Free Asparagine Research Articles

Influence of Asparaginase on Acrylamide Content, Color, and Texture in Oat, Corn, and Rice Cookies.

The safety of cereal-based baked goods can be compromised by acrylamide, a processing contaminant and class 2A carcinogen. One method to prevent acrylamide formation is by converting asparagine to aspartic acid using asparaginases. Four different asparaginases were tested using two dough incubation temperatures and dosages for oat, corn, and rice cookies. To evaluate the impact of asparaginases on product quality, color and texture were measured. Acrylamide was reduced by up to 97, 95, and 92% for oat, corn, and rice cookies, respectively, compared to the control. Asparaginase treatment resulted in minor changes in color and texture. There was a strong correlation between acrylamide concentrations in cookies and the free asparagine content of the flour. By minimizing the formation of acrylamide while maintaining product quality, the use of asparaginases offers a promising approach to enhancing food safety standards and protecting public health, potentially influencing regulatory guidelines and consumer preferences.

Carrot strips of various origins: Impact on acrylamide formation in baked goods

The study explored the effects of varied qualities of carrot strips (Q1, Q2) with similar market specification, water addition (56.5–67.8 g/100 g flour) and carrot quantities (7.5–22.5 g/100 g flour) on AA concentrations in wheat bread crumb and crust and baking quality. Analyses included reducing sugars (HPAEC–PAD) and free asparagine (HPLC) levels in doughs as well as acrylamide (HPLC-MS/MS) in crumbs and crusts and baking quality measures. Bread crusts with Q1 reached AA levels of 142 ± 13 μg/kg and significant values in the crumbs (40.8 ± 6.7 μg/kg), whereas bread crusts with Q2 reached up to 274 ± 10 μg/kg and crumbs did not contain AA. With increasing dough water addition, a significant reduction in AA was visible in the case of carrots Q1 and a significant increase in crust AA for carrots Q2. Significant and positive correlation was observed between AA levels in crusts and the addition of carrot strips (rQ1 = 0.93, p < 0.001), whereas for the crust moisture and AA a significant and negative correlation was observed (rQ1 = −0.73, p < 0.01). The precursors in the dough were significantly influenced by the quantities and qualities of ingredients, while the AA formation depended on the quality of the carrot strips and the amount of water added.

Acrylamide formation in air-fryer roasted legumes as affected by legume species and roasting degree: the correlation of acrylamide with asparagine and free sugars.

Acrylamide is a well-recognized hazardous compound with known carcinogenic, genotoxic, neurotoxic, and reproductive toxic effects. This research aimed to investigate how different legume species and roasting durations influence acrylamide formation during air-fryer roasting. The study also examined the relationship between acrylamide formation and the levels of free asparagine and free sugars in different bean species. Asparagine content varies substantially across different bean species. Sucrose was the predominant sugar across all bean species, with smaller amounts of galactose and glucose. Air-fryer-roasted Wandu kong (garden pea) showed the highest acrylamide formation, followed by Ultari kong (kidney bean) and Heoktae (black soybean), in that order. Beans roasted for longer periods in an air fryer contained significantly higher levels of acrylamide. This study revealed a strong positive correlation between acrylamide formation and the level of free asparagine in the beans, highlighting the risks associated with certain legume species and air-fryer roasting durations.

Asparaginase treatment to mitigate acrylamide formation in wheat and rye cookies

The Maillard reaction between asparagine and a carbonyl source can lead to acrylamide formation, which is classified as "probably carcinogenic to humans". This study aimed to investigate the impact of varying amounts, incubation times, and temperatures of asparaginases on reducing acrylamide in wheat and rye cookies while preserving sensory attributes. To determine if different asparaginases reduce acrylamide without negative effects on cookie quality, acrylamide was quantified using ELISA, and color, texture and sensory attributes were assessed. Adding asparaginases resulted in a reduction of acrylamide by up to 85%. Incubating cookie dough for 10 or 30 min at 60 °C or 90 °C did not impact acrylamide formation. Acrylamide concentrations were higher by an average of 78 μg/kg for an incubation temperature of 90 °C compared to 60 °C. Rye cookies exhibited higher acrylamide concentrations by 490 μg/kg compared to wheat due to higher free asparagine in rye flour. Asparaginases showed minimal effects on cookie color and texture, with no changes in sensory evaluation. Acrylamide can be reduced using asparaginases, providing a simple and effective means of ensuring safe food for consumers.

Reducing Acrylamide Formation Potential by Targeting Free Asparagine Accumulation in Seeds.

Acrylamide is a probable carcinogen in humans and is formed when reducing sugars react with free asparagine (Asn) during thermal processing of food. Although breeding for low reducing sugars worked well in potatoes, it is less successful in cereals. However, reducing free Asn in cereals has great potential for reducing acrylamide formation, despite the role that Asn plays in nitrogen transport and amino acid biosynthesis. In this perspective, we summarize the efforts aimed at reducing free Asn in cereal grains and discuss the potentials and challenges associated with targeting this essential amino acid, especially in a seed-specific manner.

Different wheat loci are associated to heritable free asparagine content in grain grown under different water and nitrogen availability.

Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential. The amount of free asparagine in grain of a wheat genotype determines its potential to form harmful acrylamide in derivative food products. Here, we explored the variation in the free asparagine, aspartate, glutamine and glutamate contents of 485 accessions reflecting wheat worldwide diversity to define the genetic architecture governing the accumulation of these amino acids in grain. Accessions were grown under high and low nitrogen availability and in water-deficient and well-watered conditions, and plant and grain phenotypes were measured. Free amino acid contents of grain varied from 0.01 to 1.02 mg g-1 among genotypes in a highly heritable way that did not correlate strongly with grain yield, protein content, specific weight, thousand-kernel weight or heading date. Mean free asparagine content was 4% higher under high nitrogen and 3% higher in water-deficient conditions. After genotyping the accessions, single-locus and multi-locus genome-wide association study models were used to identify several QTLs for free asparagine content located on nine chromosomes. Each QTL was associated with a single amino acid and growing environment, and none of the QTLs colocalised with genes known to be involved in the corresponding amino acid metabolism. This suggests that free asparagine content is controlled by several loci with minor effects interacting with the environment. We conclude that breeding for reduced asparagine content is feasible, but should be firmly based on multi-environment field trials. Different wheat QTLs were associated to the free asparagine content of grain grown in four different conditions. Environmental effects are a key factor when selecting for low acrylamide-forming potential.

Mitigation of acrylamide formation in wood oven baked pizza base using wholemeal and refined wheat flour with low free asparagine content: considerations on fibre intake and starch digestibility.

In wheat-derived bakery products, the quantity of free asparagine (fAsn) has been identified as a key factor in acrylamide (AA) formation. Based on this assumption, four varieties of common wheat (Triticum aestivum L.), Stromboli, Montecarlo, Sothys and Cosmic, selected for their different fAsn content inside the grain, were studied to evaluate their potential in the production of pizza with reduced AA levels. To this purpose, wholemeal and refined flours were obtained from each variety. The fAsn content ranged from 0.25 to 3.30 mmol kg-1, with higher values for wholemeal flours which also showed greater amount of ash, fibre and damaged starch than refined wheat flours. All types of flours were separately used to produce wood oven baked pizza base, according to the Traditional Speciality Guaranteed EU Regulation (97/2010). AA reduction in the range 47-68% was found for all the selected wheat cultivars, compared with a commercial flour, with significantly lower values registered when refined flour was used. Moreover, refined leavened dough samples showed decreased levels of fAsn and reducing sugars due to the fermentation activity of yeasts. Furthermore, it was confirmed that pizza made with wholemeal flours exhibited lower rapidly digestible starch (RDS) and rapidly available glucose (RAG) values compared to that prepared with the refined flour. This study clearly shows that a reduced asparagine content in wheat flour is a key factor in the mitigation of AA formation in pizza base. Unfortunately, at the same time, it is highlighted how it is necessary to sacrifice the beneficial effects of fibre intake, such as lowering the glycaemic index, in order to reduce AA. © 2024 Society of Chemical Industry.

Factors Influencing the Accumulation of Free Asparagine in Wheat Grain and the Acrylamide Formation in Bread

Asparagine is one of the precursors of acrylamide that can form during bread production. The aim of this work was to determine the effect of genotype, environment, sulfur fertilization, and the interaction of those factors on the asparagine content, technological value of wheat, and acrylamide level in bread. The research material consisted of five wheat cultivars grown in two locations in Poland with nitrogen fertilization of 110 kg ha−1 and sulfur fertilization of 30 kg ha−1. The standard ISO method for analyzing the milling and baking properties of wheat was used. The UHPLC-MS/MS method for analyzing the amino acids and the GC/MS method for acrylamide in bread were implemented. The analysis of variance results indicated that the location influenced the total variance in the measured asparagine content and quality of wheat the most, followed by the cultivar and then by the interaction between the environment and cultivar. Sulfur fertilization had no significant effect on the asparagine content, but slightly lowered the gluten quality and loaf volume of bread. However, sulfur fertilization in connection with the cultivar characterized by low starch damage had a positive effect on lowering the acrylamide in bread. Asparagine content in wheat and acrylamide in bread varies mostly depending on cultivar and environment.

Low asparagine wheat: Europe's first field trial of genome edited wheat amid rapidly changing regulations on acrylamide in food and genome editing of crops.

We review the undertaking of a field trial of low asparagine wheat lines in which the asparagine synthetase gene, TaASN2, has been knocked out using CRISPR/Cas9. The field trial was undertaken in 2021-2022 and represented the first field release of genome edited wheat in Europe. The year of the field trial and the period since have seen rapid changes in the regulations covering both the field release and commercialisation of genome edited crops in the UK. These historic developments are reviewed in detail. Free asparagine is the precursor for acrylamide formation during high-temperature cooking and processing of grains, tubers, storage roots, beans and other crop products. Consequently, work on reducing the free asparagine concentration of wheat and other cereal grains, as well as the tubers, beans and storage roots of other crops, is driven by the need for food businesses to comply with current and potential future regulations on acrylamide content of foods. The topic illustrates how strategic and applied crop research is driven by regulations and also needs a supportive regulatory environment in which to thrive.

Characterizing the formation of process contaminants during coffee roasting by multivariate statistical analysis.

Coffee is a relevant source of dietary exposure for neoformed furan, alkyl furans and acrylamide. In this study, different statistical methods (hierarchical cluster analysis, correlation analysis, partial least squares regression analysis) were used for characterizing the formation of these process contaminants in green coffee beans roasted under the same standardized conditions. The results displayed a strong correlation between sucrose levels and furans in relation to the other sugars analyzed, while acrylamide formation was strongly related to the free asparagine. The data suggest that a sufficiently large amino acid pool in green coffee favors Maillard-induced acrylamide formation from asparagine, while reactions amongst the carbonyl-containing sugar fragmentation products leading to furan formation are suppressed. If the pool of free amino acids is small, it is depleted faster during roasting, thus favoring the formation of furans by caramelization, basically a sugar degradation process in which reactive carbonyl substances are generated and react together.

Differential gene expression provides leads to environmentally regulated soybean seed protein content.

Soybean is an important global source of plant-based protein. A persistent trend has been observed over the past two decades that soybeans grown in western Canada have lower seed protein content than soybeans grown in eastern Canada. In this study, 10 soybean genotypes ranging in average seed protein content were grown in an eastern location (control) and three western locations (experimental) in Canada. Seed protein and oil contents were measured for all lines in each location. RNA-sequencing and differential gene expression analysis were used to identify differentially expressed genes that may account for relatively low protein content in western-grown soybeans. Differentially expressed genes were enriched for ontologies and pathways that included amino acid biosynthesis, circadian rhythm, starch metabolism, and lipid biosynthesis. Gene ontology, pathway mapping, and quantitative trait locus (QTL) mapping collectively provide a close inspection of mechanisms influencing nitrogen assimilation and amino acid biosynthesis between soybeans grown in the East and West. It was found that western-grown soybeans had persistent upregulation of asparaginase (an asparagine hydrolase) and persistent downregulation of asparagine synthetase across 30 individual differential expression datasets. This specific difference in asparagine metabolism between growing environments is almost certainly related to the observed differences in seed protein content because of the positive correlation between seed protein content at maturity and free asparagine in the developing seed. These results provided pointed information on seed protein-related genes influenced by environment. This information is valuable for breeding programs and genetic engineering of geographically optimized soybeans.

Comparison of a rapid enzymatic assay with column chromatography and nuclear magnetic resonance methods for free asparagine detection and quantification in wheat

A rapid, reliable, and user-friendly method that does not require complex sample preparation and sophisticated instrumentation will fulfill an emerging demand for free asparagine (ASN) analysis to ascertain acrylamide-related food safety issues in baked cereal products. This study validated a new method for wheat free asparagine analysis by comparing a rapid enzymatic test with two established analytical methods: UPLC and 1H-NMR using asparagine standards and whole-wheat flours with a range of free asparagine concentrations. For method validation, various quality parameters were assessed, including accuracy, precision, and sensitivity. Experimental and time costs associated with the analyses were estimated. The rapid test method had consistent accuracy, excellent precision of 1.0 or less (CV%), good sensitivity (0.0389 AU L mg−1) of free asparagine signal detection, and the least cost for experimental materials and instrument usage. A strong correlation (r = 0.997) between the results generated by the rapid enzymatic test and the UPLC method for free ASN quantification in whole-wheat flour was obtained. Overall, the rapid test method is recommended for ASN analysis in simple laboratory environments or in industrial applications.

Heat-Introduced Formation of Acrylamide in Table Olives: Analysis of Acrylamide, Free Asparagine, and 3-Aminopropionamide.

Acrylamide was detected in considerable amounts in black table olives. In this study, besides black, also green and naturally black table olives were investigated for their acrylamide, free asparagine, and 3-aminopropionamide contents before and after heat treatment. Acrylamide amount was 208-773 μg/kg in black table olives and did not change due to heat treatment. In green and naturally black table olives acrylamide was ≤24 μg/kg before heat treatment and rose to 1200 μg/kg afterward. Asparagine content was 0.35-35 mg/kg in all samples before heat treatment and after heat treatment with no considerable change in the range. 3-Aminopropionamide showed amounts of ≤56 μg/kg in the unheated samples and increased up to 131 μg/kg due to heat impact. However, quantified asparagine and 3-aminopropionamide amounts were insufficient in almost all samples to explain the acrylamide quantities formed due to heat treatment based on the formation via the Maillard reaction.

Reducing the Risk of Acrylamide and Other Processing Contaminant Formation in Wheat Products.

Wheat is a staple crop, consumed worldwide as a major source of starch and protein. Global intake of wheat has increased in recent years, and overall, wheat is considered to be a healthy food, particularly when products are made from whole grains. However, wheat is almost invariably processed before it is consumed, usually via baking and/or toasting, and this can lead to the formation of toxic processing contaminants, including acrylamide, 5-hydroxymethylfurfural (HMF) and polycyclic aromatic hydrocarbons (PAHs). Acrylamide is principally formed from free (soluble, non-protein) asparagine and reducing sugars (glucose, fructose and maltose) within the Maillard reaction and is classified as a Group 2A carcinogen (probably carcinogenic to humans). It also has neurotoxic and developmental effects at high doses. HMF is also generated within the Maillard reaction but can also be formed via the dehydration of fructose or caramelisation. It is frequently found in bread, biscuits, cookies, and cakes. Its molecular structure points to genotoxicity and carcinogenic risks. PAHs are a large class of chemical compounds, many of which are genotoxic, mutagenic, teratogenic and carcinogenic. They are mostly formed during frying, baking and grilling due to incomplete combustion of organic matter. Production of these processing contaminants can be reduced with changes in recipe and processing parameters, along with effective quality control measures. However, in the case of acrylamide and HMF, their formation is also highly dependent on the concentrations of precursors in the grain. Here, we review the synthesis of these contaminants, factors impacting their production and the mitigation measures that can be taken to reduce their formation in wheat products, focusing on the role of genetics and agronomy. We also review the risk management measures adopted by food safety authorities around the world.

Accounting for environmental variation in the free asparagine content of wheat grain

Accounting for environmental variation in the free asparagine content of wheat grain

Genetic control of grain amino acid composition in a UK soft wheat mapping population.

Wheat (Triticum aestivum L.) is a major source of nutrients for populations across the globe, but the amino acid composition of wheat grain does not provide optimal nutrition. The nutritional value of wheat grain is limited by low concentrations of lysine (the most limiting essential amino acid) and high concentrations of free asparagine (precursor to the processing contaminant acrylamide). There are currently few available solutions for asparagine reduction and lysine biofortification through breeding. In this study, we investigated the genetic architecture controlling grain free amino acid composition and its relationship to other traits in a Robigus × Claire doubled haploid population. Multivariate analysis of amino acids and other traits showed that the two groups are largely independent of one another, with the largest effect on amino acids being from the environment. Linkage analysis of the population allowed identification of quantitative trait loci (QTL) controlling free amino acids and other traits, and this was compared against genomic prediction methods. Following identification of a QTL controlling free lysine content, wheat pangenome resources facilitated analysis of candidate genes in this region of the genome. These findings can be used to select appropriate strategies for lysine biofortification and free asparagine reduction in wheat breeding programs.

Exploring Variability of Free Asparagine Content in the Grain of Bread Wheat (Triticum aestivum L.) Varieties Cultivated in Italy to Reduce Acrylamide-Forming Potential.

Acrylamide, a suspected human carcinogen, is generated during food processing at high temperatures in the Maillard reaction, which involves reducing sugars and free asparagine. In wheat derivatives, free asparagine represents a key factor in acrylamide formation. Free asparagine levels in the grain of different wheat genotypes has been investigated in recent studies, but little is known about elite varieties that are cultivated in Italy. Here, we analysed the accumulation of free asparagine in a total of 54 bread wheat cultivars that are relevant for the Italian market. Six field trials in three Italian locations over two years were considered. Wholemeal flours obtained from harvested seeds were analysed using an enzymatic method. Free asparagine content ranged from 0.99 to 2.82 mmol/kg dry matter in the first year, and from 0.55 to 2.84 mmol/kg dry matter in the second year. Considering the 18 genotypes that were present in all the field trials, we evaluated possible environment and genetic influences for this trait. Some cultivars seemed to be highly affected by environment, whereas others showed a relative stability in free asparagine content across years and locations. Finally, we identified two varieties showing the highest free asparagine levels in our analysis, representing potential useful materials for genotype x environment interaction studies. Two other varieties, which were characterized by low amounts of free asparagine in the considered samples, may be useful for the food industry and for future breeding programs aimed to reduce acrylamide-forming potential in bread wheat.

Reducing Dietary Acrylamide Exposure from Wheat Products through Crop Management and Imaging.

The nutritional safety of wheat-based food products is compromised by the presence of the processing contaminant acrylamide. Reduction of the key acrylamide precursor, free (soluble, non-protein) asparagine, in wheat grain can be achieved through crop management strategies, but such strategies have not been fully developed. We ran two field trials with 12 soft (biscuit) wheat varieties and different nitrogen, sulfur, potassium, and phosphorus fertilizer combinations. Our results indicated that a nitrogen-to-sulfur ratio of 10:1 kg/ha was sufficient to prevent large increases in free asparagine, whereas withholding potassium or phosphorus alone did not cause increases in free asparagine when sulfur was applied. Multispectral measurements of plants in the field were able to predict the free asparagine content of grain with an accuracy of 71%, while a combination of multispectral, fluorescence, and morphological measurements of seeds could distinguish high free asparagine grain from low free asparagine grain with an accuracy of 86%. The acrylamide content of biscuits correlated strongly with free asparagine content and with color measurements, indicating that agronomic strategies to decrease free asparagine would be effective and that quality control checks based on product color could eliminate high acrylamide biscuit products.

Asparagine and dough quality: Gluten strength relationships in hard red spring wheat

AbstractBackground and objectivesThe presence of acrylamide, a probable carcinogen, is a global concern for the baking industry. The reduction of free asparagine in wheat is an effective strategy to mitigate acrylamide formation. However, field‐based strategies for this purpose also affect gluten strength and bread quality. Therefore, we investigated the relationship between wheat free asparagine concentration, gluten strength and whole wheat bread quality for an extensive set of hard red spring wheat samples.FindingsGluten strength parameters negatively correlated to wheat free asparagine concentration and showed strong inverse correlations to wheat free asparagine concentration per unit mass of protein. Reducing free asparagine concentration in the wheat did not affect the quality of the bread.ConclusionsIn efforts to deliver wheat grains with a low acrylamide‐formation potential, wheat producers can apply strategies, for example, variety selection, that will reduce free asparagine without worries that gluten strength and bread quality will be impaired.Significance and noveltySafety concerns regarding free asparagine in wheat have elicited much attention from cereal scientists and the breadmaking industry. However, the effects of reducing free asparagine on gluten strength have not been systematically studied in the literature. This comprehensive study shows that there is an inverse relationship between gluten strength and wheat free asparagine concentration.

Asparagine and dough quality: Gluten strength factors in hard red spring wheat

AbstractBackground and ObjectivesSafety aspects regarding free asparagine concentration in wheat arise because of its acrylamide formation potential in baked wheat products. Free asparagine concentration in wheat is affected by environment, genotype, and nitrogen and sulfur fertilization, which also affect wheat protein content and composition. Changing protein content and composition affects gluten strength, altering product quality. This study investigated how gluten strength was affected by environment, genotype, and fertilization treatments grown under commercially relevant conditions.FindingsEnvironment predominated in affecting dough extensibility and genotype primarily affected resistance to extension, with minimal effects from fertilization treatments. Differences in extensigraph parameters at two resting times segregated genotypes according to gluten strength.ConclusionsFactors affecting free asparagine accumulation in wheat also affect gluten strength. Genotype and environment require primary attention since they play an important role in both wheat safety and quality.Significance and NoveltyThe lack of studies investigating the impact of factors affecting free asparagine concentration on the gluten strength of wheat has implications for the global wheat market. From a novel perspective on dough extensibility and resistance to extension, the effects of environment, genotype, and fertilization on the gluten strength of commercially important wheat varieties are considered in a food safety context.