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.     

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.     

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.     

Kinetic Model for the Formation of Acrylamide during the Finish-Frying of Commercial French Fries

Acrylamide is formed from reducing sugars and asparagine during the preparation of French fries. The commercial preparation of French fries is a multistage process involving the preparation of frozen, par-fried potato strips for distribution to catering outlets, where they are finish-fried. The initial blanching, treatment in glucose solution, and par-frying steps are crucial because they determine the levels of precursors present at the beginning of the finish-frying process. To minimize the quantities of acrylamide in cooked fries, it is important to understand the impact of each stage on the formation of acrylamide. Acrylamide, amino acids, sugars, moisture, fat, and color were monitored at time intervals during the frying of potato strips that had been dipped in various concentrations of glucose and fructose during a typical pretreatment. A mathematical model based on the fundamental chemical reaction pathways of the finish-frying was developed, incorporating moisture and temperature gradients in the fries. This showed the contribution of both glucose and fructose to the generation of acrylamide and accurately predicted the acrylamide content of the final fries.     

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.     

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.     

Effectiveness of methods for reducing acrylamide in bakery products

Pilot-scale bread, biscuit, and cracker doughs have been baked to assess how well recipe changes could reduce acrylamide in commercial bakery products. Removing ammonium-based raising agents was beneficial in biscuits. In doughs, long yeast fermentations were an effective way of reducing asparagine levels and hence acrylamide. At moderate fermentation times fructose levels increased, but the yeast later absorbed this, so the net effect on acrylamide was beneficial. Metal ions such as calcium reduced acrylamide when added as the carbonate or chloride. Hence, the fortification of flour with calcium carbonate, over and above its natural mineral content, has an additional benefit. However, some other possible methods of adding calcium to bakery doughs, for example, via the permitted preservative calcium propionate, were not beneficial. Amino acid addition to doughs gave modest reductions in acrylamide. Lowering the dough pH reduced acrylamide, but at the expense of higher levels of other process contaminants such as 3-monochloropropane-1,2-diol (3-MCPD).     

Impact of harvest year on amino acids and sugars in potatoes and effect on acrylamide formation during frying

Acrylamide is formed via the Maillard reaction between reducing sugars and asparagine in a number of carbohydrate-rich foods during heat treatment. High acrylamide levels have been found in potato products processed at high temperatures. To examine the impact of harvest year, information on weather conditions during growth, that is, temperature, precipitation, and light, was collected, together with analytical data on the concentrations of free amino acids and sugars in five potato clones and acrylamide contents in potato chips (commonly known as crisps in Europe). The study was conducted for 3 years (2004-2006). The contents of acrylamide precursors differed between the clones and the three harvest years; the levels of glucose were up to 4.2 times higher in 2006 than in 2004 and 2005, and the levels of fructose were 5.6 times higher, whereas the levels of asparagine varied to different extents. The high levels of sugars in 2006 were probably due to the extreme weather conditions during the growing season, and this was also reflected in acrylamide content that was approximately twice as high as in preceding years. The results indicate that acrylamide formation is dependent not only on the content and relative amounts of sugars and amino acids but also on other factors, for example, the food matrix, which may influence the availability of the reactants to participate in the Maillard reaction.     

Fermentation Reduces Free Asparagine in Dough and Acrylamide Content in Bread

Free asparagine is an important precursor for acrylamide in cereal products. The content of free asparagine was determined in 11 milling fractions from wheat and rye. Whole grain wheat flour contained 0.5 g/kg and whole grain rye flour 1.1 g/kg. The lowest content was found in sifted wheat flour (0.2 g/kg). Wheat germ had the highest content (4.9 g/kg). Fermentation (baker's yeast or baker's yeast and sourdough) of doughs made with the different milling fractions was performed to investigate whether the content of free asparagine was reduced by this process. In general, most of the asparagine was utilized after 2 hr of fermentation with yeast. Sourdough fermentation, on the other hand, did not reduce the content of free asparagineas efficiently but had a strong negative impact on asparagine utilization by yeast. This indicates that this type of fermentation may result in breads with higher acrylamide content than in breads fermented with yeast only. The effect of fermentation time on acrylamide formation inyeast‐leavened bread was studied in a model system. Doughs (sifted wheat flour with whole grain wheat flour or rye bran) were fermented for a short (15+15 min) or a long time (180+180 min). Compared with short fermentation time, longer fermentation reduced acrylamide content in bread made with whole grain wheat 87%. For breads made with rye bran, the corresponding reduction was 77%. Hence, extensive fermentation with yeast may be one possible way to reduce acrylamide content in bread.     

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.     

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.     

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.     

Rye Flour Extraction Rate Affects Maillard Reaction Development,Antioxidant Activity,and Acrylamide Formation in Bread Crisps

This report shows the effect of rye flour extraction rate on Maillard reaction, antioxidant activity, and acrylamide formation during toasting of rye bread crisps. Four rye flours with extraction rates of 70, 85, 95, and 100% were tested. Maillard reaction development was studied by measuring browning development, hydroxymethylfurfural (HMF), and glucosilisomaltol (GIM) formation, as well as antioxidant activity. Results showed that HMF and GIM concentrations in toasted bread crisps were higher as the flour extraction rate increases. Antioxidant activity increased during toasting as a consequence of antioxidant Maillard reaction product formation. Acrylamide concentration was clearly affected by free asparagine content of flour, while no effect of dietary fiber and natural antioxidant content of flours had an effect on acrylamide formation. Overall data suggest that the rate of Maillard reaction is higher in whole flours because of their higher free amino acid and protein content.     

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.     

The role of creatine in the generation of N-methylacrylamide: a new toxicant in cooked meat

Investigations of different sources of acrylamide formation in model systems consisting of amino acids and sugars have indicated the presence of two pathways of acrylamide generation; the main pathway specifically involves asparagine to directly produce acrylamide after a sugar-assisted decarboxylation step, and the second, nonspecific pathway involves the initial formation of acrylic acid from different sources and its subsequent interaction with ammonia and/or amines to produce acrylamide or its N-alkylated derivatives. Aspartic acid, beta-alanine, and carnosine were found to follow the acrylic acid pathway. Labeling studies using [(13)C-4]aspartic acid have confirmed the occurrence in this amino acid of a previously proposed sugar-assisted decarboxylation mechanism identified in the asparagine/glucose model system. In addition, creatine was found to be a good source of methylamine in model systems and was responsible for the formation of N-methylacrylamide through the acrylic acid pathway. Labeling studies using creatine (methyl-d(3)) and (15)NH(4)Cl have indicated that both the nitrogen and the methyl groups of methylamine had originated from creatine. Furthermore, analysis of cooked meat samples has also confirmed the formation of N-methylacrylamide during cooking.     

Acrylamide and pyrazine formation in model systems containing asparagine

The effect of different sugars and glyoxal on the formation of acrylamide in low-moisture starch-based model systems was studied, and kinetic data were obtained. Glucose was more effective than fructose, tagatose, or maltose in acrylamide formation, whereas the importance of glyoxal as a key sugar fragmentation intermediate was confirmed. Glyoxal formation was greater in model systems containing asparagine and glucose rather than fructose. A solid phase microextraction GC-MS method was employed to determine quantitatively the formation of pyrazines in model reaction systems. Substituted pyrazine formation was more evident in model systems containing fructose; however, the unsubstituted homologue, which was the only pyrazine identified in the headspace of glyoxal-asparagine systems, was formed at higher yields when aldoses were used as the reducing sugar. Highly significant correlations were obtained for the relationship between pyrazine and acrylamide formation. The importance of the tautomerization of the asparagine-carbonyl decarboxylated Schiff base in the relative yields of pyrazines and acrylamide is discussed.     

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.     

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.     

Sucrose metabolism and exopolysaccharide production in wheat and rye sourdoughs by Lactobacillus sanfranciscensis.

The exopolysaccharide (EPS) produced from sucrose by Lactobacillus sanfranciscensis LTH2590 is predominantly composed of fructose. EPS production during sourdough fermentation has the potential to affect rheological properties of the dough as well as the volume, texture, and keepability of bread. Its in situ production by L. sanfranciscensis LTH2590 was demonstrated during sourdough fermentation after the hydrolysis of water soluble polysaccharides. In wheat and rye doughs with sucrose addition the concentration of fructose in the hydrolysate of polysaccharides was significantly higher than that in the hydrolysate of control doughs or doughs without sucrose addition. EPS production by L. sanfranciscensis in wheat doughs was confirmed by the determination of delta (13)C values of water soluble polysaccharides after the addition of naturally labeled sucrose, originating from C(3)- and C(4)-plants. In rye doughs, evidence for EPS production with the isotope technique could be demonstrated only by the determination of delta (13)C values of fructose from water soluble polysaccharides. In addition to EPS formation from sucrose, sucrose hydrolysis by L. sanfranciscensis in wheat and rye sourdoughs resulted in an increase of mannitol and acetate concentrations and in accumulation of glucose. It was furthermore observed that flour arabinoxylans were solublized during the fermentation.     

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.