Acrylamide formation mechanism in heated foods

Recent findings of a potential human carcinogen, acrylamide, in foods have focused research on the possible mechanisms of formation. We present a mechanism for the formation of acrylamide from the reaction of the amino acid asparagine and a carbonyl-containing compound at typical cooking temperatures. The mechanism involves formation of a Schiff base followed by decarboxylation and elimination of either ammonia or a substituted imine under heat to yield acrylamide. Isotope substitution studies and mass spectrometric analysis of heated model systems confirm the presence of key reaction intermediates. Further confirmation of this mechanism is accomplished through selective removal of asparagine with asparaginase that results in a reduced level of acrylamide in a selected heated food.     

Why asparagine needs carbohydrates to generate acrylamide

Structural considerations dictate that asparagine alone may be converted thermally into acrylamide through decarboxylation and deamination reactions. However, the main product of the thermal decomposition of asparagine was maleimide, mainly due to the fast intramolecular cyclization reaction that prevents the formation of acrylamide. On the other hand, asparagine, in the presence of reducing sugars, was able to generate acrylamide in addition to maleimide. Model reactions were performed using FTIR analysis, and labeling studies were carried out using pyrolysis-GC/MS as an integrated reaction, separation, and identification system to investigate the role of reducing sugars. The data have indicated that a decarboxylated Amadori product of asparagine with reducing sugars is the key precursor of acrylamide. Furthermore, the decarboxylated Amadori product can be formed under mild conditions through the intramolecular cyclization of the initial Schiff base and formation of oxazolidin-5-one. The low-energy decarboxylation of this intermediate makes it possible to bypass the cyclization reaction, which is in competition with thermally induced decarboxylation, and hence promote the formation of acrylamide in carbohydrate/asparagine mixtures. Although the decarboxylated Amadori compound can be formed under mild conditions, it requires elevated temperatures to cleave the carbon-nitrogen covalent bond and produce acrylamide.     

Importance of oil degradation components in the formation of acrylamide in fried foodstuffs

This study investigates the importance of selected oil degradation components and some analogues in the formation of acrylamide. For this, a model system containing silica gel, PBS buffer, and oil was heated in a closed tubular reactor, under practically relevant heating conditions. Several probable acrylamide precursors were mixed together with free asparagine in the model system, such as partial glycerides, glycerol, acrolein, acrylic acid, and several aldehydes. Only the heated model system containing acrolein and asparagine showed a significantly higher acrylamide content compared to the control to which only asparagine was added. It was postulated that a nucleophilic 1,2-addition of the alpha-amino group of free asparagine to the carbonyl function of acrolein would lead to the formation of acrylamide. This hypothesis could partially be confirmed, replacing acrolein with other alpha,beta-unsaturated aldehydes. However, the contribution of acrolein to the overall formation of acrylamide appeared to be negligible in the presence of a reducing sugar, indicating that in foodstuffs the importance of acrolein and other oil degradation products is probably small.     

Acrylamide in gingerbread: critical factors for formation and possible ways for reduction

The influence of ingredients, additives, and process conditions on acrylamide formation in gingerbread was investigated. The sources for reducing sugars and free asparagine were identified, and the effect of different baking agents on acrylamide formation was evaluated. Ammonium hydrogencarbonate strongly enhanced acrylamide formation, but its N atom was not incorporated into acrylamide, nor did acrylic acid form acrylamide in gingerbread. Acrylamide concentration and browning intensity both increased with baking time and correlated with each other. The use of sodium hydrogencarbonate as baking agent reduced the acrylamide concentration by >60%. Free asparagine was a limiting factor for acrylamide formation, but the acrylamide content could also be lowered by replacing reducing sugars with sucrose or by adding organic acids. It is concluded that a significant reduction of acrylamide in gingerbread can be achieved by using sodium hydrogencarbonate as baking agent, minimizing free asparagine, and avoiding prolonged baking.     

Further insight into thermally and pH-induced generation of acrylamide from glucose/asparagine model systems

On the basis of numerous studies on the mechanism of formation of acrylamide (AA) from asparagine and reducing sugars, the decarboxylated Schiff base [ N-( d-glucos-1-yl)-3'-aminopropionamide] and its corresponding Amadori product [ N-(1-deoxy- d-fructos-1-yl)-3'-aminopropionamide) are considered to be possible direct precursors in addition to 3-aminopropionamide (AP). Furthermore, the mechanism of decarboxylation of the initially formed N-( d-glucos-1-yl)asparagine to generate the above-mentioned precursors also remains to be confirmed. To identify the relative importance of AA precursors, the decarboxylated Amadori product (AP ARP) and the corresponding Schiff base were synthesized and their relative abilities to generate AA under dry and wet heating conditions were studied. Under both conditions, the N-( d-glucos-1-yl)-3'-aminopropionamide had the highest intrinsic ability to be converted into AA. In the dry model system, the increase was almost 4-fold higher than the corresponding AP ARP or AP; however, in the wet system, the increase was 2-fold higher relative to AP ARP but >20-fold higher relative to AP. In addition, to gain further insight into the decarboxylation step, the amino acid/sugar reactions were analyzed by FTIR to monitor the formation of the previously proposed 5-oxazolidinone intermediate known to exhibit a peak in the range of 1770-1810 cm (-1). Spectroscopic studies clearly indicated the formation of an intense peak in the indicated range, the precise wavelength being dependent on the amino acid and the sugar used. The identity of the peak was verified by observing a 40 cm (-1) shift when [(13)C-1]-labeled amino acid was used.     

Quantitation of 3-aminopropionamide in potatoes-a minor but potent precursor in acrylamide formation

3-Aminopropionamide (3-APA) has recently been suggested as a transient intermediate in acrylamide (AA) formation during thermal degradation of asparagine initiated by reducing carbohydrates or aldehydes, respectively. 3-APA may also be formed in foods by an enzymatic decarboxylation of asparagine. Using a newly developed method to quantify 3-APA based on liquid chromatography/tandem mass spectrometry, it could be shown that the biogenic amine was present in several potato cultivars in different amounts. Further experiments indicated that 3-APA is formed during storage of intact potatoes (20 or 35 degrees C) or after crushing of the cells. The heating of 3-APA under aqueous or low water conditions at temperatures between 100 and 180 degrees C in model systems always generated more AA than in the same reaction of asparagine, thereby pointing to 3-APA as a very effective precursor of AA. While the highest yields measured were about 28 mol % in the presence of carbohydrates (170 degrees C; aqueous buffer), in the absence of carbohydrates, 3-APA was even converted by about 63 mol % into AA upon heating at 170 degrees C under aqueous conditions. Propanoic acid amides bearing an amino or hydroxy group in the alpha-position, such as 2-hydroxypropionamide and l-alaninamide, were ineffective in AA generation indicating that elimination occurs only from the beta-position.     

Determination of acrylamide during roasting of coffee

In this study different Arabica and Robusta coffee beans from different regions of the world were analyzed for acrylamide after roasting in a laboratory roaster. Due to the complex matrix and the comparably low selectivity of the LC-MS at m/ z 72, acrylamide was analyzed after derivatization with 2-mercaptobenzoic acid at m/ z 226. Additionally, the potential precursors of acrylamide (3-aminopropionamide, carbohydrates, and amino acids) were studied. The highest amounts of acrylamide formed in coffee were found during the first minutes of the roasting process [3800 ng/g in Robusta ( Coffea canephora robusta) and 500 ng/g in Arabica ( Coffea arabica)]. When the roasting time was increased, the concentration of acrylamide decreased. It was shown that especially the roasting time and temperature, species of coffee, and amount of precursors in raw material had an influence on acrylamide formation. Robusta coffee contained significantly larger amounts of acrylamide (mean = 708 ng/g) than Arabica coffee (mean = 374 ng/g). Asparagine is the limiting factor for acrylamide formation in coffee. 3-Aminopropionamide formation was observed in a dry model system with mixtures of asparagine with sugars (sucrose, glucose). Thermal decarboxylation and elimination of the alpha-amino group of asparagine at high temperatures (>220 degrees C) led to a measurable but low formation of acrylamide.     

In-depth mechanistic study on the formation of acrylamide and other vinylogous compounds by the maillard reaction

The formation of acrylamide was studied in low-moisture Maillard model systems (180 degrees C, 5 min) based on asparagine, reducing sugars, Maillard intermediates, and sugar degradation products. We show evidence that certain glycoconjugates play a major role in acrylamide formation. The N-glycosyl of asparagine generated about 2.4 mmol/mol acrylamide, compared to 0.1-0.2 mmol/mol obtained with alpha-dicarbonyls and the Amadori compound of asparagine. 3-Hydroxypropanamide, the Strecker alcohol of asparagine, generated only low amounts of acrylamide ( approximately 0.23 mmol/mol), while hydroxyacetone increased the acrylamide yields to more than 4 mmol/mol, indicating that alpha-hydroxy carbonyls are much more efficient than alpha-dicarbonyls in converting asparagine into acrylamide. The experimental results are consistent with the reaction mechanism based on (i) a Strecker type degradation of the Schiff base leading to azomethine ylides, followed by (ii) a beta-elimination reaction of the decarboxylated Amadori compound to afford acrylamide. The beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the nitrogen atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under low moisture conditions decarboxylated model Amadori compounds obtained by synthesis. The corresponding vinylogous compounds were only generated if a beta-proton was available, for example, styrene from the decarboxylated Amadori compound of phenylalanine. Therefore, it is suggested that this thermal pathway may be common to other amino acids, resulting under certain conditions in their respective vinylogous reaction products.     

Gas chromatographic investigation of acrylamide formation in browning model systems

Acrylamide formed in browning model systems was analyzed using a gas chromatograph with a nitrogen-phosphorus detector. Asparagine alone produced acrylamide via thermal degradation at the level of 0.99 microgram/g of asparagine. When asparagine was heated with triolein-which produced acrolein at the level of 1.82 +/- 0.31 (n = 5) mg/L of headspace by heat treatment-acrylamide was formed at the level of 88.6 microgram/g of asparagine. When acrolein gas was sprayed onto asparagine heated at 180 degrees C, a significant amount of acrylamide was formed (114 microgram/g of asparagine). On the other hand, when acrolein gas was sprayed onto glutamine under the same conditions, only a trace amount of acrylamide was formed (0.18 microgram/g of glutamine). Relatively high levels of acrylamide (753 microgram/g of ammonia) were formed from ammonia and acrolein heated at 180 degrees C in the vapor phase. The reaction of acrylic acid, which is an oxidation product of acrolein and ammonia, produced a high level of acrylamide (190 000 microgram/g of ammonia), suggesting that ammonia and acrolein play an important role in acrylamide formation in lipid-rich foods. Acrylamide can be formed from asparagine alone via thermal degradation, but carbonyl compounds, such as acrolein, promote its formation via a browning reaction.     

Review of methods for the reduction of dietary content and toxicity of acrylamide

Potentially toxic acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose in cereals, potatoes, and other plant-derived foods. This overview surveys and consolidates the following dietary aspects of acrylamide: distribution in food originating from different sources; consumption by diverse populations; reduction of the acrylamide content in the diet; and suppression of adverse effects in vivo. Methods to reduce adverse effects of dietary acrylamide include (a) selecting potato, cereal, and other plant varieties for dietary use that contain low levels of the acrylamide precursors, namely, asparagine and glucose; (b) removing precursors before processing; (c) using the enzyme asparaginase to hydrolyze asparagine to aspartic acid; (d) selecting processing conditions (pH, temperature, time, processing and storage atmosphere) that minimize acrylamide formation; (e) adding food ingredients (acidulants, amino acids, antioxidants, nonreducing carbohydrates, chitosan, garlic compounds, protein hydrolysates, proteins, metal salts) that have been reported to prevent acrylamide formation; (f) removing/trapping acrylamide after it is formed with the aid of chromatography, evaporation, polymerization, or reaction with other food ingredients; and (g) reducing in vivo toxicity. Research needs are suggested that may further facilitate reducing the acrylamide burden of the diet. Researchers are challenged to (a) apply the available methods and to minimize the acrylamide content of the diet without adversely affecting the nutritional quality, safety, and sensory attributes, including color and flavor, while maintaining consumer acceptance; and (b) educate commercial and home food processors and the public about available approaches to mitigating undesirable effects of dietary acrylamide.     

Acrylamide formation from asparagine under low moisture Maillard reaction conditions. 2. Crystalline vs amorphous model systems

The formation of acrylamide was investigated in model systems based on asparagine and glucose under low moisture Maillard reaction conditions as a function of reaction temperature, time, physical state, water activity, and glass transition temperature. Equimolar amorphous glucose/asparagine systems with different water activities were prepared by freeze drying and were shown to quickly move to the rubbery state already at room temperature and a water activity of above 0.15. The acrylamide amounts were correlated with physical changes occurring during the reaction. Pyrolysis and kinetics of acrylamide release in amorphous and crystalline glucose/asparagine models indicated the importance of the physical state in acrylamide formation. In amorphous systems, acrylamide was generated in higher concentrations and at lower temperatures as compared to the crystalline samples. Time and temperature are covariant parameters in both systems affecting the acrylamide formation by thermal processes. On the other side, the water activity and glass transition temperature do not seem to be critical parameters for acrylamide formation in the systems studied.     

Changes in free amino acids and sugars in potatoes due to sulfate fertilization and the effect on acrylamide formation

To examine how sulfur deprivation may affect acrylamide formation in cooked potatoes, three varieties of potato were grown under conditions of either severe sulfur deprivation or an adequate supply of sulfur. In all three varieties sulfur deprivation led to a decrease in acrylamide formation, even though the levels of sugars, which are acrylamide precursors, were higher in tubers of the sulfur-deprived plants. In one variety the concentration of free asparagine, the other precursor for acrylamide, was also higher. There was a very close correlation between the concentration of asparagine in the tubers expressed as a proportion of the total free amino acid pool and the formation of acrylamide upon cooking, whereas sugars were poorly correlated with acrylamide. In potatoes, where concentrations of sugars are usually limiting, competition between asparagine and other amino acids participating in the Maillard reaction may be a key determinant of the amount of acrylamide that is formed during processing.     

Reducing acrylamide precursors in raw materials derived from wheat and potato

A review of agronomic and genetic approaches as strategies for the mitigation of acrylamide risk in wheat and potato is presented. Acrylamide is formed through the Maillard reaction during high-temperature cooking, such as frying, roasting, or baking, and the main precursors are free asparagine and reducing sugars. In wheat flour, acrylamide formation is determined by asparagine levels and asparagine accumulation increases dramatically in response to sulfur deprivation and, to a much lesser extent, with nitrogen feeding. In potatoes, in which sugar concentrations are much lower, the relationships between acrylamide and its precursors are more complex. Much attention has been focused on reducing the levels of sugars in potatoes as a means of reducing acrylamide risk. However, the level of asparagine as a proportion of the total free amino acid pool has been shown to be a key parameter, indicating that when sugar levels are limiting, competition between asparagine and the other amino acids for participation in the Maillard reaction determines acrylamide formation. Genetic approaches to reducing acrylamide risk include the identification of cultivars and other germplasm in which free asparagine and/or sugar levels are low and the manipulation of genes involved in sugar and amino acid metabolism and signaling. These approaches are made more difficult by genotype/environment interactions that can result in a genotype being "good" in one environment but "poor" in another. Another important consideration is the effect that any change could have on flavor in the cooked product. Nevertheless, as both wheat and potato are regarded as of relatively high acrylamide risk compared with, for example, maize and rice, it is essential that changes are achieved that mitigate the problem.     

Acrylamide in roasted almonds and hazelnuts

The influences of composition and roasting conditions on acrylamide formation in almonds and hazelnuts were investigated. Eighteen samples of almonds originating from the U.S. and Europe were analyzed for sugars and free amino acids, and acrylamide formed during roasting was determined. Asparagine was the main free amino acid in raw almonds and correlated with the acrylamide content of dark roasted almonds. Roasting temperature was another key factor and had a very strong influence on acrylamide formation. Almonds of European origin contained significantly less free asparagine and formed significantly less acrylamide during roasting as compared to the almonds from the U.S. Roasted hazelnuts contained very little acrylamide because of the low content of free asparagine in the raw nut. Reducing sugars, although being consumed much faster than free amino acids in both types of nuts, were not decisive for the extent of acrylamide formation during roasting.     

Thermally generated 3-aminopropionamide as a transient intermediate in the formation of acrylamide

On the basis of the recent findings that "biogenic amines" can also be formed during thermal food processing from their parent amino acids in a Strecker-type reaction, the formation of 3-aminopropionamide, the biogenic amine of asparagine, was investigated in model systems as well as in thermally processed Gouda cheese. The results of model studies revealed that, besides acrylamide, 3-aminopropionamide was also formed in amounts of 0.1-0.4 mol % when asparagine was reacted in the presence of either glucose or 2-oxopropionic acid. Results of a second series of model experiments in which [(13)C(4)(15)N(2)]-asparagine ([(13)C(4)(15)N(2)]-Asn) and unlabeled 3-aminopropionamide were reacted together in the presence of glucose revealed a >12-fold higher efficacy of 3-aminopropionamide in acrylamide generation as compared to asparagine. Both [(13)C(3)(15)N(2)]-3-aminopropionamide and [(13)C(3)(15)N(1)]-acrylamide were formed during [(13)C(4)(15)N(2)]-Asn degradation in a ratio of about 1:4, supporting the idea that 3-aminopropionamide is a transient intermediate in acrylamide formation. In this study, 3-aminopropionamide was identified and quantified for the first time in foods, namely, in Gouda cheese. Although the fresh cheese contained low amounts of 3-aminopropionamide, its concentrations were much increased to approximately 1300 mug/kg after thermal processing. In isotope labeling studies, performed by administering to the cheese [(13)C(4)(15)N(2)]-Asn in a ratio of 1:2 as compared to the "natural" concentrations of asparagine, similar ratios of unlabeled/labeled 3-aminopropionamide and unlabeled/labeled acrylamide were determined. Thus, 3-aminopropionamide could be verified as a transient intermediate of acrylamide formation during food processing.     

Influence of agronomic factors and extraction rate on the acrylamide contents in yeast-leavened breads

Because the impact of agronomical factors on bakery products quality is still an insufficiently studied field, acrylamide contents of breads produced from flours of nine wheat, two rye, and two spelt varieties harvested in 2003 and 2004 were investigated. It could be demonstrated that acrylamide content in bread strongly depends on the cultivar, with extremes differing by a factor of 5.4 due to marked differences in free asparagine and crude protein contents. Nitrogen fertilization also resulted in elevated amino acid and protein contents, thus increasing acrylamide levels from 10.6 to 55.6 mug/kg. Independent of fertilization, harvest year turned out to be another factor influencing acrylamide formation. Breads produced from 2003 flours showed significantly higher acrylamide contents than those of 2004, which was ascribed to favorable light and temperature conditions during the cultivation period, thus enhancing amino acid and protein contents. Sprouting of the grain also resulted in significantly higher acrylamide levels, which was attributed to elevated enzyme activities and the formation of precursors from protein and starch. Furthermore, bakery products made from flours with higher extraction rates were shown to contain higher acrylamide levels resulting from extracted free asparagine and protein from the aleuron layers of the cereal grain.     

Potential of acrylamide formation,sugars, and free asparagine in potatoes: a comparison of cultivars and farming systems

Glucose, fructose, sucrose, free asparagine, and free glutamine were analyzed in 74 potato samples from 17 potato cultivars grown in 2002 at various locations in Switzerland and different farming systems. The potential of these potatoes for acrylamide formation was measured with a standardized heat treatment. These potentials correlated well with the product of the concentrations of reducing sugars and asparagine. Glucose and fructose were found to determine acrylamide formation. The cultivars showed large differences in their potential of acrylamide formation which was primarily related to their sugar contents. Agricultural practice neither influenced sugars and free asparagine nor the potential of acrylamide formation. It is concluded that acrylamide contents in potato products can be substantially reduced primarily by selecting cultivars with low concentrations of reducing sugars.     

Contribution of lipid oxidation products to acrylamide formation in model systems

The reactions of asparagine with methyl linoleate ( 1), methyl 13-hydroperoxyoctadeca-9,11-dienoate ( 2), methyl 13-hydroxyoctadeca-9,11-dienoate ( 3), methyl 13-oxooctadeca-9,11-dienoate ( 4), methyl 9,10-epoxy-13-hydroxy-11-octadecenoate ( 5), methyl 9,10-epoxy-13-oxo-11-octadecenoate ( 6), 2,4-decadienal ( 7), 2-octenal ( 8), 4,5-epoxy-2-decenal ( 9), and benzaldehyde ( 10) were studied to determine the potential contribution of lipid derivatives to acrylamide formation in heated foodstuffs. Reaction mixtures were heated in sealed tubes for 10 min at 180 degrees C under nitrogen. The reactivity of the assayed compounds was 7 > 9 > 4 > 2 > 8 approximately 6 > 10 approximately 5. The presence of compounds 1 and 3 did not result in the formation of acrylamide. These results suggested that alpha,beta,gamma,delta-diunsaturated carbonyl compounds were the most reactive compounds for this reaction followed by lipid hydroperoxides, more likely as a consequence of the thermal decomposition of these last compounds to produce alpha,beta,gamma,delta-diunsaturated carbonyl compounds. However, in the presence of glucose this reactivity changed, and compound 1/glucose mixtures showed a positive synergism (synergism factor = 1.6), which was observed neither in methyl stearate/glucose mixtures nor in the presence of antioxidants. This synergism is proposed to be a consequence of the formation of free radicals during the asparagine/glucose Maillard reaction, which oxidized the lipid and facilitated its reaction with the amino acid. These results suggest that both unoxidized and oxidized lipids are able to contribute to the conversion of asparagine into acrylamide, but unoxidized lipids need to be oxidized as a preliminary step.     

Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts

The relations between the formation of acrylamide and color, pyrazines, or antioxidants in an asparagine/d-glucose browning model system under various conditions were investigated. The highest level of acrylamide was produced in the asparagine/glucose (1:3) system heated at 170 degrees C for 30 min (2629 microg/g asparagine). Color intensity increased with temperature and heating time. The formation of pyrazines increased steadily with an increase of temperature (140-170 degrees C) and heating time (15-60 min). Antioxidant formation varied among the samples heated under different conditions. A clear correlation between formation of acrylamide and browning color was obtained. The formation of acrylamide was linearly correlated with the formation of total pyrazines during the initial stages of the Maillard reaction. No obvious correlation between formation of acrylamide and antioxidants was observed. However, excess amounts of asparagine increased the formation of antioxidants, whereas excess amounts of glucose reduced its formation.     

Investigations on the promoting effect of ammonium hydrogencarbonate on the formation of acrylamide in model systems

NH4HCO3 is known to promote acrylamide formation in sweet bakery products. This effect was investigated with respect to sugar fragmentation and formation of acrylamide from asparagine and sugar fragments in model systems under mild conditions. The presence of NH4HCO3 led to increases in acrylamide and alpha-dicarbonyls from glucose and fructose, respectively. As compared to glucose or fructose, sugar fragments such as glyoxal, hydroxyethanal, and glyceraldehyde formed much higher amounts of acrylamide in reaction with asparagine. The enhancing effect of NH4HCO3 is explained by (1) the action of NH3 as base in the retro-aldol reactions leading to sugar fragments, (2) facilitated retro-aldol-type reactions of imines in their protonated forms leading to sugar fragments, and (3) oxidation of the enaminols whereby glyoxal and other reactive sugar fragments are formed. These alpha-dicarbonyl and alpha-hydroxy carbonyl compounds may play a key role in acrylamide formation, especially under mild conditions.