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Chapter 4

Reducing the Acrylamide-Forming Potential of Wheat, Rye and Potato: A Review Downloaded by PURDUE UNIV on November 24, 2016 | http://pubs.acs.org Publication Date (Web): November 18, 2016 | doi: 10.1021/bk-2016-1237.ch004

Nigel G. Halford* and Tanya Y. Curtis Plant Biology and Crop Science Department, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom *E-mail: [email protected]

The Maillard reaction, which produces a plethora of color and flavor compounds, is also responsible for the formation of acrylamide, an undesirable processing contaminant. Acrylamide is a Group 2a carcinogen and was discovered in a variety of popular foods, notably those derived from potatoes and cereals, as well as coffee, in 2002. It forms from free asparagine and reducing sugars, with free asparagine concentration being the main determinant of acrylamide-forming potential in cereal products but reducing sugar concentration being more important in potatoes, the difference arising from the relative concentrations of free asparagine and reducing sugars in the different raw materials. The European Commission set ‘indicative’ levels for acrylamide in food in 2011 and 2013, and is currently reviewing its options for further measures, making the issue one of the most difficult facing the cereal and potato supply chains. Here we review research into agronomic and genetic approaches to reducing the acrylamide-forming potential of wheat, rye and potato.

Introduction The Maillard reaction, which produces the brown colors and flavors that are the major focus of this book, is also responsible for the formation of acrylamide (Figure 1), an undesirable processing contaminant: processing contaminants may be defined as 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 © 2016 American Chemical Society Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by PURDUE UNIV on November 24, 2016 | http://pubs.acs.org Publication Date (Web): November 18, 2016 | doi: 10.1021/bk-2016-1237.ch004

food, and are undesirable either because they have an adverse effect on product quality or because they are potentially harmful (1).

Figure 1. Structure of acrylamide Acrylamide is classed as a probable (Group 2a) carcinogen by the International Agency for Research on Cancer (2) and also has reproductive and neurotoxicological effects at high doses (3). The European Food Safety Authority (EFSA) Expert Panel on Contaminants in the Food Chain (CONTAM) stated in its 2015 report that the margins of exposure for acrylamide indicate a concern for neoplastic effects based on animal evidence (4). The European Commission has already issued ‘indicative’ levels for the presence of acrylamide in food in 2011 and revised them downwards for many products in 2013 (5). It is currently reviewing additional options for risk management measures in response to the CONTAM report. Acrylamide forms during cooking and processing at temperatures above 120 °C, usually during the processes of frying, roasting and baking, and levels of several thousand µg kg-1 (parts per billion (ppb)) have been reported in some foods. The predominant route for its formation is via a Strecker-type degradation of free asparagine by highly reactive carbonyl compounds produced within the Maillard reaction (6–8), although other routes for its formation have been proposed, for example with 3-aminopropionamide as a possible transient intermediate (9) or through pyrolysis of gluten (10). The production of carbonyl compound intermediates within the Maillard reaction involves reducing sugars and other free amino acids, which means that the concentrations of these metabolites as well as free asparagine may affect acrylamide formation. Indeed, the concentration of reducing sugars is the major determinant of acrylamide-forming potential in potato (11–15). Acrylamide intake due to different food groups in the three most populous European countries, France, Germany and the UK, is shown in Table 1 (1, 16). In France and Germany, the biggest single contributor to dietary acrylamide intake is bread, while in the UK the contribution of bread and cereal products overall is lower. This reflects differences in dietary preferences between the three countries rather than variation in acrylamide levels in similar products, with more fried potato products being consumed in the UK, for example, than in France and Germany. Bread contains relatively low levels of acrylamide, but this is outweighed with respect to its contribution to acrylamide intake by its high consumption. It is also important to note that the acrylamide level will increase 36 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

considerably if the bread is toasted (17), highlighting the problem of how foods are cooked in the home. The simplest advice is to toast the bread to a light color, since color development and acrylamide formation are closely linked, but there is little or no awareness of this amongst consumers.


Table 1. Contribution (%) of different food groups to dietary acrylamide intake for adults (18-64) in the three most populous states of the European Union (16)

Risk Assessment and Monitoring by International Authorities The CONTAM report of 2015 (4) concluded that although the results of epidemiological studies on the effects of dietary acrylamide were inconsistent, there was a risk of neoplastic effects (abnormal growth of tissue, such as a tumor), based on the animal evidence and the margin of exposure (defined by 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 observable effect levels to the estimated exposure dose). The Food and Agriculture Organisation of the United Nations and the World Health Organisation (FAO/WHO) Joint Expert Committee of Food Additives (JECFA) has also concluded that the presence of acrylamide in the human diet is a concern (18). In 2011 and 2013, the European Commission set ‘indicative’ levels for acrylamide in different food categories (5), based on the results of EFSA’s monitoring program for the presence of acrylamide in different foodstuffs across Europe, and these are shown in Table 2. Indicative levels are not maximum levels or an indication of safety or lack of it: rather they are the levels that the 37 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Commission believes the food industry should be able to achieve, based on the monitoring data. The publication of the CONTAM report, however, has forced the European Commission to consider further options for managing the risk of acrylamide in the diet, which may include the imposition of obligatory codes of practice (a hazard analysis, critical control point (HACCP) approach) and the setting of maximum levels of acrylamide in food, both backed up by new regulations.


Table 2. Indicative values set by the European Commission for acrylamide content in cereal- and potato-based foods in 2011 and revised indicative values issued in 2013 (5)

In the US, the Food and Drug Administration (FDA) has issued an ‘action plan’ on acrylamide with the goals of developing screening methods, identifying means to reduce exposure, assessing dietary exposure of American consumers, increasing understanding of acrylamide toxicology to enable quantitative risk assessment, and informing consumers. To date, the FDA has stopped short of issuing advice or restrictions on levels, but in 2005 the Attorney General of the State of California filed a lawsuit against four food companies for not putting a Proposition 65 warning label on their products to make consumers aware that the products contained acrylamide (the State of California requires that a Proposition 65 warning be included in the labeling of any product that contains a compound that may cause cancer, birth defects, or reproductive harm). The lawsuit was settled in 2008 when the companies committed to cutting the level of acrylamide in their products to below 275 µg kg-1 and paid $3 million in fines. Note that, as with Europe’s indicative levels, there is no evidence that the 275 µg kg-1 figure is safe or unsafe; it is simply the figure that the two sides agreed should be achievable, after lengthy negotiation.

38 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The Acrylamide Toolbox The food industry responded rapidly to the discovery of acrylamide in its products and the methods that have been devised to reduce acrylamide formation have been compiled in the Acylamide Toolbox, published by Food Drink Europe (19). Approaches include selecting varieties that contain low levels of free asparagine and reducing sugars; removing free asparagine and reducing sugars before cooking or processing, for example by treating with the enzyme asparaginase to hydrolyze asparagine to aspartic acid; controlling the pH, temperature and time of cooking and processing; ensuring optimum storage conditions for the raw material before processing (particularly important for potato); and adding food ingredients that have been reported to inhibit acrylamide formation, such as amino acids, antioxidants, non-reducing carbohydrates, garlic compounds, protein and metal salts. One modification that has been introduced with considerable effect in the manufacture of bakery products is the replacement of ammonium hydrogen carbonate with sodium hydrogen carbonate (also known as sodium bicarbonate or bicarbonate of soda) as a leavening agent. This has been shown to reduce the acrylamide content in biscuits by about 70% (20). The Acrylamide Toolbox is a rare example of the food industry sharing knowledge to address a common problem, and shows how seriously the industry takes the acrylamide issue. The challenge for the food industry is to continue working to reduce acrylamide levels to as low as reasonably achievable (the ALARA principle) while retaining the colors, flavors and aromas that define products and brands and are demanded by consumers. This has been more successful for some food types than others, because many of the acrylamide mitigation tools are food system-specific and show large variations in effectiveness across food categories. It is important for the food industry to be able to demonstrate that the approaches described in the Toolbox are effective and that progress is being made in reducing acrylamide levels in food. One sector of the industry that has been able to show a clear reduction in the levels of acrylamide in its products is European potato chip (UK crisp) manufacturing (21). A dataset was compiled of manufacturers’ measurements of acrylamide levels in 40,455 samples of fresh sliced potato chips from 20 European countries for years 2002 to 2011. Analysis of the data showed a clear, significant downward trend for mean levels of acrylamide, from 763 µg kg-1 in 2002 to 358 µg kg-1 in 2011 (Figure 2A); this was a decrease of 53%. The effect of seasonality arising from the influence of potato storage on acrylamide levels was also evident, with acrylamide in the first six months of the year being significantly higher than in the second six months (Figure 2B). This illustrates very clearly that the food industry has to deal with a highly variable raw material while complying with current indicative levels and possibly future maximum levels set by the European Commission. Even so, the proportion of samples containing acrylamide at a level above the indicative value for potato crisps of 1000 µg kg-1 introduced by the European Commission in 2011 fell from 23.8% in 2002 to 3.2% in 2011 (21).



Figure 2. A. Overall mean acrylamide levels (µg kg-1) in samples of potato chips (crisps) shown over years 2002-2011 (bars), with standard errors and with trend in 95% (Q95) quantiles (line). B. Mean acrylamide levels in samples of potato chips over time with standard errors, plotted monthly. Reproduced from reference (21).

Acrylamide-Forming Potential in Potato: Effects of Variety, Crop Management and Storage Enabling potatoes to be produced with lower and more predictable acrylamide-forming potential is now a target for potato breeders and agronomists. This requires the development of genotypes that stay consistently low in acrylamide-forming potential through a range of environments and conditions, and the development of best crop management practice (22). In the meantime, variety selection has already been shown to be a powerful tool in keeping acrylamide formation in potato products to as low as reasonably achievable. Halford et al. (2012), for example, reported the results of a study of nine varieties (French fry varieties Maris Piper, Pentland Dell, King Edward, Daisy, and Markies; and chipping (UK crisping) varieties Lady Claire, Lady Rosetta, Saturna, and Hermes) grown in the United Kingdom in 2009 (11). Tubers were analyzed at monthly intervals through storage from November 2009 to July 40 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


2010 and acrylamide formation was measured in heated flour and crisps fried in oil. Crisps produced from Lady Claire and Saturna were consistently below the 1000 µg kg-1 acrylamide level, as were crisps from Lady Rosetta during early storage and Markies in late storage. The effect of seasonality arising from the influence of potato storage on acrylamide levels was evident in the study, consistent with the data shown in Figure 2B, and a number of previous and subsequent studies (12, 13, 23). This has led to the advice that potatoes should only be used for chipping (crisping), frying and roasting within their recommended storage window (22). Both nutrition and water availability have also been shown to affect the acrylamide-forming potential of potatoes (12, 13, 24, 25), with increased nitrogen and irrigation generally leading to more acrylamide-forming potential, although there are differences in the ways that the types of potato (boiling, chipping (crisping) and French-fry) respond and in the responses of varieties within each type. Muttucumaru et al. (2013), for example, analyzed 13 varieties of potato grown in a field trial in 2010 and treated with different combinations of nitrogen and sulfur (12). The study showed that nitrogen application can affect acrylamide-forming potential in potatoes but that the effect is type- (French fry, chipping and boiling) and variety-dependent, with most varieties showing an increase in acrylamide formation in response to increased nitrogen but some showing a decrease. Sulfur application, on the other hand, reduced glucose concentrations and mitigated the effect of high nitrogen application on the acrylamide-forming potential of some of the French fry-type potatoes. Advice on both nitrogen and sulfur application would therefore have to be carefully tailored for specific varieties, and with sulfur application having no significant effect on yield it may be difficult to convince farmers that expenditure on sulfur fertilizer represented good value for money anyway. The effect of water supply has been studied in both field- and glasshousegrown potatoes (25) and is an important factor because irrigation is frequently used in potato cultivation to maximize yield. Water availability was shown to have profound effects on free amino acid and sugar concentration in tubers, leading to the conclusion that farmers should irrigate potatoes only if necessary to maintain the health and yield of the crop, because irrigation may increase the acrylamide-forming potential of the tubers. Even mild drought stress caused significant changes in composition, but these differed from those caused by more extreme drought stress. Free proline concentration, for example, increased in the field-grown potatoes of one variety by almost 15-fold in response to lack of irrigation, whereas free asparagine concentration was not affected significantly in the field but almost doubled in response to more severe drought stress in the glasshouse. Furthermore, the different genotypes were affected in dissimilar fashion by the same treatment, indicating that there is no single, unifying potato tuber drought stress response. These studies have all contributed to our understanding of the relationship between free asparagine and reducing sugar concentration in potatoes and the formation of acrylamide during cooking and processing, something that is extremely important in enabling food producers to achieve optimal quality control. Breeders are also more likely to invest in programs aimed at reducing 41 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


acrylamide-forming potential in potato if they are confident that the correct target traits have been identified. Muttucumaru et al. (2014) (13) considered this issue in the light of these studies and others that had shown reducing sugar concentration, free asparagine concentration or free asparagine concentration as a proportion of the total free amino acid pool to be the determining factor for acrylamide formation (12, 14, 15, 26–28). Additional data was analyzed from a controlled field trial, and as with the studies reported by Halford et al. (2012) (11) and Muttucumaru et al. (2013) (12), glucose and fructose concentrations showed the best correlations with acrylamide formation (Figure 3A and B), but free asparagine concentration contributed to the variance in the French fry varieties (Figure 3C), consistent with the conclusion that free asparagine concentration is an important contributor to acrylamide-forming potential in French fry varieties but not chipping (crisping) varieties (13). The difference between types is possibly explained by the higher concentration of reducing sugars in the French fry varieties compared with the chipping (crisping) varieties and the consequent lower ratio of free asparagine to reducing sugars. Consistent with this, another recent study modeled the kinetics of acrylamide formation in French fry production and concluded that both the fructose/glucose ratio and the ratio of asparagine to total free amino acids could affect acrylamide formation (29). Very low free asparagine concentration has been achieved in genetically modified (GM) potatoes in which asparagine synthetase gene expression in the tubers has been reduced by RNA interference (RNAi) (30, 31). These potatoes were reported to give good color when fried, supporting the hypothesis that targeting free asparagine concentrations could enable acrylamide-forming potential to be reduced without compromising the characteristics that consumers demand in fried and roasted potato products. Indeed, Simplot has recently begun to market a low-acrylamide biotech (GM) potato variety carrying the RNAi low asparagine trait. The variety, called Innate, also has reduced activity of two genes encoding enzymes of starch breakdown, phosphorylase L (PhL) and starch-associated R1 (R1), as well as a gene (PPO5) encoding polyphenol oxidase, an enzyme involved in bruising, all as a result of RNAi. The commercialization of low acrylamide biotech potatoes is an interesting development because no market has yet been established for biotech potatoes, even in the US, which is the only country in which Innate is currently available. The strategy of targeting asparagine synthesis specifically in the tuber makes sense because asparagine has been shown not to be a major transported amino acid in potato, so the free asparagine that accumulates in tubers must be synthesized there (32). Potatoes with low free asparagine rather than reducing sugar concentration could be particularly suitable for home cooking, where most consumers use color development to assess when roasted or fried potatoes have been cooked sufficiently. Low reducing sugar concentration is also important but chipping (crisping) varieties in particular have been bred for low sugar concentration for many years and the lowest concentration may already be close to the minimum for the potatoes to be ‘fryable’. Furthermore, the concentration of reducing sugars affects all products of the Maillard reaction, and the color, flavor and aroma that those products provide. 42 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 3. Graphs showing correlations between acrylamide formation in potato flour heated to 160 °C for 20 minutes and the concentration of: A. Glucose; B. Fructose; C. Free asparagine. Correlation coefficients and p values are given for the complete dataset, and for French fry types (F) and chipping (crisping) types. Lettering at the data points derives from the name of the variety and whether it was a stored (S) or unstored (U) sample. Reproduced (adapted) from reference (13). The link between acrylamide-forming potential and color, flavor and aroma development is an important consideration for the food industry. Correlations between color development and acrylamide formation were shown by Halford et al. (2012) (11) (Figure 4), while Elmore et al. (2010) quantified approximately 50 compounds in the headspace extracts of potato flour heated at 180 °C for 20 43 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


min, of which over 40 were affected by the fertilization regime and/or variety (27, 33). It seems inevitable, therefore, that acrylamide mitigation strategies that cause large changes in free amino acid and/or sugar concentration are likely to lead to significant effects on product quality.

Figure 4. Acrylamide formation in chips (crisps) and color: Hunter L (top) and a (bottom). Data points from French fry varieties are designated F, those from chipping (crisping) varieties C. Reproduced with permission from reference (11). Copyright 2012 American Chemical Society. There is, of course, no prospect of a GM variety being developed for the European market in the foreseeable future, and breeders in Europe will have to use conventional breeding methods backed up by the latest genomics tools: Potato genome data is now available, for example, and genes encoding key enzymes can be mapped. Some progress has also been made towards identifying quantitative trait loci for acrylamide-forming potential (28, 34). In conclusion to this section on potato, we encourage potato breeders to engage on the acrylamide issue. The food industry has worked hard to reduce the levels of acrylamide in its products and there has been impressive progress in 44 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


some sectors, as we have described for potato chips (crisps) in Europe (21) (Figure 2). There is certainly an expectation within the food industry that potato breeders will engage with the issue with the same determination, and it is important that potato breeders make reduced acrylamide-forming potential a priority and that they have sufficient information on the genetics underlying what is undoubtedly a complex trait to make progress. The important conclusion from recent studies is that free asparagine as well as reducing sugar concentration should be a target. Indeed, we concur with the conclusion of Parker et al. (2012) (29) that reducing the concentration of free asparagine as a proportion of the total free amino acid pool would be the most likely way of reducing acrylamide formation in potato products while retaining the characteristics that define products and are demanded by consumers, bearing in mind again that compounds responsible for color, flavor and aroma form by similar pathways to acrylamide.

Acrylamide-Forming Potential in Wheat and Rye: Effects of Variety, Environment and Crop Management In contrast to potato, the relationship between free asparagine and reducing sugar concentration and acrylamide formation in wheat and rye is relatively simple, with free asparagine concentration being the major determinant of acrylamide-forming potential (35–39) (Figure 5). This is because cereal grain contains approximately one tenth the concentration of free asparagine typical of potato tubers but comparable concentrations of reducing sugars (40), meaning that the ratio of free asparagine to reducing sugars is much lower.

Figure 5. Free asparagine concentration and acrylamide formation in wheat and rye flour heated for 20 min at 180 °C. Reproduced (adapted) from reference (1). 45 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Significant variation in free asparagine concentration in the grain of different wheat genotypes has been shown in several studies. Muttucumaru et al. (2006), for example, analyzed the grain of three wheat varieties, Solstice, Malacca and Claire, grown under glass (35) When the plants were fully supplied with nutrients, free asparagine concentration in the grain ranged from 4.12 mmol kg-1 in Claire to 5.20 mmol kg-1 in Malacca, and acrylamide in flour heated to 160 °C for 20 minutes ranged from 679 µg kg-1 to 934 µg kg-1, a difference of 38%. Grain from six different wheat varieties grown at six different locations around the United Kingdom over two harvest years also showed significant differences (36). The concentrations of free asparagine in the grain samples varied from 0.6 mmol kg-1 to 4.4 mmol kg-1, representing a more than 7-fold difference. Notably, two well-known biscuit wheat varieties, Claire and Robigus, showed a marked difference, with Robigus having an average free asparagine concentration across the sites and years of 2.59 mmol kg-1, while the average for Claire was 1.89 mmol kg-1, a difference of 37% with respect to the lower value (Figure 6A). Claire was also much more consistent and therefore predictable across the sites and harvest years. In the same study (36), two varieties, Spark and Rialto, and four doubled haploid lines from a Spark × Rialto mapping population were analyzed. The lines differed significantly in free asparagine concentration in the grain, with one doubled haploid line having a lower concentration than either parent. The lowest and highest free asparagine genotypes also differed in the ratio of free aspartic acid to free asparagine, and the concentration of the total free amino acid pool, implicating asparagine synthetase, which converts glutamine and aspartic acid to glutamic acid and asparagine, as being responsible. Significant varietal differences have also been observed in a study of old and new rye varieties grown for the HEALTHGRAIN program (37), and in a separate study of five current UK commercial rye varieties (Agronom, Askari, Festus, Fugato and Rotari) (38). In the latter study, the highest accumulator, Askari, contained 8.08 mmol kg-1 free asparagine, while the lowest, Agronom, contained only 5.42 mmol kg-1, a difference of approximately 50% with respect to the lower value (Figure 6B). The variation in free asparagine content of grain from different varieties and genotypes grown under the same conditions shows the potential for varietal selection in addressing the acrylamide problem. However, free asparagine accumulates in plants in response to a variety of stresses and is also affected by the availability of nutrients (41, 42). Wheat is especially responsive to sulfur availability in this respect, particularly when nitrogen is readily available. Indeed, in some of the studies described in the previous section, varietal effects were dwarfed by the effect of sulfur supply. In the study by Muttucumaru et al. (2006) (35), for example, free asparagine concentration increased by more than 11-fold in variety Claire, 16-fold in Solstice and almost 30-fold in Malacca as a result of sulfur deficiency, with the acrylamide formed in heated Malacca flour rising to 5198 µg kg-1. The effect of sulfur availability was also evident in field-grown wheat cv. Hereward: A field trial was conducted at a site with very poor nutrient retention and grain from two plots treated with 40 kg sulfur per hectare contained 4.43 and 46 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


3.07 mmol kg-1 of free asparagine, compared with 7.8 mmol kg-1 in grain from a plot treated with 10 kg sulfur per hectare and 75.7 and 55.5 mmol kg-1 in grain from two plots that did not receive any sulfur fertilizer (35). Acrylamide formation in heated flour ranged from 723 µg kg-1 in a high-sulfur plot to 5286 µg kg-1 in one of the plots that did not receive any sulfur fertilizer, a more than 7-fold difference. Indeed, even the application of 10 kg sulfur per hectare resulted in 60% more acrylamide formation in heated flour than application of 40 kg sulfur per hectare.

Figure 6. A. Concentration of free asparagine (mmol per kg) in grain from wheat cv. Claire and Robigus grown at six different locations in 2006 (light grey) and 2007 (dark grey). Average for Claire: 1.89 mmol per kg; average for Robigus: 2.59 mmol per kg; difference 37%. Reproduced with permission from reference (36). Copyright 2009 American Chemical Society. B. Mean free asparagine concentration in grain of five commercially used rye varieties grown in a field trial in 2009-2010. Reproduced (adapted) from reference (39). 47 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Granvogl et al. (2007) (38) also showed the dramatic effect of sulfur deficiency on free asparagine concentration and acrylamide-forming potential in wheat, with acrylamide in heated flour from one cultivar, Star, ranging from only 94 µg kg-1 with sulfur feeding to 3124 µg kg-1 with sulfur deprivation, a 33-fold difference. Similar results were obtained with varieties Spark and Rialto and the Spark × Rialto doubled haploid lines analyzed by Curtis et al. (2009) (36). The importance of ensuring that wheat be supplied with sufficient sulfur was demonstrated recently by analyzing grain from field trials of four different varieties of winter wheat, grown at three different locations in the UK over three harvest years (2010-2012) (43). In each trial, sulfur had been applied at rates of 0, 5, 10, 20, and 30 kg per hectare. The results averaged over the trials showed a clear and statistically significant (p < 0.05) effect of sulfur application in reducing the acrylamide-forming potential of wheat (Figure 7, top panel). The level of application that showed the best, statistically significant (p < 0.05) reduction in acrylamide formation differed between the trials, but at some sites some was as high as 20 kg sulfur (50 kg SO3 equivalent) per hectare (Figure 7, bottom panel, shows the result from one of the sites where this was the case). This application rate has therefore been adopted by the UK’s Agriculture and Horticulture Development Board as the recommended rate for all wheat destined for human consumption in order to keep acrylamide-forming potential as low as reasonably achievable, regardless of yield and other quality issues (43). There is evidence that free asparagine is predominantly accumulated in the embryo and aleurone layer under sulfur-sufficiency but also accumulates at high levels in the endosperm under sulfur-deficient conditions (44). This means that there is more risk of acrylamide formation in wholegrain products than white flour products, but that sulfur deficiency will disproportionately affect white flour products. The distribution of free asparagine in the grain is reflected to some extent in the expression of asparagine synthetase genes. There are four asparagine synthetase genes in wheat, TaASN1-4 (45), as there are in maize (Zea mays) (46) and barley (Hordeum vulgare) (47). TaASN4 has only been identified in genome data and has not been analyzed in detail. Of the other three, TaASN1 appears to be the most responsive to both sulfur deficiency and nitrogen supply (45, 48). However, the expression of TaASN2 in the embryo and to a lesser extent the endosperm during mid-development is much higher than the expression of any of the genes in any other tissue. Indeed, the high levels of expression of TaASN2 in the grain suggest that most of the free asparagine that accumulates in the grain is synthesized there, making TaASN2 a potential target for genetic intervention. However, more work needs to be done to confirm this and to show that it is true under both sulfursufficient and -deficient conditions, given the sulfur-responsiveness of TaASN1. The signaling pathway that is responsible for the sulfur-response of TaASN1 also requires further investigation as a potential target for genetic intervention. There is evidence that it involves the protein kinase, general control nonderepressible-2 (GCN2) (48), and potentially a putative N-motif or GCN4-like regulatory motif in the TaASN1 gene promoter (45). In addition, a full picture of the role of the different asparagine synthetases will only emerge when the kinetic parameters of the enzymes have been measured, and it is notable that the enzymes encoded by 48 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


three of the maize genes have already been shown to have significant differences in that respect (49).

Figure 7. Top panel: Acrylamide formation averaged over four varieties of wheat (Alchemy, Viscount, Oakley and Panorama) produced at three different sites (Brockhampton, Frostenden and Woburn, UK) over three years (2009/10, 2010/11 and 2011/12) with standard errors (n = 6) for five levels of applied sulfur. Bottom panel: Acrylamide formation in flour from wheat cv. Oakley grown at a single site in Woburn, UK, in 2010/2011 with five different levels of sulfur application. Samples were wholemeal flour heated for 20 minutes at 170 °C. Data from Curtis et al. (2014) (43).

Concluding Remarks The acrylamide issue is a difficult problem facing the food industry in Europe and worldwide. It is made more difficult by the fact that many of the compounds that define product types and brands, and which are demanded by consumers, are formed by similar pathways to acrylamide; changes in acrylamide-forming potential may therefore affect other aspects of the processing properties of grains and tubers. Nevertheless, developing varieties of potato, wheat, rye and other crops with reduced potential for acrylamide formation would enable the food 49 Granvogl et al.; Browned Flavors: Analysis, Formation, and Physiology ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

industry to comply with an evolving regulatory framework without costly changes to processing methods. We therefore urge plant breeders to take the acrylamide issue on board or risk losing market share to those that do.

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