Downloaded via BETHEL UNIV on April 3, 2019 at 13:23:17 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Chapter 3
Acrylamide: New European Risk Management Measures and Prospects for Reducing the Acrylamide-Forming Potential of Wheat Nigel G. Halford* and Sarah Raffan Plant Sciences Department, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom *E-mail:
[email protected] Acrylamide (C3H5NO) is a processing contaminant formed from free asparagine and reducing sugars during high-temperature cooking and processing. It is a Group 2A carcinogen, and the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM Panel) has expressed concern for the potential tumor-inducing effects of dietary exposure. Potato, coffee, and cereal products are the major contributors to dietary acrylamide intake. The European Commission recently introduced strengthened risk management regulations for acrylamide in food, including compulsory mitigation measures and new benchmark levels. Measures adopted to reduce acrylamide formation in potato chips in Europe resulted in a 53% decrease from 2002 to 2011. However, since 2011 there has been a leveling off, suggesting that the easy gains have already been made. Acrylamide levels in chips are influenced by seasonal and geographical factors, making regulatory compliance more difficult. In cereals, acrylamide formation is determined by free asparagine concentration, and this differs substantially between varieties. We would support the inclusion of information on grain asparagine concentration in variety descriptions. However, crop management, including ensuring good disease control and sulfur sufficiency, is also important. A key enzyme in asparagine synthesis is asparagine synthetase. Wheat has four asparagine synthetase genes, TaASN1–4. Gene expression and biochemical data have
© 2019 American Chemical Society Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
identified TaASN2 as a prime target for genetic interventions to reduce wheat’s acrylamide-forming potential.
Introduction Acrylamide is a white, odorless, crystalline, water-soluble solid, with the chemical formula C3H5NO. In its monomeric form it is regarded as a hazardous chemical, affecting the nervous system, male reproduction, and development, as well as being carcinogenic in laboratory animals. It is readily absorbed through the skin and by inhalation and is one of the carcinogens in tobacco smoke. Acrylamide forms a polymer, polyacrylamide, that is not considered toxic and has a variety of industrial uses as a flocculant, binding agent, and thickener in wastewater and sewage treatment; in the production of paper, plastic products, grout, cement, pesticides, food packaging, dyes, and cosmetics; soil treatment; ore processing; and sugar manufacturing. It is, of course, also familiar to biochemists and molecular biologists due to its use in polyacrylamide gel electrophoresis (PAGE). Polyacrylamide may contain a small amount of soluble, monomeric acrylamide as an impurity, and since polyacrylamide is used in water treatment, monomeric acrylamide is a recognized potential water pollutant. The World Health Organization has set a guideline value for the presence of acrylamide in water of 0.5 µg/L, with the proviso that exposure should be reduced to as low a level as technically achievable. Fortunately, acrylamide is biodegraded by microorganisms and so does not persist in soil or lakes. The use of acrylamide in industrial processes means that there is a risk of occupational exposure and exposure to acrylamide as a pollutant. Acrylamide is metabolized to produce glycidamide (C3H5NO2), and it may be glycidamide rather than acrylamide itself that is responsible for the genotoxic and carcinogenic effects that result from exposure to acrylamide (1). Both acrylamide and glycidamide react with glutathione, and this represents the primary detoxification and excretion route, the glutathione adducts that are formed being converted in the liver to mercapturic acids, which are then excreted in urine (1). Both acrylamide and glycidamide also form adducts with proteins, notably hemoglobin in the blood, and the detection of acrylamide-hemoglobin adducts in blood tests is the favored method for obtaining quantitative measurements of acrylamide exposure (1). The adduct forms with the valine residues at the Ntermini of the globin chains, with the ratio of adducts to globin chains providing the measure of exposure. The adducts are detected using mass spectrometry after removal from the N-terminal end of the protein by a modification of the Edman degradation method (1). Toxicological studies with acrylamide have been conducted in a range of animal species, including rats, mice, guinea pigs, monkeys, cats, and dogs. The results of these studies were analyzed by the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM Panel) and summarized in the CONTAM Panel’s “Scientific Opinion on Acrylamide in Food” report of 2015 (1). The Panel concluded that high doses of acrylamide caused a range of effects on development and the nervous system. It also concluded 28 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
that both acrylamide and glycidamide had been shown to be genotoxic and carcinogenic, with tumors induced in multiple tissues in both male and female mice and rats. Early data on the effects of acrylamide in humans came from studies on the consequences of occupational exposure. In 1993, for example, Emma Bergmark and colleagues at the University of Washington in Seattle examined workers employed by an acrylamide-manufacturing factory in China (2). Several of the workers showed signs of peripheral neuropathy, and the effects correlated with the levels of acrylamide-hemoglobin adducts detectable in blood samples. In 1997, by then at the University of Stockholm, Bergmark reported the results of a study of laboratory personnel who were regularly using PAGE (3). Acrylamide-hemoglobin adducts were again detectable in blood samples collected from the subjects, with those using PAGE and smokers in the study showing significantly increased levels of adducts compared with controls. Bergmark noted that there were surprisingly high levels of adducts in the control group (i.e., non-smokers who did not use PAGE) and remarked “the origin of this background is not known (3).” Other studies on occupational exposure to acrylamide also reported a high level of background adducts in control groups. The explanation came in 2000 when a team led by Margareta Törnqvist at the University of Stockholm published the results of a study investigating whether cooked food could contain acrylamide (4). Rats that were fed fried animal feed for 1 or 2 months showed much higher levels of acrylamide-hemoglobin adducts than control rats fed unfried feed. The team showed that acrylamide formed in the heating of the feed, and the amount of acrylamide that formed was consistent with the measured levels of adducts. They concluded that cooked food was probably a major source of acrylamide exposure. Törnqvist’s team followed that study up with an analysis of acrylamide levels in common, heated foodstuffs. It was the paper resulting from that study, published in 2002, that alerted the world to the presence of acrylamide in common foods (5). The study reported high levels of acrylamide in potato products and crispbread, but it was soon discovered that other cereal products and coffee were also important contributors to dietary intake (Table 1) (6). Acrylamide is not detectable in raw foods and can therefore be classified as a processing contaminant. Processing contaminants are substances that are produced in a food when it is cooked or processed, are not present or are present at much lower concentrations in the raw, unprocessed food, and are undesirable either because they have an adverse effect on product quality or because they are potentially harmful (7). Törnqvist’s 2002 study also found that acrylamide did not form to detectable levels in any of the boiled foods that were analyzed, and acrylamide continues to be associated predominantly with fried, baked, roasted, and toasted foods (5).
Acrylamide Formation Following the discovery of acrylamide in popular foods (5), teams led by Don Mottram at the University of Reading in the United Kingdom and Richard 29 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Stadler at the Nestlé Research Center in Lausanne, Switzerland, showed that acrylamide could form from reducing sugars and free (soluble, non-protein) asparagine, within the Maillard reaction (8, 9). The Maillard reaction takes its name from the French chemist, Louis Camille Maillard, who first described it in 1912 (10), although the steps in the reaction as they are understood today were first proposed by an American chemist, John Hodge, in 1953 (11). It comprises a series of nonenzymatic reactions between reducing sugars and amino groups, principally those of free amino acids, and any amino acid can participate, not just asparagine. It is promoted by high temperature and low moisture content, and its products include melanoidin pigments, which are responsible for the brown color in fried, baked, roasted, and toasted foods, and complex mixtures of compounds that impart flavor and aroma (12).
Table 1. Contribution (%) of Different Food Groups to Dietary Acrylamide Intake for Adults in the Three Most Populous States of the European Uniona Food Group Country
Biscuits
Crisp bread
Breadb
Breakfast cereals
Muesli
Cereal products total
France
7.6
5.3
25.7
1.3
1.0
40.9
Germany
6.1
4.0
32.0
1.2
2.1
45.4
United Kingdom
6.3
2.0
15.0
5.0
3.6
31.9
Food Group
a
Country
French fries
Potato chips
Oven potatoes
Potato products total
Coffee
France
12.9
1.6
3.8
18.3
40.4
Germany
26.7
5.3
2.0
34.0
20
United Kingdom
41.3
8.5
17.3
67.1
0.7
Source: Data from ref (6).
b
Includes “soft” and “unspecified” bread types.
The most abundant reducing sugars in plants are the monosaccharides, glucose and fructose, and the disaccharide, maltose. These sugars can adopt ring or linear structures; in the linear form, glucose and maltose have a free aldehyde group (CHO), while fructose has a free keto group (C-CO-C). Sucrose, on the other hand, which is the most abundant sugar of all in most plant tissues, is a non-reducing sugar. The Maillard reaction is very complex and is not described in detail in this review. A key point to note is that it is multistep, with amino groups participating in the early and later stages, and is not one reaction but many. This means that the relationship between precursor concentration and different products is not a 30 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
simple one; for instance, the concentrations of free amino acids as a whole may affect the rate and scale of the first part of the reaction, but the ratios of different free amino acids to each other in the pool may also be important, determining the relative amounts of different products that are made in the final step if sugars are limiting. It is also important to note that other routes have been proposed for the formation of acrylamide, for example with 3-aminopropionamide as a possible transient intermediate (13) or through pyrolysis of gluten (14). Nevertheless, the Maillard reaction appears to be the predominant route, and free asparagine and reducing sugars can therefore be regarded as the precursors for acrylamide formation.
Risk Posed by Dietary Acrylamide and the Response of Regulatory Authorities The 2015 EFSA CONTAM report estimated the mean exposure of humans to acrylamide in their diet to be between 0.4 and 1.9 μg/kg body weight per day, across all age groups, with the 95th percentile at 0.6 to 3.4 μg/kg body weight per day (1). This level of exposure is three orders of magnitude or so lower than the doses used in rodent toxicology tests and also substantially lower than the occupational exposure that has been used to study the effects of acrylamide on people. In addition to toxicology studies, the CONTAM Panel considered the results of a wide range of epidemiological studies. After assessing all of the evidence, the Panel concluded that “the current levels of dietary exposure to acrylamide are not of concern with respect to non-neoplastic effects.” In other words, the Panel did not consider the level of exposure to acrylamide in the diet to be sufficient to cause neurological, reproductive, or developmental effects. However, while the Panel considered that epidemiological studies had not conclusively demonstrated acrylamide to be a human carcinogen, the margins of exposure indicated “a concern for neoplastic effects.” Margin of exposure is defined by the EFSA as the ratio of the level at which a small but measurable effect is observed to the estimated exposure dose; it is also sometimes defined as the ratio of the maximum no-adverse-effect-level to the estimated exposure dose. The European Commission issued indicative values for the presence of acrylamide in foods in 2011 and revised them downward for many product types in 2013 (Table 2) (15). These were not meant to be safety limits; indeed, widely different indicative values were set for different products. Rather, indicative values were set at a level that the European Commission believed the food industry should be able to achieve, based on the results of screening programs carried out by European Union member states and reported to the EFSA (6). If a product was found to exceed the indicative value, the relevant food safety authority was expected to take action to ensure that the manufacturer addressed the problem. The CONTAM Panel report of 2015 forced the European Commission to take more action (1), and in April 2018 Commission Regulation (EU) 2017/2158 came into force across the European Union, introducing compulsory risk management measures that apply to all food businesses and the renaming of indicative values as 31 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
benchmark levels, with the benchmark level reduced in many cases (Table 2) (16). The regulation also states that the setting of maximum levels (MLs) for acrylamide (levels above which it would be illegal to sell a food product) should be considered for some foods in the future. There should be no doubt, therefore, of the direction of travel of the European Commission with respect to its measures to reduce the presence of acrylamide in food. We urge all stakeholders in the food supply chain to consider not only the current regulatory scenario, but also possible scenarios that could be put in place in the future. This is particularly important for plant breeders because of the long timescale of variety development.
Table 2. Indicative Values and Benchmark Levels (Parts per Billion) for Acrylamide in Food, Set by the European Commissiona Food
Indicative Value 2011
Indicative Value 2013
Benchmark Level 2018
French fries
600
600
500
Potato chips (U.K. crisps)
1000
1000
750
80
50
150
100
200–400
300
500
350
500
400
450
350
Soft bread (wheat) Soft bread (other) Breakfast cereals
150 400
Biscuits Crackers
500
Crispbread
a
Gingerbread
1000
1000
800
Cereal-based baby foods
100
50
40
Roast coffee
450
450
400
Instant coffee
900
900
850
Source: Data from refs (15) and (16).
Other food safety authorities around the world have generally been slower to act on acrylamide than the European Commission. The U.S. Food and Drug Administration, for example, has not set restrictions on levels of acrylamide in food, although it has issued an “action plan” with the goals of developing screening methods, assessing dietary exposure and identifying means to reduce it, and increasing understanding of acrylamide toxicology (17). On the other hand, in 2005, the attorney general of California filed a lawsuit against four food companies for not putting a Proposition 65 warning label on their products to alert consumers to the presence of acrylamide, as required by the state of California for any product that contains a compound that may cause cancer, birth defects, 32 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
or reproductive harm. The lawsuit was settled in 2008 when the companies committed to cutting the level of acrylamide in their products and paid $3 million in fines.
The FoodDrinkEurope Toolbox Since acrylamide was discovered in food in 2002, a range of measures have been developed to reduce its formation. These have been compiled in a “Toolbox” by FoodDrinkEurope, the latest update of which was published in 2014 (18). In potato chips (U.K. crisps), for example, the measures that have been implemented include improved control of cooking temperature and duration, monitoring of moisture levels in the finished product, post-frying quality control based on color, switching to very low-sugar potato varieties (used only within their optimum storage window), and the careful control of storage temperature and conditions. Manufacturers have also introduced checks on potato sugar concentrations at time of harvest, during storage, and at the factory gate. The success of these measures is evident from analyses of European manufacturers’ data on acrylamide in potato chips from 2002 to 2011 (19) and subsequently from 2002 to 2016 (20). Studies showed a 53% reduction in mean acrylamide levels from 763 parts per billion (ppb) (µg kg-1) in 2002 to 358 ppb in 2011 (Figure 1). However, after 2011 there was a leveling off, with the mean level for 2016 being 412 ppb, suggesting that the most effective acrylamide reduction measures had been devised and implemented by 2011. The two studies also revealed a marked seasonality in acrylamide, with highest levels found in the first half of the year when potatoes were being used from storage, and lowest levels from July to September when potatoes were being harvested (Figure 2). The second of these two studies found that higher values for acrylamide were recorded in the north of Europe (comprising Denmark, Finland, Lithuania, Latvia, Norway, and Sweden) than in the south, west, or east (20). It is not clear why this should be, but this geographical effect coupled with the seasonality of acrylamide levels meant that over that period, more than 30% of potato chips produced in northern Europe during the first half of the year exceeded the benchmark level of 750 ppb set for potato chips by the European Commission (Figure 3). The figure for May was approaching 50%, and even in the other regions the failure rate with respect to the current benchmark level would have been over 10%. Indeed, it could be argued that the benchmark level of 750 ppb is unrealistic and that setting it at a lower level than the previous indicative value of 1000 ppb was not justified. The fact that this study showed acrylamide levels in potato chips in Europe to have leveled off in recent years is particularly important given the threat from the European Commission to impose MLs on sectors of the food industry that do not show “sufficient improvement.” It is not clear what the European Commission would consider sufficient improvement to be or whether reductions from 2002 will be taken into account or further reductions expected over the next few years. Clearly, imposing an ML for potato chips at 750 ppb would have severe consequences for manufacturers. Possible additional tools for acrylamide reduction are available but not yet widely applied, including blanching, vacuum 33 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
frying, or par vacuum frying (18), and the application of pulsed electric field technology (personal communication), all of which may be effective in some product types but also impact product cost and quality. However, even with these additional processing measures, further substantial reductions may not be achievable without a step change in the acrylamide-forming potential of the raw material—in other words, the breeding of potato varieties with much lower concentrations of reducing sugars and/or free asparagine.
Figure 1. Acrylamide concentrations in potato chips in Europe from 2002 to 2016. Overall means (bars) with standard errors and with trend in 90th and 95th quantiles. Reproduced with permission from ref (20).
Figure 2. Mean acrylamide levels in samples of potato chips from 2002 to 2016, with standard errors, plotted monthly. Reproduced with permission from ref (20). 34 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 3. Proportion of potato chip samples with more than the benchmark level of 750 ppb acrylamide for each month over the period 2011–2016 for geographic regions of Europe. Source: Data from ref (20).
No data of the sort shown in Figures 1–3 have been published for other food types, including any cereal products, and it is difficult to see how sufficient improvement on acrylamide for those products could be demonstrated without such data.
Acrylamide-Forming Potential of Wheat The rest of this chapter is devoted to a review of the acrylamide-forming potential of wheat and the agronomic and genetic approaches being taken to reduce it. In potatoes, the major determinant of acrylamide-forming potential is the concentration of reducing sugars, but in cereal grain it is free asparagine concentration, the difference arising from the lower ratio of free asparagine to reducing sugar concentration in cereal grain compared with potato tubers (21–28). Measurements of free asparagine concentration in wheat varieties on the U.K.’s Agriculture and Horticulture Development Board Recommended List have been carried out over several seasons, and one dataset, from 2012–2013, is shown graphically in Figure 4 (28).
35 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 4. Free asparagine concentration in grain of winter (fall-sown) wheat varieties grown in a field trial at Woburn, Bedfordshire, U.K., 2012–2013. One of several field trials that have been conducted. Source: Replotted from data in ref (28).
Free asparagine concentration in wheat grain and many other plant tissues is very responsive to abiotic and biotic stress (29–31). Nevertheless, some wheat varieties have been in the bottom half of the rankings on asparagine concentration over several field trials, while some have consistently been in the top half (28). Most of the “low” varieties are soft milling wheats, used predominantly for biscuits, breakfast cereals, and non-food uses, but selecting varieties for low asparagine simply on the basis that they are soft would be simplistic and potentially counterproductive because some soft wheat varieties are high in asparagine. We would support the inclusion of information on grain free asparagine concentration in the U.K.’s Recommended List variety descriptions, but there may still be too much uncertainty for end users to trust variety rankings. Adding to the difficulty, the turnover in varieties in the United Kingdom is so rapid that, by the time enough is known about free asparagine concentrations, the variety may no longer be available. Conducting testing during variety development offers a possible solution, but this is currently not performed. Another problem is that biscuit (soft) wheat cultivation in the United Kingdom has declined dramatically in recent years, partly because the variety Claire, a stalwart of that market, is susceptible to new strains of rust. Furthermore, the food industry operates across European borders, and we are not aware of similar data being available in other countries.
36 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 5. (a) Acrylamide formation in flour from wheat grown with or without fungicide treatment. Reproduced with permission from ref (27). Copyright 2016 American Chemical Society. (b) Effects of sulfur availability on acrylamide-forming potential in wheat. Reproduced with permission from ref (7). Crop management is also extremely important, and ensuring good disease control and sulfur sufficiency (Figure 5) are both included in the compulsory mitigation measures for wheat (16). Sulfur deficiency causes a massive increase in free asparagine concentration in wheat grain (21–23, 27, 28), with a concomitant effect on acrylamide formation upon heating (Figure 5a). Consequently, we recommend that 20 kg of sulfur per hectare should be supplied to all wheat being grown for human consumption. Poor disease control also causes free asparagine concentration to increase, in some varieties by several fold, again with a concomitant effect on acrylamide formation (Figure 5b) (27). Both sulfur deficiency and disease also cause the variety ranking on free asparagine 37 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
concentration to change. This means that a farmer who invested in a low asparagine variety would have to ensure good crop management practice in order to benefit from the outlay on the seed.
Wheat Asparagine Synthetases Asparagine synthesis occurs by the adenosine triphosphate (ATP)-dependent transfer of the amino group of glutamine to a molecule of aspartate to generate glutamate and asparagine and is catalyzed by the enzyme asparagine synthetase. Wheat has four asparagine synthetase genes (32, 33), called TaASN1–4 (Figure 6). TaASN1, 2, and 4 are present as single copies on chromosomes 5, 3, and 4, respectively, although some varieties lack a TaASN2 gene on chromosome 3B, while there are two copies of TaASN3 on chromosome 1 (33). TaASN4 has not been characterized in any detail. Of the others, the expression of TaASN2 in the embryo and endosperm during mid to late grain development is the highest of any of the genes in any tissue (32).
Figure 6. Diagrammatic representation of asparagine synthetase genes from wheat. Reproduced with permission from ref (33).
38 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Enzymes TaASN1 and TaASN2 have been expressed heterologously and characterized biochemically (33). Both were able to produce asparagine and glutamate from aspartate and glutamine, confirming that they were asparagine synthetases, as shown for TaASN2 in Figure 7. A continuous Petri net model based on mass-action kinetics was constructed using SNOOPY® software to describe the reaction (Figure 8) (33). The model comprises eleven molecules (species): adenosine monophosphate (AMP), asparagine (Asn), asparagine synthetase enzyme (for the purpose of the modelling annotated as ASNe), asparagine synthetase enzyme complexed with glutamine (ASNe-Gln), asparagine synthetase enzyme complexed with ammonia (ASNe-NH3), aspartate (Asp), ATP, β-aspartyl-complex (βAsp-AMP-ASNe-NH3), glutamine (Gln), glutamate (Glu), and magnesium ions (Mg2+). The experimental data showed that the concentration of glutamate increased at a faster rate than the concentration of asparagine (Figure 7), and the product concentrations plateaued with the concentration of glutamate more than double that of asparagine. This indicated that the early stages of the reaction (r1 and r2 in Figure 8) could proceed faster than and independently of the later stages (r3 and r4), consistent with the hypothesis proposed by Gaufichon et al. (34) that steps r1 to r4 occur sequentially rather than simultaneously. So, despite the overall equation of the reaction being Glutamine + Aspartate + ATP → Glutamate + Asparagine + AMP + PPi, glutamate synthesis can proceed independently of asparagine synthesis when aspartate is not available.
Figure 7. Plot showing the synthesis of asparagine and glutamate by TaASN2. Purified TaASN2 was added to an assay mix of 100 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer (pH 7.6), 1.6 mM aspartate, 10 mM glutamine, 10 mM ATP, 10 mM MgCl2, and 1 mM DTT and incubated at 30°C. Aliquots (100 µL) were removed and mixed with 100 µL of 10% trichloroacetic acid to stop the reaction. The free asparagine and glutamate produced in the reaction were detected by high-performance liquid chromatography after derivatization with o-phthalaldehyde reagent. Reproduced with permission from ref (33). 39 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
The similarity in the biochemical data obtained for the two asparagine synthetases, coupled with the gene expression data showing TaASN2 to be the most highly expressed asparagine synthetase gene in wheat grain, confirmed TaASN2 to be an appropriate target for genetic interventions.
Figure 8. Model representing the reaction catalyzed by asparagine synthetase, comprising metabolites (circles) and reactions (squares). The concentrations of metabolites are indicated abstractly by numbers in the circles. Note that for clarity, water and pyrophosphate are not included. The asparagine synthetase enzyme is shown as ASNe. The model was generated assuming that the reactions follow mass action kinetics and includes a dissociation step, D, for the ASNe-NH3 complex. Reproduced with permission from ref (33).
Concluding Remarks Genetic and agronomic approaches to solving the acrylamide problem could eventually lead to massive savings for the food industry by avoiding the necessity of costly modifications to manufacturing lines or the loss of valuable product types and brands, dwarfing the cost of the research involved. The development of modern techniques for genetic interventions, including chemical mutagenesis coupled with genomics, and genome editing, may make step reductions in the acrylamide-forming potential of potatoes and cereals possible, effectively solving the problem, at least in the context of current regulations and benchmark levels. However, the success of this approach will depend on how the plants respond to the interventions that are made and any undesirable effects on nitrogen metabolism and protein synthesis. 40 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
In the meantime, all stakeholders in the food supply chain in Europe face an increasingly difficult regulatory scenario. While awareness of the acrylamide issue is high among large food producers, it appears to be low to nonexistent among small companies, particularly in the restaurant/catering sector. This must change because the mitigation measures described in Commission Regulation (EU) 2017/ 2158 are compulsory.
Acknowledgments Sarah Raffan is supported by a BBSRC SWBio iCASE DTP Studentship, with partners: Keith Edwards (University of Bristol), AHDB, KWS UK Ltd, Saaten Union UK Ltd, RAGT Seeds Ltd, Syngenta UK Ltd, and Limagrain UK Ltd. Nigel Halford is supported at Rothamsted Research by the BBSRC via the Designing Future Wheat Program (BB/P016855/1).
References 1.
European Food Safety Authority Panel on Contaminants in the Food Chain (CONTAM). Scientific opinion on acrylamide in food. EFSA J. 2015, 13, 4104. 2. Bergmark, E.; Calleman, C. J.; He, F.; Costa, L. G. Hemoglobin adducts in humans occupationally exposed to acrylamide. Toxicol. Appl. Pharmacol. 1993, 120, 45–54. 3. Bergmark, E. Hemoglobin adducts of acrylamide and acrylonitrile in laboratory workers, smokers and nonsmokers. Chem. Res. Toxicol. 1997, 10, 78–84. 4. Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Törnqvist, M. Acrylamide: a cooking carcinogen? Chem. Res. Toxicol. 2000, 13, 517–522. 5. Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Törnqvist, M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem. 2002, 50, 4998–5006. 6. European Food Safety Authority. Results on acrylamide levels in food from monitoring years 2007−2009 and exposure assessment. EFSA J. 2011, 9, 2133. 7. Curtis, T. Y.; Postles, J.; Halford, N. G. Reducing the potential for processing contaminant formation in cereal products. J. Cereal Sci. 2014, 59, 382–392. 8. Mottram, D. S.; Wedzicha, B. L.; Dodson, A. T. Acrylamide is formed in the Maillard reaction. Nature 2002, 419, 448–449. 9. Stadler, R. H.; Blank, I.; Varga, N.; Robert, F.; Hau, J.; Guy, P. A.; Robert, M.C.; Riediker, S. Acrylamide from Maillard reaction products. Nature 2002, 419, 449–450. 10. Maillard, L. C. Action des acides aminés sur les sucres: formation des mélanoïdines par voie méthodique. (in French). C. R. Acad. Sci. 1912, 154, 66–68. 11. Hodge, J. E. Chemistry of the Browning reaction in model systems. J. Agric. Food Chem. 1953, 1, 928–943. 41 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
12. Mottram, D. S. The Maillard reaction: source of flavor in thermally processed foods. In Flavors and Fragrances: Chemistry, Bioprocessing and Sustainability; Berger, R. G., Ed.; Springer-Verlag: Berlin, 2007; pp 269–284. 13. Granvogl, M.; Schieberle, P. Thermally generated 3-aminopropionamide as a transient intermediate in the formation of acrylamide. J. Agric. Food Chem. 2006, 54, 5933–5938. 14. Claus, A.; Weisz, G. M.; Schieber, A.; Carle, R. Pyrolytic acrylamide formation from purified wheat gluten and gluten-supplemented wheat bread rolls. Molec. Nutr. Food Res. 2006, 50, 87–93. 15. Commission Recommendation of 8 November 2013 on Investigations into the Levels of Acrylamide in Food; European Commission, Brussels, 2013. 16. Commission Regulation (EU) 2017/2158 of 20 November 2017 Establishing Mitigation Measures and Benchmark Levels for the Reduction of the Presence of Acrylamide in Food; European Commission, Brussels, 2017. 17. Guidance for Industry, Acrylamide in Foods; U.S. Food and Drug Administration, Silver Spring, MD, 2016. 18. Acrylamide Toolbox 2013; FoodDrinkEurope, Brussels, 2013. 19. Powers, S. J.; Mottram, D. S.; Curtis, A.; Halford, N. G. Acrylamide concentrations in potato crisps in Europe from 2002 to 2011. Food Addit. Contam., Part A 2013, 30, 1493–1500. 20. Powers, S. J.; Mottram, D. S.; Curtis, A.; Halford, N. G. Acrylamide levels in potato crisps in Europe from 2002 to 2016. Food Addit. Contam., Part A 2017, 34, 2085–2100. 21. Muttucumaru, N.; Halford, N. G.; Elmore, J. S.; Dodson, A. T.; Parry, M.; Shewry, P. R.; Mottram, D. S. The formation of high levels of acrylamide during the processing of flour derived from sulfate-deprived wheat. J. Agric. Food Chem. 2006, 54, 8951–8955. 22. Granvogl, M.; Wieser, H.; Koehler, P.; von Tucher, S.; Schieberle, P. 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. J. Agric. Food Chem. 2007, 55, 4271–4277. 23. Curtis, T. Y.; Muttucumaru, N.; Shewry, P. R.; Parry, M. A.; Powers, S. J.; Elmore, J. S.; Mottram, D. S.; Hook, S.; Halford, N. G. Effects of genotype and environment on free amino acid levels in wheat grain: implications for acrylamide formation during processing. J. Agric. Food Chem. 2009, 57, 1013–1021. 24. Curtis, T. Y.; Powers, S. J.; Balagiannis, D.; Elmore, J. S.; Mottram, D. S.; Parry, M. A. J.; Raksegi, M.; Bedö, Z.; Shewry, P. R.; Halford, N. G. Free amino acids and sugars in rye grain: implications for acrylamide formation. J. Agric. Food Chem. 2010, 58, 1959–1969. 25. Postles, J.; Powers, S.; Elmore, J. S.; Mottram, D. S.; Halford, N. G. Effects of variety and nutrient availability on the acrylamide forming potential of rye grain. J. Cereal Sci. 2013, 57, 463–470. 26. Postles, J.; Curtis, T. Y.; Powers, S. J.; Elmore, J. S.; Mottram, D. S.; Halford, N. G. Changes in free amino acid concentration in rye grain in 42 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
27.
28.
29. 30.
31.
32.
33.
34.
response to nitrogen and sulfur availability, and expression analysis of genes involved in asparagine metabolism. Frontiers Plant Sci. 2016, 7, 917. Curtis, T. Y.; Powers, S. J.; Halford, N. G. Effects of fungicide treatment on free amino acid concentration and acrylamide-forming potential in wheat. J. Agric. Food Chem. 2016, 64, 9689–9696. Curtis, T. Y.; Powers, S. J.; Wang, R.; Halford, N. G. Effects of variety, year of cultivation and sulfur supply on the accumulation of free asparagine in the grain of commercial wheat varieties. Food Chem. 2018, 239, 304–313. Lea, P. J.; Sodek, L.; Parry, M. A.; Shewry, P. R.; Halford, N. G. Asparagine in plants. Ann. Appl. Biol. 2007, 150, 1–26. Halford, N. G.; Curtis, T. Y.; Chen, Z.; Huang, J. Effects of abiotic stress and crop management on cereal grain composition: implications for food quality and safety. J. Exp. Bot. 2015, 66, 1145–1156. Curtis, T. Y.; Bo, V.; Tucker, A.; Halford, N. G. Construction of a network describing asparagine metabolism in plants and its application to the identification of genes affecting asparagine metabolism in wheat under drought and nutritional stress. Food Energy Sec. 2018, 7, e00126. Gao, R.; Curtis, T. Y.; Powers, S. J.; Xu, H.; Huang, J.; Halford, N. G. Food safety: structure and expression of the asparagine synthetase gene family of wheat. J. Cereal Sci. 2016, 68, 122–131. Xu, H.; Curtis, T. Y.; Powers, S. J.; Raffan, S.; Gao, R.; Huang, J.; Heiner, M.; Gilbert, D.; Halford, N. G. Genomic, biochemical and modelling analyses of asparagine synthetases from wheat. Frontiers Plant Sci. 2018, 8, 2237. Gaufichon, L.; Reisdorf-Cren, M.; Rothstein, S. J.; Chardon, F.; Suzuki, A. Biological functions of asparagine synthetase in plants. Plant Sci. 2010, 179, 141–153.
43 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
