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.     

Influence of sulfur fertilization on the amounts of free amino acids in wheat. correlation with baking properties as well as with 3-aminopropionamide and acrylamide generation during baking

Sulfur (S) fertilization has been long-known to influence the amounts of total free amino acids in plants. To determine the impact of S deficiency in wheat on the concentration of, in particular, free asparagine, the spring wheat cultivar 'Star' was grown in a laboratory scale (5 L pot) at five different levels of S fertilization. After maturity, the kernels were milled into white flours (1-5) and analyzed for their contents of total S and total nitrogen as well as for free amino acids and glucose, fructose, maltose, and sucrose. Extremely high concentrations of free asparagine (Asn; 3.9-5.7 g/kg) were determined in flours 1 and 2 (30 and 60 mg of S), whereas much lower amounts (0.03-0.4 g/kg) were present in flours grown at higher S levels. The amounts of the reducing carbohydrates were, however, scarcely affected by S fertilization. In agreement with the high amount of Asn in flours 1 and 2, heating of both flours led to the generation of very high amounts of acrylamide (1.7-3.1 mg/kg) as well as of 3-aminopropionamide (40-76 mg/kg). Similar concentrations were measured in crispbread prepared from both flours. Application of rheological measurements on doughs prepared from each flour and a determination of the loaf volume of bread baked therefrom clearly indicated that flours 1 and 2 would be excluded from commercial bread processing due to their poor technological properties. Two commercial flours showed relatively low concentrations of acrylamide after a thermal treatment.     

New insights into the formation of aroma-active strecker aldehydes from 3-oxazolines as transient intermediates

2-Substituted-5-methyl-3-oxazolines, a novel class of aroma precursors that are able to release the respective Strecker aldehydes by hydrolysis, were identified. Hydrolysis can take place after the addition of water or with human saliva during mastication, respectively. 2-Isobutyl-, 2-sec-isobutyl-, 2-isopropyl, and 2-benzyl-5-methyl-3-oxazolines were synthesized and structurally identified by means of gas chromatography-mass spectrometry (GC-MS) in the electron impact mode and in the chemical ionization mode as well as by one- and two-dimensional NMR experiments. With these compounds at hand, a variety of stability experiments were performed using headspace-GC-MS or proton transfer reaction-MS techniques on the basis of stable isotope dilution assays, proving the ability to release the respective Strecker aldehydes was dependent on the pH value as well as on the hydrolysis time. After the addition of water at 37 °C, for example, >70 mol % of 3-methylbutanal or >40 mol % of phenylacetaldehyde was liberated from a solution of 2-isobutyl-5-methyl-3-oxazoline or 2-benzyl-5-methyl-3-oxazoline, respectively, after 5 min. Furthermore, the presence of 2-isobutyl-5-methyl-3-oxazoline in dark chocolate containing 70% cocoa was proven by GC-MS.     

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.     

Influence of oil type on the amounts of acrylamide generated in a model system and in French fries

Acrylamide formation was studied by use of a new heating methodology, based on a closed stainless steel tubular reactor. Different artificial potato powder mixtures were homogenized and subsequently heated in the reactor. This procedure was first tested for its repeatability. By use of this experimental setup, it was possible to study the acrylamide formation mechanism in the different mixtures, eliminating some variable physical and chemical factors during the frying process, such as heat flux and water evaporation from and oil ingress into the food. As a first application of this optimized heating concept, the influence on acrylamide formation of the type of deep-frying oil was investigated. The results obtained from the experiments with the tubular reactor were compared with standardized French fry preparation tests. In both cases, no significant difference in acrylamide formation could be found between the various heating oils applied. Consequently, the origin of the deep-frying vegetable oils did not seem to affect the acrylamide formation in potatoes during frying. Surprisingly however, when artificial mixtures did not contain vegetable oil, significantly lower concentrations of acrylamide were detected, compared to oil-containing mixtures.     

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.     

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.     

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.     

Tests for the depolymerization of polyacrylamides as a potential source of acrylamide in heated foods

This study was conducted to test the possibility that polyacrylamides used in agriculture may contribute to acrylamide formation in heated foods by thermal depolymerization. Two samples of polyacrylamide with low molecular weights of 1.5 and 10 kDa were used to test for in-chain and end-chain depolymerization. They were added as aqueous solutions to filter paper for heating in a drying/dry environment and added to a cooking oil for heating in a fatty environment. The heating conditions were 175 degrees C for 15 and 30 min, respectively. Both regimens were tested in the absence and presence of the redox-active metal ions Fe(III) and Cu(II), and all tests were conducted without the exclusion of atmospheric oxygen. There was no evidence of any significant depolymerization of polyacrylamide to free acrylamide monomer, <0.04%. In fact, residual levels of acrylamide present already in the low molecular weight polymers were seen to fall by 50-80% on heating. Consequently, it is concluded that even if polyacrylamides were to contaminate agricultural crops and foods derived therefrom (which itself is an unproven suggestion), there is no evidence that the polymers would depolymerize on heating of the food to form acrylamide in any significant amount.     

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.     

Influence of storage practices on acrylamide formation during potato frying

A number of parameters linked to storage of potatoes were evaluated with regard to their potential to influence the acrylamide formation in French fries. Acrylamide, which is a potential human carcinogen, is reported to be formed during the frying of potatoes as a result of the reactions between asparagine and reducing sugars. This study was conducted using three potato varieties (Bintje, Ramos, and Saturna) typically used in Belgium, The Netherlands, and the northern part of France for French fry and crisp production. Saturna, mainly used in crisp production, appeared to be the least susceptible for acrylamide formation during frying. Especially storage at low temperatures (4 degrees C) compared to storage at 8 degrees C seemed to enhance acrylamide formation due to a strong increase in reducing sugars caused by low-temperature storage. Because of the reversible nature of this physiological reaction, it was possible to achieve a significant reduction of the reducing sugars after a reconditioning of the cold-stored potatoes for 3 weeks at 15 degrees C. All changes in acrylamide concentrations could mainly be explained by the reducing sugar content of the potato (R2 = 0.84, n = 160). This means that, by ensuring a low reducing sugar content of the potato tuber, the risk for acrylamide formation will largely be reduced. Finally the use of a sprout inhibitor did not influence the composition of the potato, and thus acrylamide formation was not susceptible to this treatment.     

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.     

Impact of pH on the kinetics of acrylamide formation/elimination reactions in model systems

The effect of pH on acrylamide formation and elimination kinetics was studied in an equimolar (0.1 M) asparagine-glucose model system in phosphate or citrate buffer, heated at temperatures between 120 and 200 degrees C. To describe the experimental data, a simplified kinetic model was proposed and kinetic parameters were estimated by combined nonlinear regression and numerical integration on the data obtained under nonisothermal conditions. The model was subsequently validated in a more realistic potato-based matrix with varying pH. By increasing acidity, the reaction rate constants at T(ref) (160 degrees C) for both acrylamide formation and elimination can significantly be reduced, whereas the temperature dependence of both reaction rate constants increases. The introduction of a lyophilized potato matrix (20%) did not affect the acrylamide formation reaction rate constant at reference temperature (160 degrees C) as compared to the asparagine-glucose model system; the elimination rate constant at T(ref), on the contrary, was almost doubled.     

Influence of fertilization on acrylamide formation during frying of potatoes harvested in 2003

The quality of the potato has been found to vary, when grown under different agricultural and environmental conditions, such as the level of fertilization. Consequently these factors may influence the acrylamide formation during the preparation of French fries. These assumptions were studied on three varieties: Bintje, Ramos, and Saturna from the harvest of 2003. Decreasing N fertilization caused increases in the reducing sugar concentration from 60% up to 100% on DM for all varieties studied. Due to a high correlation between the reducing sugar content and the generation of acrylamide during frying, this resulted in a parallel increase in the acrylamide concentration of the French fries. Thus by lowering the amount of N fertilizer, an increase of 30-65% of the acrylamide generation during frying could be observed. It seems of extreme importance to find an appropriate balance between the level of N fertilizer in order to diminish acrylamide formation but on the other hand to obtain an acceptable tuber and to consider the environmental impact. All results reported should be seen in the perspective of the warm growing season of 2003.     

Influence of processing parameters on acrylamide formation during frying of potatoes

Consistent evidence suggests that the probable human carcinogen acrylamide is formed in starch-rich foodstuffs through heat-induced interaction of asparagine and reducing sugars during Maillard browning. However, information regarding the influence of processing parameters on acrylamide formation is scarce. We investigated the impact of temperature, heating time, browning level, and surface-to-volume ratio (SVR) on acrylamide generation in fried potatoes. Acrylamide content was determined by liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). In potato shapes with low SVR, acrylamide content consistently increased with increasing temperature and processing times. By contrast, in shapes with intermediate to high SVR, maximal acrylamide formation occurred at 160-180 degrees C, while higher temperatures or prolonged processing times caused a decrease of acrylamide levels. Moreover, browning levels were not a reliable measure of acrylamide content in large-surface products.     

Relationship between virgin olive oil phenolic compounds and acrylamide formation in fried crisps

In this paper the relationship between virgin olive oil (VOO) phenol compounds and the formation of acrylamide in potato crisps was investigated. The phenol compositions of 20 VOO samples were screened by LC-MS, and 4 oils, characterized by different phenol compound patterns, were selected for frying experiments. Slices of potatoes were fried at 180 degrees C for 5, 10, and 15 min, and acrylamide content was determined by LC-MS. Results demonstrated that VOO phenolic compounds are not degraded during frying, and crisp color was not significantly different among the four VOOs. Acrylamide concentration in crisps increased during frying time, but the formation was faster in the oil having the lowest concentration of phenolic compounds. Moreover, the VOO having the highest concentration of ortho-diphenolic compounds is able to efficiently inhibit acrylamide formation in crisps from mild to moderate frying conditions. It was concluded that the use of ortho-diphenolic-rich VOOs can be proposed as a reliable mitigation strategy to reduce acrylamide formation in domestic deep-frying.     

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.     

Kinetics of acrylamide formation and elimination during heating of an asparagine-sugar model system

The kinetics of acrylamide (AA) was analyzed by heating a simple model system consisting of asparagine and glucose, fructose, or sucrose (0.01 M, pH 6) at temperatures between 140 and 200 degrees C. The AA concentration appeared to be the net result of simultaneous formation and elimination. A general kinetic model describing the AA yield was identified, and kinetic parameters were obtained by nonlinear regression on the nonisothermally derived data. On the basis of kinetic parameters, the AA formation appeared to proceed faster and to be more temperature sensitive in the asparagine-glucose than in the asparagine-fructose model system. The AA elimination kinetics, on the other hand, was similar. Significantly less AA was formed in the asparagine-sucrose model system as compared to the model systems with glucose or fructose.     

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.     

Carcinogenicity of acrylamide: a computational study

This paper reports a series of ab initio, density functional theory (DFT), and semiempirical molecular orbital (MO) calculations concerning the reaction between the ultimate carcinogen of acrylamide and guanine. Acrylamide--a product of the Maillard reaction--is present in a variety of fried and oven-cooked food. After intake, it is epoxidized by cytochrome P450 2E1 to yield the ultimate carcinogen--glycidamide. Effects of solvation were considered using the Langevin dipoles (LD) model of Florian and Warshel and the solvent reaction field (SCRF) model of Tomasi and co-workers. In silico activation free energies are in very good agreement with the experimental value of 22.8 kcal/mol. This agreement presents strong evidence in favor of the validity of the proposed S N2 reaction mechanism and points to the applicability of quantum chemical methods to studies of reactions associated with carcinogenesis. In addition, insignificant stereoselectivity of the studied reaction was predicted. Finally, the competing reaction of glycidamide with adenine was simulated, and the experimentally observed regioselectivity was successfully reproduced.