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.     

Comparison of volatile generation in serine/threonine/glutamine-ribose/glucose/fructose model systems

Thermal generation of volatiles in nine model reactions was studied and compared. Each of the model systems contained one amino acid and one monosaccharide. The amino acid was serine, threonine, or glutamine, and the monosaccharide was ribose, glucose, or fructose. More unsubstituted pyrazine was generated in serine-sugar systems than threonine-sugar systems. The formation of several furfuryl-substituted pyrazines and pyrroles was observed in some of the studied systems. Total pyrazines were generated more in glutamine-containing systems than in serine- and threonine-containing systems, and the reverse was true for generation of furfuryl-substituted compounds. Acetylpyrazine was generated in serine/threonine/glutamine-glucose and serine/glutamine-fructose systems.     

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.     

Formation of Strecker aldehydes and pyrazines in a fried potato model system

Sugars and amino acids were removed from potato slices by soaking in water and ethanol. They were then infused with various combinations of sugars (glucose and/or fructose) and amino acids (asparagine, glutamine, leucine, isoleucine, phenylalanine, and/or methionine) and fried. Volatile compounds were trapped onto Tenax prior to gas chromatography-mass spectrometry. Relative amounts of compounds (relative to the internal standard) and relative yields (per mole of amino acid infused into the slices) were determined. Amounts of 10 pyrazines, 4 Strecker aldehydes, and 4 other compounds were monitored. Relative amounts and relative yields of compounds varied according to the composition of the system. For the single amino acid-glucose systems, leucine gave the highest relative amount and relative yield of its Strecker aldehyde. Asparagine and phenylalanine gave the highest total relative amount and total relative yield, respectively, of pyrazines. In the system containing all of the amino acids and glucose, the relative amount of 3-methylbutanal was higher, whereas the amounts of the other monitored Strecker aldehydes were lower. Most of the relative amounts of individual pyrazines were lower compared to the glucose-asparagine system, whereas the total relative yield of pyrazines was lower, compared to all of the single amino acid-glucose mixtures. Addition of fructose to the mixed amino acid-glucose model system generated Strecker aldehydes and pyrazines in ratios that were more similar to those of untreated potato chips than to those from the same system but without fructose. Both the sugars and the amino acids present in potato are crucial to the development of flavor compounds in fried potato slices.     

Impact of the N-terminal amino acid on the formation of pyrazines from peptides in Maillard model systems

Only a minor part of Maillard reaction studies in the literature focused on the reaction between carbohydrates and peptides. Therefore, in continuation of a previous study in which the influence of the peptide C-terminal amino acid was investigated, this study focused on the influence of the peptide N-terminal amino acid on the production of pyrazines in model reactions of glucose, methylglyoxal, or glyoxal. Nine different dipeptides and three tripeptides were selected. It was shown that the structure of the N-terminal amino acid is determinative for the overall pyrazine production. Especially, the production of 2,5(6)-dimethylpyrazine and trimethylpyrazine was low in the case of proline, valine, or leucine at the N-terminus, whereas it was very high for glycine, alanine, or serine. In contrast to the alkyl-substituted pyrazines, unsubstituted pyrazine was always produced more in the case of experiments with free amino acids. It is clear that different mechanisms must be responsible for this observation. This study clearly illustrates the capability of peptides to produce flavor compounds such as pyrazines.     

Identification and monitoring of intermediates and products in the acrylamide pathway using online analysis

Acrylamide formation under controlled processing conditions was studied in a starch matrix by analyzing volatile compounds in the gas phase using online mass spectrometry. Compounds were identified using mass spectral analysis, authentic standards, and the labeling patterns from isotopically labeled asparagine and sugars. Acrylamide, 3-aminopropanamide, methylpyrazine, 3-oxopropanamide, and aminopropan-2-one were assigned to the ions at m/ z 72, 89, 95, 88, and 74, respectively. Ion m/ z 60 was proposed as the transamination product of glyoxal, but labeling experiments did not support this assignment. Temporal formation of acrylamide and related compounds was studied in 51 samples containing asparagine and selected sugars or carbonyls. Data from the experiments were analyzed to investigate correlations between the amounts of acrylamide, intermediates, and pyrazines formed. A strong correlation between 3-aminopropanamide and acrylamide was found in all samples, whereas other correlations were reactant specific. Preliminary multiway analysis of the data identified temporal similarities in the ion profiles and showed that dynamic monitoring can follow the production and utilization of intermediates leading to acrylamide.     

Added versus Accumulated Sugars on Color Development and Acrylamide Formation in French-Fried Potato Strips

Added (glucose addition) versus accumulated (in situ sugar development via cold-temperature storage) sugar treatments were investigated in relation to acrylamide formation within fried potato strips at standardized levels of finish-fried color (Agtron color scores ranged from 36 to 84). The added sugar treatment exhibited a relatively reduced rate of acrylamide formation and generally possessed a lower and less variable acrylamide content (61-1290 ng/g) than the accumulated sugar scheme (61-2191 ng/g). In a subsequent experiment, added fructose applied to strip surfaces via dipping prior to frying favored acrylamide formation over color development relative to added glucose, for which the reverse trend was observed. Thus, where acrylamide differences were noted between added and accumulated sugar treatments (given equivalent Agtron color scores), this result was likely aided by the relative higher fructose content in strips of the accumulated sugar scheme rather than simply a greater relative concentration of total reducing sugars.     

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.     

Mechanisms of alkylpyrazine formation in a potato model system containing added glycine

The use of glycine to limit acrylamide formation during the heating of a potato model system was also found to alter the relative proportions of alkylpyrazines. The addition of glycine increased the quantities of several alkylpyrazines, and labeling studies using [2-13C]glycine showed that those alkylpyrazines which increased in the presence of glycine had at least one 13C-labeled methyl substituent derived from glycine. The distribution of 13C within the pyrazines suggested two pathways by which glycine, and other amino acids, participate in alkylpyrazine formation, and showed the relative contribution of each pathway. Alkylpyrazines that involve glycine in both formation pathways displayed the largest relative increases with glycine addition. The study provided an insight into the sensitivity of alkylpyrazine formation to the amino acid composition in a heated food and demonstrated the importance of those amino acids that are able to contribute an alkyl substituent. This may aid in estimating the impact of amino acid addition on pyrazine formation, when amino acids are added to foods for acrylamide mitigation.     

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.     

Formation of pyrazines and a novel pyrrole in Maillard model systems of 1,3-dihydroxyacetone and 2-oxopropanal

Alkylpyrazines are a very important class of Maillard flavor compounds, but their mechanism of formation is complex and consists of different pathways. The model reaction of 20 different amino acids with 1,3-dihydroxyacetone, as a precursor of 2-oxopropanal, was studied by means of SPME-GC-MS to investigate the involvement of the amino acid side chain in the substitution pattern of the resulting pyrazines. 2,5-Dimethylpyrazine was quantitatively the most important pyrazine formed from all of the amino acids. The amino acid side chain is not involved in its formation. The substituents of other less abundant pyrazines resulted mainly from the incorporation of the Strecker aldehyde or aldol condensation products in the intermediate dihydropyrazine. The importance of different reaction mechanisms was evaluated, taking into account the pattern of pyrazines identified. In the solvent extracts of aqueous model reactions of 2-oxopropanal with amino acids, the main reaction product was not a pyrazine but a novel pyrrole. This pyrrole was identified as 2,5-diacetyl-3-methyl-1 H-pyrrole by means of spectral analysis, secured by chemical synthesis. A reaction mechanism for its formation was proposed and evaluated. The influence of various reaction conditions on the formation of 2,5-diacetyl-3-methyl-1 H-pyrrole and 2,5-dimethylpyrazine in the model reaction of alanine with 2-oxopropanal was studied. These results underscore the importance of the ratio of the different reagents and the presence of water in the resulting flavor formation in the Maillard reaction.     

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.     

Structure-reactivity relationships of flavan-3-ols on product generation in aqueous glucose/glycine model systems

Ring structure-reactivity relationships of three flavan-3-ols [epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG)] and three simple phenolic compounds (1,3,5-trihydroxybenzene, 1,2,3-trihydroxybenzene, and methylgallate as the analogous individual A, B, and C benzene rings of EGCG) on product generation in an aqueous glucose-glycine reaction model system (125 degrees C and 30 min) were investigated. The addition of EC, ECG, or EGCG to a glucose-glycine model was reported to similarly significantly reduce the formation of pyrazine, methyl-substituted pyrazines, and cyclotene. All three flavan-3-ols were also reported to generate phenolic-C2, C3, C4, and C6 sugar fragment adducts and to statistically reduce the concentration of glyoxal, glycolaldehyde, methylglyoxal, hydroxyacetone, diacetyl, acetoin, and 3-deoxyglucosone during the reaction time course, except for the EGCG reaction where 3-deoxyglucosone was not statistically different from the control after 20 min. For the simple phenolic compounds, methylgallate followed by 1,2,3-trihydroxybenzene was the least reactive, while 1,3,5-trihydroxybenzene was reported as the most reactive phenolic structure for quenching or reducing the concentration of the alpha-hydroxy- and alpha-dicarbonyl sugar fragments during the reaction time course. These results imply that the main mechanism flavan-3-ols reduced product generation was phenolic-sugar fragment carbonyl trapping reactions primarily on the A ring (the meta-polyhydroxylated benzene ring) or not due to the alteration of the reaction reduction potential.     

Volatile compounds produced from monosodium glutamate in common food cooking

A mixture of soybean oil and/or water, sugar and/or monosodium glutamate (MSG) was water-boiled or oil-heated at 100, 120, 140, 160, or 170 degrees C, respectively, and volatile compounds produced were isolated by solid-phase microextraction and then identified by GC and GC/MS. Oxidative thermal degradation products of fatty acids (OTDPFA) were the major volatile compounds detected for all water-boiled samples. When MSG and sugar were heated together, 2, 5-dimethyl pyrazine and methyl pyrazine were also detected at or after 160 min heating. Water added in soybean oil increased OTDPFA production. In oil-heated samples of soybean oil alone, soybean oil with MSG, and soybean oil with sugar, OTDPFA were also the major compounds found. The samples containing MSG also produced 2-pyrrolidone, and the samples containing sugar also produced furfural and 5-hydroxy methyl furfural. The samples containing soybean oil, sugar, and MSG produced 23 pyrazines, OTDPFA, and gamma-butyrolactone as the major volatile compounds. The contents of OTDPFA were lower in MSG plus sugar and MSG-added samples, and it is postulated that antioxidant activities were produced in cooking.     

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.     

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.     

Pyrazine formation from serine and threonine.

The formation of pyrazines from L-serine and L-threonine has been studied. L-Serine and L-threonine, either alone or combined, were heated at 120 degrees C as low temperature for 4 h or at 300 degrees C as high temperature for 7 min. The pyrazines formed from each reaction were identified by GC/MS, and the yields (to the amino acid used, as parts per million) were determined by GC/FID. It was found that pyrazine, methylpyrazine, ethylpyrazine, 2-ethyl-6-methylpyrazine, and 2,6-diethylpyrazine were formed from serine, whereas 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, trimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine, and 2-ethyl-3, 5-dimethylpyrazine were formed from threonine. Mechanistically, it is proposed that the thermal degradation of serine or threonine is composed of various complex reactions. Among these reactions, decarbonylation followed by dehydration is the main pathway to generate the alpha-aminocarbonyl intermediates leading to the formation of the main product, such as pyrazine from serine or 2, 5-dimethylpyrazine from threonine. Also, deamination after decarbonylation generates more reactive intermediates, alpha-hydroxycarbonyls. Furthermore, aldol condensation of these reactive intermediates provides alpha-dicarbonyls. Subsequently, these alpha-dicarbonyls react with the remaining serine or threonine by Strecker degradation to form additional alpha-aminocarbonyl intermediates, which then form additional pyrazines. In addition, decarboxylation and retroaldol reaction may also involve the generation of the intermediates.     

Effect of carbohydrate substrate on fermentation by kefir yeast supported on delignified cellulosic materials

The suitability of delignified cellulosic (DC) material supported kefir yeast to ferment raw materials that contain various single carbohydrates, for the production of potable alcohol and alcoholic drinks, is examined in this investigation. Results are reported of fermentations carried out with sucrose, fructose, and glucose in synthetic media. Repeated batch fermentations at various initial sugar concentrations of sucrose, fructose, and glucose were performed at 30 degrees C in the presence of the aforementioned biocatalyst. The results clearly show feasible yields in the range of 0.38-0.41 g/g, alcohol concentrations of 7.6-8.2% v/v, fermentation time of 90-115 h, and conversion of 92-96%. DC material supported kefir fermented 11-fold more rapidly than free cells and 9-fold more rapidly in comparison to kissiris supported kefir. The main volatile byproducts such as amyl alcohols (mixture of 2-methyl-1-butanol and 3-methyl-1-butanol), ethanal, and ethyl acetate were formed in all sugar fermentation products. The formation of 65-110 ppm of ethyl acetate is as high and even higher than that obtained with traditional wine yeasts. The increase of the initial concentration of sugar in the fermentation media resulted in an increase in contents of volatiles. The fine aroma that was obtained in the product of fructose could be attributed to the high percentage of ethyl acetate on total volatiles. The efficiency of DC material supported kefir was the same for the fermentations of individual sugars or a mixture of fructose, sucrose, and glucose. When whey with raisin extracts was fermented, lower yields were obtained but the aroma of the product was even better.     

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.     

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.