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.     

Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread

A repeatable procedure for studying the effects of internal and external factors on acrylamide content in yeast-leavened wheat bread has been developed. The dough contained wheat endosperm flour with a low content of precursors for acrylamide formation (asparagine and reducing sugars), dry yeast, salt, and water. The effects of asparagine and fructose, added to the dough, were studied in an experiment with a full factorial design. More than 99% of the acrylamide was found in the crust. Added asparagine dramatically increased the content of acrylamide in crusts dry matter (from about 80 microg/kg to between 600 and 6000 microg/kg) while added fructose did not influence the content. The effects of temperature and time of baking were studied in another experiment using a circumscribed central composite design. Mainly temperature (above 200 degrees C) but also time increased the acrylamide content in crust dry matter (from below 10 to 1900 microg/kg), and a significant interaction was found between these two factors. When baked at different conditions with the same ingredients, a highly significant relationship (P < 0.001) between color and acrylamide content in crust was found. Added asparagine, however, did not increase color, showing that mainly other amino compounds are involved in the browning reactions.     

Acrylamide formation from asparagine under low-moisture Maillard reaction conditions. 1. Physical and chemical aspects in crystalline model systems

The formation of acrylamide in crystalline model systems based on asparagine and reducing sugars was investigated under low-moisture reaction conditions. The acrylamide amounts were correlated with physical changes occurring during the reaction. Molecular mobility of the precursors turned out to be a critical parameter in solid systems, which is linked to the melting behavior and the release of crystallization water of the reaction sample. Heating binary mixtures of asparagine monohydrate and anhydrous reducing sugars led to higher acrylamide amounts in the presence of fructose compared to glucose. Differential scanning calorimetry measurements performed in open systems indicated melting of fructose at 126 degrees C, whereas glucose and galactose fused at 157 and 172 degrees C, respectively. However, glucose was the most reactive and fructose the least efficient sugar in anhydrous liquid systems, indicating that at given molecular mobility the chemical reactivity of the sugar was the major driver in acrylamide formation. Furthermore, reaction time and temperature were found to be covariant parameters: acrylamide was preferably formed by reacting glucose and asparagine at 120 degrees C for 60 min, whereas 160 degrees C was required at shorter reaction time (5 min). These results suggest that, in addition to the chemical reactivity of ingredients, their physical state as well as reaction temperature and time would influence the formation of acrylamide during food processing.     

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.     

Acrylamide in foods: occurrence,sources, and modeling

Acrylamide in food products-chiefly in commercially available potato chips, potato fries, cereals, and bread-was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were homogenized with water/dichloromethane, centrifuged, and filtered through a 5 kDa filter. The filtrate was cleaned up on mixed mode, anion and cation exchange (Oasis MAX and MCX) and carbon (Envirocarb) cartridges. Analysis was done by isotope dilution ([D(3)]- or [(13)C(3)]acrylamide) electrospray LC-MS/MS using a 2 x 150 mm (or 2 x 100 mm) Thermo HyperCarb column eluted with 1 mM ammonium formate in 15% (or 10% for the 2 x 100 mm column) methanol. Thirty samples of foods were analyzed. Concentrations of acrylamide varied from 14 ng/g (bread) to 3700 ng/g (potato chips). Acrylamide was formed during model reactions involving heating of mixtures of amino acids and glucose in ratios similar to those found in potatoes. In model reactions between amino acids and glucose, asparagine was found to be the main precursor of acrylamide. Thus, in the reaction between nitrogen-15 (amido)-labeled asparagine and glucose, corresponding (15)N-labeled acrylamide was formed. The yield of the model reaction is approximately 0.1%.     

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.     

Analysis of acrylamide in green tea by gas chromatography-mass spectrometry

Optimization of the solid-phase extraction cleanup procedure enabled the GC-MS analysis of acrylamide in tea samples without the interference of bromination by tea catechins. Although polyvinylpolypyrrolidone (PVPP) is available for removing tea catechins from tea extract, the peaks derived from PVPP had the same retention time as brominated acrylamide in mass chromatograms obtained by GC-MS. A considerable amount of acrylamide was formed at roasting temperatures of > or =120 degrees C; the highest acrylamide level was observed when tea samples were roasted at 180 degrees C for 10 min. Higher temperatures and longer processing times caused a decrease in the acrylamide content. Furthermore, an analysis of 82 tea samples showed that rather than the reducing sugar content, the asparagine content in tea leaves was a significant factor related to acrylamide formation in roasted products. The acrylamide level in roasted tea products was controlled by asparagine in the presence of reducing sugars.     

Toward a kinetic model for acrylamide formation in a glucose-asparagine reaction system

A kinetic model for the formation of acrylamide in a glucose-asparagine reaction system is pro-posed. Equimolar solutions (0.2 M) of glucose and asparagine were heated at different temperatures (120-200 degrees C) at pH 6.8. Besides the reactants, acrylamide, fructose, and melanoidins were quantified after predetermined heating times (0-45 min). Multiresponse modeling by use of nonlinear regression with the determinant criterion was used to estimate model parameters. The proposed model resulted in a reasonable estimation for the formation of acrylamide in an aqueous model system, although the behavior of glucose, fructose, and asparagine was slightly underestimated. The formation of acrylamide reached its maximum when the concentration of sugars was reduced to about 0. This supported previous research, showing that a carbonyl source is needed for the formation of acrylamide from asparagine. Furthermore, it is observed that acrylamide is an intermediate of the Maillard reaction rather than an end product, which implies that it is also subject to a degradation reaction.     

Effectiveness of ionizing radiation in reducing furan and acrylamide levels in foods

Furan and acrylamide are two possible carcinogens commonly found in many thermally processed foods. The possibility of using ionizing radiation to reduce the levels of thermally induced furan and acrylamide in water and selected foods was investigated. Aqueous furan solutions, and foods (frankfurters, sausages, infant sweet potatoes) that contained furan were irradiated to various doses of gamma-rays. Water and oil spiked with acrylamide and potato chips (a known acrylamide-containing food) were also irradiated. In addition, possible irradiation-induced formation of acrylamide in glucose and asparagine solutions was analyzed. Results showed that irradiation at 1.0 kGy destroyed almost all furan in water. In frankfurters, sausages, and infant sweet potatoes, the rate of irradiation-induced destruction of furan was much lower than the rate in water, although significant reductions in furan levels were observed in all foods. Irradiation at 2.5-3.5 kGy, doses that can inactivate 5-log of most common pathogens, reduced furan levels in the food samples by 25-40%. Similarly to furan, acrylamide in water was also sensitive to irradiation. After 1.5 kGy of irradiation, most of the acrylamide was degraded. Irradiation, however, had a very limited effect on acrylamide levels in oil and in potato chips, even at a dose of 10 kGy. No detectable acrylamide was formed in the mixture of asparagine and glucose upon irradiation. These results suggest that a low dose of irradiation easily destroys furan and acrylamide in water. In real foods, however, the reduction of furan was less effective than in water, whereas the reduction in acrylamide was minimal.     

Investigation of the influence of different moisture levels on acrylamide formation/elimination reactions using multiresponse analysis

The influence of water activity on the formation and elimination reactions of acrylamide was examined by means of multiresponse modeling on two different levels of complexity: basic equimolar asparagine-glucose systems and equimolar potato-based asparagine-glucose systems. To this end, model systems were first equilibrated to initial water activities in the range of 0.88-0.99 (corresponding roughly to the moisture gradient observed in French fries) and then heated at temperatures between 120 and 200 degrees C during different reaction times. For each sample, the concentration of acrylamide, glucose, asparagine, and aspartic acid was measured, as well as the extent of browning. A mechanistic model was proposed to model the five measured responses simultaneously. For both types of model systems, the model prediction was quite adequate, with the exception of the extent of browning, especially in the case of the potato-based model system. Moreover, the corresponding estimated kinetic parameters for acrylamide formation and elimination did not change significantly (based on a 95% confidence level) within the range of water activities tested, nor between the systems in the absence or presence of the potato matrix. The only remarkable difference was observed for the activation energy of acrylamide elimination, which was lower in the presence of the potato matrix, although not always significant. In general, these results confirm the generic nature of the model proposed and show that the influence of different moisture levels on acrylamide formation and elimination is minimal and that the addition of a potato matrix has little or no influence on the kinetic model and corresponding kinetic parameters.     

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.     

Role of water upon the formation of acrylamide in a potato model system

The moisture sorption isotherms of a commercial potato powder were investigated at 20 degrees C for water activities ranging from 0.11 to 0.97. The sorption isotherms were typical type-II sigmoidal curves, with a steep increase in moisture content for water activities above 0.9 and exhibiting hysteresis over the whole water activity range. On the basis of the isotherms, the influence of the initial water activity and moisture content on both Maillard browning and acrylamide formation was determined by heating oil containing potato powder mixtures in a closed stainless-steel tubular reactor. The Maillard browning, as determined spectrophotometrically, showed an optimum at intermediate water activities. The yields of acrylamide, expressed relatively to the molar amount of asparagine, remained constant below 0.8 aw and below moisture contents of about 20% (on a dry basis). For the more intense heat treatments, an increased acrylamide yield was however observed at higher moisture contents, with an optimum at water contents of about 100% (on a dry basis). However, this increase and optimum was not observed at less intense heat treatments. At moisture contents above 100%, a significant decrease in acrylamide yields was assessed, although the water activity increased only marginally in this area of the sorption isotherms. It was thus observed that the acrylamide content was rather dependent upon the moisture content than upon the water activity in the high-moisture potato powder model system.     

Acrylamide in French fries: influence of free amino acids and sugars

The free amino acid profile and sugar (fructose, glucose, and sucrose) composition were determined in potato samples selected to give a large range of variation (a total of 66 samples). From these samples French fries were produced in a laboratory-scale simulation of an industrial process followed by a finish fry at 180 degrees C for 3.5 min using a restaurant fryer. The final product was blast frozen and analyzed for acrylamide. Acrylamide was detected in all samples, but its concentration varied significantly from 50 to 1800 ng/g. For isotope dilution (13C3) acrylamide analysis, samples were extracted with water, cleaned up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analyzed by LC-MS/MS. Statistical analysis of the data indicates that the effect of sugars and asparagine on the concentration of acrylamide in French fries is positive and significant (p < 0.001). It appears that one of the ways acrylamide formation in French fries can be effectively controlled is by the use of raw products with low sugar (and to a lesser degree, asparagine) content.     

Measurement of acrylamide and its precursors in potato, wheat, and rye model systems

The relationship between acrylamide and its precursors, namely, free asparagine and reducing sugars, was studied in cakes made from potato flake, wholemeal wheat, and wholemeal rye, cooked at 180 degrees C, from 5 to 60 min. Between 5 and 20 min, major losses of asparagine, water, and total reducing sugars were accompanied by large increases in acrylamide, which maximized in all three products between 25 and 30 min, followed by a slow linear reduction. Acrylamide formation did not occur to a large degree until the moisture contents of the cakes fell below 5%. Linear relationships were observed for acrylamide formation with the residual levels of asparagine and reducing sugars for all three food materials.     

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.     

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.     

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.     

Acrylamide formation in almonds (Prunus dulcis): influences of roasting time and temperature, precursors, varietal selection, and storage

Acrylamide is a probable human carcinogen that is found in many roasted and baked foods. This paper describes two sensitive and reliable LC-(ESI)MS/MS methods for the analysis of (1) acrylamide and (2) common acrylamide precursors (i.e., glucose, fructose, asparagine, and glutamine) in raw and roasted almonds. These methods were used to evaluate the impact of roasting temperatures (between 129 and 182 °C) and times on acrylamide formation. Controlling the roasting temperature at or below 146 °C resulted in acrylamide levels below 200 ppb at all roasting times evaluated. Six varieties of almonds collected in various regions of California over two harvest years and roasted at 138 °C for 22 min had acrylamide levels ranging from 117 ± 5 μg/kg (Sonora) to 221 ± 95 μg/kg (Butte) with an average of 187 ± 71 μg/kg. A weak correlation between asparagine content in raw almonds and acrylamide formation was observed (R(2) = 0.6787). No statistical relationship was found between acrylamide formation and almond variety, orchard region, or harvest year. Stability studies on roasted almonds indicated that acrylamide levels decreased by 12.9-68.5% (average of 50.2%) after 3 days of storage at 60 °C. Short-term elevated temperature storage may be another approach for mitigating acrylamide levels in roasted almonds.     

Investigations of factors that influence the acrylamide content of heated foodstuffs

The acrylamide content of heated foodstuffs should be considered to be the net result of complex reactions leading to the formation and elimination/degradation of this compound. The present study, involving primarily homogenized potato heated in an oven, was designed to characterize parameters that influence these reactions, including the heating temperature, duration of heating, pH, and concentrations of various components. Higher temperature (200 degrees C) combined with prolonged heating times produced reduced levels of acrylamide, due to elimination/degradation processes. At certain concentrations the presence of asparagine or monosaccharides (in particular, fructose and also glucose and glyceraldehyde) was found to increase the net content of acrylamide. Addition of other free amino acids or a protein-rich food component strongly reduced the acrylamide content, probably by promoting competing reactions and/or covalently binding acrylamide formed. The dependence on pH of the acrylamide content exhibited a maximum around pH 8; in particular, lower pH was shown to enhance elimination and decelerate formation of acrylamide. In contrast, the effects of additions of antioxidants or peroxides on acrylamide content were small or nonexistent.     

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.