+ charge on carbon atom, and steric effects. For example, reactivity of carbonyl compound is affected by the presence of neighboring groups. However, under dry conditions, melting point of precursors is the determining factor for the reactivity, as melting of reactants 49 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
provides the molecular mobility that is crucial for a reaction. As reported by Robert et al. (31), fructose, having a lower melting point compared to glucose, tended to more efficiently convert asparagine into acrylamide than glucose. Similarly, Hamzalioğlu and Gökmen (30) also found a direct relation between the melting points of bioactive carbonyls and their efficiency in reacting with asparagine. From the bioactive carbonyl compounds tested, vanillin, with the lowest melting point (84 °C), caused the rapid conversion of asparagine into acrylamide, whereas dehydroascorbic acid had a comparably lower conversion rate since it has the highest melting point (Figure 4).
Figure 3. Proposed mechanism for the contribution of curcumin on acrylamide formation from asparagine. Adapted with permission from ref (29). Copyright 2013 Springer Nature.
Process Conditions Apart from the precursors, process conditions have a strong influence on acrylamide formation. It is a fact that boiling does not lead to acrylamide formation, indicating the prerequisite of high temperature processing and low moisture conditions for its formation (14, 15, 32). It was stated that a temperature of >120 °C is required for acrylamide formation (16). Baking and roasting are high-temperature food processes leading to acrylamide formation. In addition to these processes, such high temperatures could also be reached during the frying process; for this reason, time and temperature have been reported as important for acrylamide formation in fried potatoes. According to the study of 50 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Gökmen et al. (33), acrylamide levels in fried potato strips increased within both increased frying time and temperature, with a higher influence of temperature compared to time. Similarly, it was reported that increased baking temperature significantly caused higher levels of acrylamide formation in biscuits during baking. Expectedly, prolonging the baking time at a constant temperature led to an increase in acrylamide content of biscuits as well (34). In a similar study, more acrylamide was formed after baking biscuits at higher temperatures for short times, whereas more time is needed to reach the same levels in the case of baking at lower temperatures (35). Besides, it was reported that acrylamide in fried products is mostly formed toward the end of the frying process (36). According to this, it was suggested that lowering the oil temperature at the final stage of the frying process could lower the acrylamide levels in potato fries as well (37). In a conducting study, acrylamide levels of French fries could be reduced by 50% within the application of temperature program for the frying process, including lower oil temperature at the final stages (38). On the other hand, at high temperatures during baking or roasting, acrylamide tends to degrade after reaching a maximum value, indicating the predomination of degradation reactions at temperatures about 200 °C (16).
Figure 4. Relationship between the melting point of carbonyl compounds and the conversion rate of asparagine into acrylamide during heating at 180 °C (ASN = asparagine; VAN = vanillin; FRU = fructose; CUR = curcumin; SIL = silymarin; ASC = ascorbic acid; DHA = dehydroascorbic acid). Adapted with permission from ref (30). Copyright 1993 CRC Press. As mentioned previously in this chapter, acrylamide formation is affected by the moisture conditions. For instance, acrylamide is formed in a food having water activity below 0.8, whereas its content reaches maximum when the water activity is about 0.4. In a conducting study in biscuits, acrylamide could not be detected in biscuits having 10% moisture even baked at 200 °C; however, highest 51 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
levels of acrylamide were observed at 2% moisture content (39). Moreover, it was reported that crumbs were free of acrylamide in bakery products while acrylamide is mostly concentrated in crust layers (40). Additionally, acrylamide levels in different regions of potato slices were monitored during frying, and the results showed that acrylamide mainly formed at the surface and regions close to surface (33). It was indicated that this was due to more favorable conditions for acrylamide formation at these regions as a result of drying, and thus, reaching of higher temperatures.
Levels of Acrylamide in Thermally Processed Foods Since its discovery in heated foods, many efforts have been undertaken by the European Commission to collect data about acrylamide levels in different foods. In June 2015, the European Food Safety Agency (EFSA) reported the acrylamide levels in monitored foods, indicating that French fries, potato chips, breads, biscuits, and coffee are the foods containing highest amounts of acrylamide (41). In addition to these foods, acrylamide is also reported to be found in breakfast cereals, roasted nuts, and vegetable chips (41). Acrylamide levels of some thermally processed foods are summarized in Table 1. Indicative values, which are not legal limits but further investigations should be carried out if foods contain higher levels of acrylamide than these values, for most of the foods are also specified in Table 1. In 2018, the EU commission decided to set benchmark levels for the industry to test the performance of their acrylamide mitigation strategies. These values trigger the food operators to both monitor and redesign the critical heat-treatment steps in the manufacturing process of thermally processed food products. Additionally, proper sampling and use of analysis methods by the food industry was also indicated in the regulation.
Bakery Products Baking includes the application of a high amount of heat load mostly at temperatures above 200 °C. As a result of such high-temperature processing, simultaneous reactions take place in foods that might result in accumulation of toxic compounds, including acrylamide, as well as color and flavor development. According to a report published by the EFSA (41), bread, biscuits, and crackers were mostly consumed by children as well as adults and adolescents, contributing to 20–60% of total acrylamide intake. From the bakery products, biscuits, crackers, and gingerbread were found to have the highest acrylamide content (Table 1). As mentioned before, acrylamide levels in baked foods mostly depend on the presence of precursors in raw material and heating conditions applied. No acrylamide is detected in breadcrumbs, whereas higher levels are reached in crust or toasted breads (43).
52 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 1. Acrylamide Levels (μg kg-1) of Foods. Adapted with permission from refs (41) and (42). Copyright 2015 and 2017 John Wiley & Sons. n
Median (μg kg-1)
Mean (μg kg-1)
P95 (μg kg-1)
Indicative Value (μg kg-1)
Benchmark Level (μg kg-1)
-Wheat-based bread
302
15
38
120
80
50
-Other
99
25
46
203
150
100
Biscuits and wafers
682
103
201
810
500
350
Crackers
162
183
231
590
500
400
Crisp bread
437
89
149
428
450
350
Ginger bread
693
155
407
1600
1000
800
French fries
378
196
332
1115
600
500
Potato crisps and snacks
800
389
580
1841
1000
750
Roasted coffee
566
203
244
563
450
400
Instant coffee
116
620
674
1333
900
850
20
522
510
–
2000
500
Bakery products Soft bread
53
Potato products
Coffee products
Coffee substitutes -Based on cereals
Continued on next page.
Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 1. (Continued). Acrylamide Levels (μg kg-1) of Foods n
Median (μg kg-1)
Mean (μg kg-1)
P95 (μg kg-1)
Indicative Value (μg kg-1)
Benchmark Level (μg kg-1)
37
3100
2942
–
4000
4000
-Bran products and whole grain cereals
151
135
164
413
400
300
-non-whole grain or non-bran based wheat and rye based products
33
140
142
–
300
300
-non-whole grain or non-bran based maize, oat, spelt, barley, and rice based products
149
50
73
230
200
150
Cereal-based baby foods
394
15
103
200
50
40
Roasted nuts
40
25
93
–
–
–
-Chicory Other products Breakfast cereals
54
Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Potato Products From the given acrylamide concentrations of different food products, fried potato products is one of the food categories with the highest acrylamide levels. According to a report published by the EFSA containing the results of a wide variety of samples analyzed, potato crisps and French fries were the foods reported to contain acrylamide levels exceeding the indicative values. It is a fact that potatoes are rich in asparagine and, thus, formation of acrylamide in French fries is limited by the concentration of reducing sugars (44–46). As acrylamide formation is highly affected by precursor concentration, varying levels of reducing sugars among the potato varieties lead to a wide range for acrylamide formation in potato products. The study indicated that the genotype of potato determines the potential of the acrylamide formation and recommended selecting potato cultivars with low concentrations of reducing sugars to mitigate the acrylamide formation. Then, reducing sugar content of potato cultivar was correlated with the final acrylamide content of the product (47, 48). Moreover, storage conditions of potatoes prior to processing also have an impact on reactant concentrations as well as final acrylamide concentration for these products. It was reported that increased reducing sugars in potatoes as a result of starch break down during storage (temperature below 8 °C); in particular, high concentrations of reducing sugar was found in potatoes stored at 4 °C (48). In addition to storage conditions, the effect of nitrogen and sulfur fertilization on the acrylamide-forming potential of different potato varieties was investigated, and the study revealed that nitrogen application increased acrylamide-forming potential in most potato varieties studied due to the increased asparagine content (49). The studies also showed that portion size had a high impact on acrylamide formation in potato fries. For instance, frying potatoes in smaller portions achieved shorter cooking times, whereas it took longer time to fry big portions of potatoes while maintaining desired texture and aroma. However, despite the extended frying time, acrylamide levels were found to be comparably lower in fried potatoes in big portions (50). Coffee Coffee, as one the most consumed beverages in the world, attracts more attention owing to its health-promoting potential. However, provided by high caffeine concentrations in coffee, its consumption is related with adverse effects on health (51). Accordingly, coffee substitutes, non-coffee products without caffeine, gained popularity, and more studies on this topic have been carried out recently. Production of both coffee and coffee substitutes includes a roasting process at temperatures exceeding 200 °C; thus, caramelization, Maillard reaction, and pyrolysis occur (52). Acrylamide concentration exponentially increases during roasting; however, it gradually decreases after reaching a maximum level (39, 53–56). Acrylamide formation in coffee products is highly affected by the type of bean species and roasting conditions (39, 54, 55). It was confirmed that Robusta coffee beans (Coffea canephora robusta) lead to acrylamide formation in higher amounts compared to Arabica coffee beans (Coffea arabica). From 55 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
the coffee substitutes, chicory has been reported to contain the highest levels of acrylamide, even higher than in coffee, as it contains high amounts of fructose (57). Other Thermally Processed Foods Extrusion cooking, a high-temperature, short-time process, is generally used in the production of snacks, ready-to-eat cereals, confectioneries, and crisp breads (58). As indicated before, acrylamide is mostly formed in low moisture systems, and extruded food products are one of the potential foods containing acrylamide since such extruded products have a moisture content lower than 5% (59). As a consequence, extruded breakfast cereals were found to contain acrylamide around 150 μg kg-1 (41). In addition to breakfast cereals, roasted nuts were indicated to contain acrylamide. From the roasted nuts, roasted almonds were the ones with highest acrylamide levels owing to high content of precursors in almonds. It was reported that acrylamide concentration in roasted almonds varied between 260 and 2147 μg kg-1 depending on roasting conditions (60, 61). However, acrylamide content of roasted hazelnuts was found to be 15 times lower than the content in roasted almonds (62).
Reactivity and Possible Reactions of Acrylamide Acrylamide is highly electrophilic due to its α, β-unsaturated carbonyl group, as shown in Figure 1. Hence, it is possible for acrylamide to be involved in 1,2 nucleophilic addition or Michael addition (1,4 nucleophilic addition) (63). Nucleophilic Addition and Michael Addition Nucleophiles, which have a free pair of electrons, are electron donors to electrophiles, thus forming a chemical bond. On the other hand, electrophiles, which are reactive species since they have free orbitals attached to electron rich center, are electron acceptors in order to form a bond with nucleophiles. In other words, nucleophiles are Lewis bases whereas electrophiles are Lewis acids. Carbonyl compounds are one of the well-known electrophiles, whereas amines, ammonia, or amides are important examples of nitrogen-containing nucleophiles. Besides, due to high nucleophilicity of sulfur, thiol compounds are the most reactive sulfur-containing nucleophiles (64). Nucleophilic addition is the addition reaction of a nucleophile with an electrophile. This process has two main steps: (1) nucleophilic attack on the electrophile’s carbon atom and (2) formation of an intermediate and the proton transfer. Finally, when the electrophilic molecule has a double bond, the double bond is substituted with a single bond. Moreover, if the carbonyl compound is an α, β-unsaturated carbonyl (Figure 5), which has conjugated double bonds, electrophilic character of carbonyl carbon is substituted with double bond of β-carbon. 56 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 5. α, β-Unsaturated carbonyl. In this case, the nucleophilic molecule may directly attack the carbonyl carbon as well as the β-carbon. Therefore, nucleophilic addition is called 1,2-addition when the reaction proceeds from carbonyl carbon, otherwise it is called 1,4-addition. 1,4-Addition is generally known as “Michael addition” (Figure 6).
Figure 6. 1,4-Addition (Michael addition) between α, β-unsaturated carbonyl and a thiol compound. Nucleophile competes in attacking at carbonyl or β-carbon, and the reactivity of nucleophile gives direction to this reaction. In the case of amines, Michael addition is more favorable since it keeps the carbonyl group. The main difference from 1,2-addition is the addition of hydrogen to oxygen in 4 position in a protonation step (Figure 7).
Figure 7. Protonation step of 1,4-addition (Michael addition) between α, β-unsaturated carbonyl and a thiol compound. The following step is tautomerization, which results in formation of the molecule having the same carbon skeleton, but positions of protons and electrons differ (Figure 8). According to its highly electrophilic character, acrylamide easily reacts with nucleophilic groups such as amino, thiol, and imidazolic NH groups found mostly in biologic molecules (16, 63). Michael addition of different amino and thiol sources to acrylamide was reported to be a useful strategy for acrylamide removal in foods, as well (65–67). For instance, it was revealed by Hidalgo et al. (66) that thiol compounds including N-acetylcysteine, glutathione, and cysteine were rapidly added to the carbon–carbon double bond of acrylamide in the absence of 57 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
amino compounds. They indicated that the addition of thiol compounds were comparably rapid as activation energy for this reaction was quite low. It was also confirmed that acrylamide removal was promoted by a reaction of sulfhydryl group when both amino and sulfhydryl group were present in the same molecule. In addition to thiol compounds, addition of amino compounds to acrylamide is also possible. In the study investigating the reactions of acrylamide with different amino compounds such as butylamine, glycine, lysine, and polylysine, one or two molecules of acrylamide were reported to be added to amino compounds through Michael-type addition, forming 3-propionamides (67).
Figure 8. Tautomerization step of 1,4-addition (Michael addition) between α, β-unsaturated carbonyl and a thiol compound.
Reactions of Acrylamide during in Vitro Digestion Ingested food enters the digestive tract, which significantly alters its structure and chemical composition by varying pH and the action of several enzymes in the mouth, stomach, and intestine. Each step of digestion might be considered as a reaction media with different specific conditions. In addition to changing pH in each step, enzymes acting on food components lead to the hydrolysis and transformations of these components. Under these conditions, some of these may interact with certain components released from the food matrix in the gastrointestinal tract. As a result of these interactions, the amount of food components may be reduced, as well as new compounds may be formed, which might also behave as reactants, depending on their reactivity. Owing to its potential reactivity, reactions of acrylamide present in foods with certain components released from the food matrix under the varying conditions in the gastrointestinal tract is possible (68, 69). Hamzalioğlu and Gökmen (70) designed an in-depth study to understand the reactions of acrylamide in thermally processed foods during digestion. For this purpose, different heated foods were subjected to a multistep in vitro digestion process, and acrylamide was monitored during digestion. As given in Table 2, acrylamide content of both digested sweet and non-sweet biscuits were significantly decreased at the end of gastric and duodenal phases. It was reported that acrylamide reduction ranged between 49.2% and 73.4% for biscuit samples at the end of the digestive process.
58 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 2. Changes in the Amount of Acrylamide in the Digests of Heated Foods during in Vitro Digestiona−d. Adapted with permission from ref (70). Copyright 2015 Royal Society of Chemistry. Acrylamide, nmol Initial
Gastric Phase
Sample 1
16.32±0.40a
10.90±0.10b
6.68±1.39c
5.73±1.09c
Sample 2
24.48±1.79a
12.27±0.45b
7.95±0.50c
7.60±0.56c
Sample 1
18.96±0.45a
13.33±2.44b
5.59±0.05c
5.24±0.35c
Sample 2
22.33±0.15a
11.57±2.44b
7.28±0.25c
5.94±0.35c
Sample 3
12.13±0.25a
10.02±0.25b
7.63±0.45c
6.16±0.25d
18.47±1.74a
73.02±6.17b
2.32±0.40c
0.34±0.18d
Sample 1
12.49±0.75a
14.98±1.29b
1.20±0.70c
0.62±0.10c
Sample 2
16.81±1.79a
24.41±0.10b
5.31±1.04c
0.52±0.03d
Duodenal Phase
Colon Phase
Non-sweet biscuits
Sweet biscuits
Potato fry Sample 1 Potato chips
a−d
Values marked with different letters in each row are significantly different (p < 0.05).
Since hydrolysis of proteins into smaller peptides or amino acids during the digestive process took place, a simulated digestive process created a pool of amino acids that might be interacting with acrylamide. Due to the highly electrophilic nature of acrylamide, each molecule of amino acid could form adducts with one or two molecules of acrylamide (67). In order to understand the mechanism of acrylamide reduction, model systems composed of amino acids and acrylamide were subjected into a simulated digestive process. Accordingly, significant reductions were reported both in acrylamide–lysine and acrylamide–cysteine model systems during the digestive process. From the tested amino acids, cysteine was reported to lead more elimination of acrylamide compared to the acrylamide–lysine model system. Owing to its highly nucleophilic -SH group, cysteine was thought to be more favorable in reacting toward acrylamide. As mentioned before, thiol compounds were more effective in Michael-type addition to acrylamide. Through analysis by high-resolution mass spectrometry in the digests of acrylamide–cysteine model system, Michael addition of one or two molecules of acrylamide to cysteine during in vitro digestion was confirmed (Figure 9).
59 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 9. Proposed mechanism for the reduction of acrylamide during in vitro digestion through the formation of Michael adducts with cysteine. Reproduced with permission from ref (70). Copyright 2015 Royal Society of Chemistry Despite significant decreases observed in acrylamide contents of biscuits during the digestive process, a dramatic increase was observed in acrylamide levels of fried potato products at the end of the gastric phase (Table 2). It was indicated that this increase might be provided by conversion of intermediates accumulated in potato products as a result of frying under gastric conditions of the in vitro digestive process. However, as digestion proceeded, most of the acrylamide disappeared in these fried potato products.
Figure 10. Proposed mechanism for the formation of acrylamide during in vitro gastric digestion from the precursors in fried potato. Reproduced with permission from ref (70). Copyright 2015 Royal Society of Chemistry. To understand better the fate of acrylamide in fried potato products during digestion, conducting model systems composed of acrylamide precursors were prepared. For this, model systems containing asparagine and glucose were heated at 180 °C for 10 min and were subsequently digested under in vitro 60 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
digestion conditions. It was revealed that acrylamide content of heated model asparagine-glucose system significantly increased at the end of gastric phase, as in fried potato products. As a consequence, most of the acrylamide formed in the asparagine–glucose model system was eliminated at the end of the intestinal stage of digestion procedure. Furthermore, intermediates formed in the asparagine–glucose model system as a result of heating were monitored during different stages of the in vitro digestion process. Disappearance of Schiff base, which is one of the most important intermediates responsible from acrylamide formation, through gastric phase was also confirmed. Through this confirmation, conversion of intermediates in fried potato products into acrylamide during digestion was summarized as given in Figure 10. According to a recent study about the reactions of acrylamide during digestion, gastric conditions could be considered as reactive media for the further conversion of intermediates into acrylamide (70). On the other hand, intestinal conditions could be effective for the reactions of acrylamide with nucleophilic groups provided by the accumulating hydrolyzed amino acids. However, the balance between the elimination and potential formation of acrylamide during the in vitro digestion process suggests that ingested amounts with foods may not directly indicate the net amount available for the body. In addition to the digestion conditions, importance of food composition and process conditions should be considered since they directly affect the formation and accumulation of intermediates, which might possess a potential threat for human health during digestion.
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