Free Asparagine Concentration 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.

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.

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.

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.

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.

Diurnal accumulation of K+-dependent L-asparaginase in leaf of common bean (Phaseolus vulgaris L.)

L-Asparaginase (EC 3.5.1.1) activity has been previously reported to fluctuate with the photoperiod in young pea leaves, with higher activity in the light. The present research sought to investigate this phenomenon in developing leaves of common bean (Phaseolus vulgaris L.). There are two genes coding for K+-dependent asparaginase in this species. Expression of PvASPG1 predominates over PvASPG2 in all tissues. The catalytic efficiency of recombinant PvASPG2 was approximately 2-fold lower than that of PvASPG1. Polyclonal antibodies were raised against a specific peptide present in PvASPG1 to use in immunoblotting. In developing seed, asparaginase protein levels in the seed coat stayed constant, whereas levels in cotyledon were lower and progressively declined. In young leaf, asparagine protein levels showed diurnal variation, increasing at the end of the dark period and slowly decreasing during the light period. This was paralleled by changes in activity levels in leaf extracts. These changes accompanied a transient increase in free asparagine concentration at the beginning of the light period. The present results demonstrated that K+-dependent asparaginase activity reaches a maximum level at the transition from dark to light, anticipating dawn, in young leaves of common bean.

Induced mutations in ASPARAGINE SYNTHETASE‐A2 reduce free asparagine concentration in the wheat grain

AbstractAcrylamide is a neurotoxin and probable carcinogen formed as a processing contaminant during baking and production of different foodstuffs, including bread products. The amino acid asparagine is the limiting substrate in the Maillard reaction that produces acrylamide, so developing wheat (Triticum aestivum L.) cultivars with low free asparagine concentrations in the grain is a promising approach to reduce dietary acrylamide exposure. A candidate gene approach was used to identify chemically induced genetic variation in ASPARAGINE SYNTHETASE 2 (ASN2) genes that exhibit a grain‐specific expression profile. In field trials, durum and common wheat lines carrying asn‐a2 null alleles exhibited reductions in free asparagine concentration in their grains of between 9 and 34% compared with wildtype sister lines. These plants showed no significant differences in spikelet number, grain size and weight, germination or baking quality traits. These nontransgenic variants can be deployed without regulatory oversight in elite wheat germplasm to reduce acrylamide‐forming potential with no negative effects on quality or agronomic performance.

Acrylamide in Corn-Based Thermally Processed Foods: A Review

Widely consumed thermally processed corn-based foods can have a great contribution to acrylamide dietary intake, thus bearing a high public health risk and requiring attention and application of strategies for its reduction. This paper reviews the literature on the acrylamide content of corn-based food products present in the market around the world. The potential of corn for acrylamide formation due to its content of free asparagine and reducing sugars is described. Human exposure to acrylamide from corn-based foods is also discussed. The content of acrylamide in corn/tortilla chips, popcorn, and corn flakes, as widely consumed products all over the world, is reported in the literature to be between 5 and 6360 μg/kg, between <LOD and 2220 μg/kg and between <LOD and 1186 μg/kg, respectively. Although these products are important acrylamide sources in the common diet of all age populations, higher intake values occurred among younger generations.

Understanding the Relationships between Free Asparagine in Grain and Other Traits to Breed Low-Asparagine Wheat.

Since the discovery of acrylamide in food, and the identification of free asparagine as the key determinant of acrylamide concentration in wheat products, our understanding of how grain asparagine content is regulated has improved greatly. However, the targeted reduction in grain asparagine content has not been widely implemented in breeding programmes so far. Here we summarise how free asparagine concentration relates to other quality and agronomic traits and show that these relationships are unlikely to pose major issues for the breeding of low-asparagine wheat. We also outline the strategies that are possible for the breeding of low-asparagine wheat, using both natural and induced variation.

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality

BackgroundUnderstanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties.ResultsNatural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency.ConclusionsSelecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

SummaryFree asparagine is the precursor for acrylamide, which forms during the baking, toasting and high‐temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4‐gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

Effects of growing environment, genotype, and commercial fertilization levels on free asparagine concentration in Western Canadian wheat

AbstractBackground and objectivesFree asparagine (ASN) is the precursor to the formation of acrylamide, which is a probable neurotoxic and carcinogenic compound formed during high‐temperature (&gt;120°C) processing of starchy foods, such as cereal‐based products. Controlling the acrylamide concentration of cereal‐based products, for example, bread, within the allowable levels established by the European Commission, is necessary for food safety purposes. One effective measure recommended by the European Commission to mitigate acrylamide in cereal‐based products is to reduce free ASN levels in raw ingredients, for example, wheat. Therefore, knowledge of free ASN levels in Canadian commercial wheat and the strategies to reduce its formation is necessary for the Canadian wheat industry to secure global market access for Canadian wheat. The objective of this study was to understand the effects of genotype, growing environment, and fertilization on free ASN concentration of whole‐wheat flour from Western Canadian wheat varieties.FindingsThe free ASN concentration of whole‐wheat flours in this study ranged from 281 to 1,014 µg/g (dry basis). The variation in free ASN levels in wheat was mainly influenced by the growing environment (44%), followed by genotype (31%) and the interaction between genotype and environment (18%). Although being significant, the effects of fertilization and interactions involving fertilization on free ASN concentration in whole‐wheat flour were minimal (0.7%–2.2%).ConclusionGrowing wheat genotypes in suitable environments, along with the selection of wheat genotypes with lower potential for free ASN formation, are the most effective strategies to control free ASN levels in Canadian wheat.Significance and noveltyLimited knowledge in regard to free ASN concentration and the strategies to reduce its formation in Canadian wheat may have a serious impact on Canadian wheat access to global markets, especially the European market. This study provides an understanding of the effects of growing environment, genotype, and fertilization on free ASN concentration of select Western Canadian commercial wheat varieties.

Roasted Rye as a Coffee Substitute: Methods for Reducing Acrylamide.

Acrylamide is assumed to be a potential carcinogen, and reference values have therefore been implemented in EU legislation. Thus, the food industry needs to reduce the acrylamide content in consumer products to the lowest possible value. In this study, roasted rye was evaluated for its suitability as a coffee substitution product with respect to its acrylamide content. The influence of process modifiers, free asparagine content, storage, and rye type on the final content of acrylamide was investigated. Changes in carbohydrate composition and brightness caused by the roasting process were described. Sample analysis was conducted via GC-MS and HPLC-CAD. Existing methods were adapted to roasted rye as the sample matrix. CaCl2 and asparaginase treatment as well as pH adjustments prior to roasting did not prove to reduce the acrylamide content. A significantly (* p < 0.027) lower free asparagine content in the raw material resulted in a lower formation of acrylamide in the final product. The acrylamide content significantly decreased (**** p < 0.0001) after 3 (1100 ± 18 µg kg−1) and 6 (490 ± 7 µg kg−1) months of long-term storage. Only samples stored for 6 months (490 ± 7 µg kg−1) met the EU acrylamide content requirements (<500 µg kg−1) for grain-based coffee substitution products.

Processing strategies to decrease acrylamide formation, reducing sugars and free asparagine content in potato chips from three commercial cultivars

Potato chips are among the highest contributors to the dietary intake of acrylamide, a potent neurotoxin and likely carcinogen in heat-processed foods. The present study aimed to determine the effects of frying conditions and additive treatments on reducing sugars, asparagine levels, and acrylamide formation in fried potato chips. Three commercially important chipping cultivars (Atlantic, Snowden, and Vigor) were tested using different frying times (3, 5, and 7 min) and temperatures (160, 170, 180, and 190 °C). Acrylamide formation in chips was found to be cultivar-specific and increased with increasing frying time and temperature. The acrylamide levels were significantly lower in chips processed at high temperatures for short frying times than in those processed at low temperatures for long frying times. In all cultivars, acrylamide synthesis was accompanied by significant decreases in the levels of reducing sugars and asparagine. The cultivar exhibiting the lowest acrylamide levels in the processed potato chips was Snowden, while the conditions most conducive to acrylamide formation in all cultivars were frying at 190 °C for 7 min. Using a 7 min frying time, decreasing the frying temperature from 190 to 160 °C mitigated the acrylamide formation in potato chips processed from Atlantic, Snowden and Vigor by 84, 67, and 78%, respectively. We also examined the effects of additives, such as 1% acetic acid, 1% citric acid, 0.1 M sodium chloride, 0.1 M calcium chloride, 0.5% ascorbic acid, or 1 M l-glycine, during the blanching of potato slices prior to frying. Blanching in distilled water led to the greatest decreases (19–59%) in acrylamide formation in all cultivars. Our findings provide the basis for new processing strategies to mitigate acrylamide formation and improve the quality of chips from these, and possibly other, potato cultivars.