Abstract
Acrylamide has neurotoxicity, carcinogenicity, and genotoxicity in experimental animals and cellular systems. Fried potato is one of the major intake sources of acrylamide in food, and fried onion was reported to contain up to 100 ng/g level of acrylamide. To determine acrylamide concentration in potato and onion stir-fried prior to boiling for simmered dishes such as curry, stew, and Niku-jaga, a typical Japanese meat/potato/onion cuisine, we collected samples stir-fried at homes of volunteers who intended voluntarily to cook these simmered dishes. Acrylamide level was analyzed by GC-MS after the xanthydrol derivatization. Among 53 stir-fried potato samples, median and average values of acrylamide were found to be 2.0 ng/g and 11 ng/g, respectively. Acrylamide levels of 27 samples (51%) were less than limit of detection (LOD) (4 ng/g), and those of 13 samples (25%) were less than limit of quantification (LOQ) (10 ng/g). In cases with less than LOD and less than LOQ of acrylamide levels, one-half of LOD and average of LOD and LOQ were adopted, respectively, to calculate the median and average. This median was markedly lower than those of fried potato (180 ng/g) and potato snacks including potato chips (550 ng/g) reported in monitoring in 2013 fiscal year in Japan. Among 58 stir-fried onion samples, acrylamide level of only one sample (2%) was less than LOD (3 ng/g), and those of 15 samples (26%) were less than LOQ (8 ng/g). The median and average values in the stir-fried onion were 14 ng/g and 36 ng/g, respectively. These values are comparable to those for stir-fried onion reported by Ministry of Agriculture, Forestry and Fisheries, Japan (median 19 ng/g, average 25 ng/g). But the maximum value of stir-fried onion 420 ng/g in the present study is much higher than the reported maximum value (70 ng/g).
Key words: : burn, curry, Maillard reaction, Niku-jaga, stew
1. Introduction
Acrylamide is reported to have neurotoxicity, carcinogenicity, and genotoxicity in experimental animals and cellular systems1,2). Since Swedish and British researches revealed the presence of acrylamide in wide range of high-temperature cooked foods3,4), quantitative analyses of this compound in food have been carried out in various countries. As acrylamide is formed in Mallard reaction from asparagine in the presence of carbonyl compounds such as reducing sugars, it is detected in wide ranges of foods heated at high temperature > 120 °C through frying, baking, grilling, and roasting. Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated the risk of acrylamide in their 64th meeting in 2005 based on analytical results of 6,752 samples from 24 countries, 67.6% of which are from Europe and 21.9% of which are from North America, with their national dietary intake data1). Later, JECFA re-evaluated the risk in their 72th meeting in 2010 based on data of acrylamide in foods from 31 countries analyzed between years of 2004 and 2009. The total number of analytical results (single or composite samples) was 12,582, with 61% coming from Europe, 28% from Asia, 9% from North America, 1% from the Pacific and 1% from Latin America2). JECFA thus estimated both the general population and the high consumers intake as 1 µg/kg bw per day and 4 µg/kg bw per day of acrylamide in their averages, respectively. Major contributing foods to the total exposure included processed root and tubers such as French fries (potato chips in Europe) and potato chips (potato crisps in Europe), processed cereals such as cookies and bread and rolls/toasts, and coffee.
Extensive analyses of acrylamide have been continued mainly in Europe and North America. Considerable amounts of data have been accumulated for exposures in these regions. Data from Asia, South America and Africa are much less despite of large regional variations of consumed food category and of processing and cooking habit. Ministry of Agriculture, Forestry and Fisheries (MAFF) Japan continued survey of acrylamide in commercial processed and cooked foods since 20045). These data suggest the contributions of rice cracker besides wheat flour based cookies, and also of roasted green tea and roasted barley grain besides coffee in Japan. Vegetables fried at home may also be a significant source of acrylamide. Intake estimation of acrylamide in Japan is possible with compilations of the analytical data of MAFF Japan5), of National Institute of Health Sciences6) and in some scientific journals7–11) in conjunction with food intake data from the National Health and Nutrition Survey by Ministry of Health, Labour and Welfare (MHLW) Japan12).
Potato is known to have high levels of asparagines, and thus fried potato may contain acrylamide up to 5,000 µg/kg level as one of the major sources of acrylamide oral intake1). Potato is consumed by boiling in addition to frying. Although the cooking through boiling hardly yields acrylamide, potato is often stir-fried prior to boiling for popular simmered dishes in Japan such as curry, stew, and Niku-jaga, which may lead to formation of acrylamide. Niku-jaga, which means literally meat-potato in Japanese, is beef or pork boiled with potato and onion in soup stock seasoned with soy source, sugar, rice wine and/or sweet cooking rice-wine. No data on acrylamide content in these stir-fried potato for cooking of the simmered dishes were reported while large numbers of data have been obtained for French fries and potato chips. The data on the stir-fried potato for the popular simmered dishes are necessary for acrylamide intake estimation in Japan. For survey of the stir-fried potato for the simmered dishes, sampling from home cookings is necessary because of necessity of picking up during initial cooking before boiling. Samples from restaurants and delicatessen shops may be available, but the samples from home cooking are used in the present experiments to verify the expected variations for the home-cooking conditions.
Acrylamide formation depends on cultivars and storage condition of raw potato, size and shape of potato pieces, quantity of meat and vegetables fried together, stirring way and frequency during stir-frying, size and material of pan, heat level, heating time etc. Extent of these contributions may vary among individual home-cookings. No precise data on the home cooking condition was available, and thus simulated estimation of acrylamide formation was difficult from the model cooking procedure.
Therefore, we have planned two-step procedure for the survey of home cooked samples. The first step is done by questionnaire about cooking of curry, stew and Niku-jaga, and then the second step consists of the sampling from the home cooking by selected cooperators. Potato and onion samples, which were stir-fried in familiar ways of individual home cooking for curry, stew or Niku-jaga, were collected. Onion, which is reported to contain 100 ng/g level of acrylamide after frying13), is often stir-fried with potato in cooking of these dishes. The present results afford an estimation for the contribution of the potato and onion in the dishes on acrylamide intake in Japan.
2. Materials and Methods
2.1. Questionnaire and Selection of Cooperators
A questionnaire shown as Supplementary material 1 was carried out to staffs and students of five different universities (two in Tokyo Metropolitan, one in Chiba Prefecture, one in Kyoto Prefecture, and one in Nagasaki Prefecture in Japan) considering the expected regional variation of cooking ways and population. In the questionnaire we asked whether potato and onion were stir-fried or not stir-fried in cooking of curry, stew, and Niku-Jaga at their homes. We also asked the intention of offering of their home-cooked materials as cooperators of the survey for food component analyses to persons who replied to stir-fry potato and/or onion for at least one of the three dishes.
2.2. Preparation of Samples
The questionees, replying that they or their family members were able to offer cook-survey samples, cooked curry, stew or Niku-jaga at home using their accustomed recipe and cooking wares (Supplementary material 2). They were not informed on the target component of the survey cooking as acrylamide. They were just informed that the samples they cooked were used for food component analysis. They were asked to take photos of potato and onion just after cutting and after stir-frying. The whole stir-fried potato and onion were sent to laboratory under refrigeration within three days after cooking or under freezing if it took more than three days. The potato and onion samples were stored at −20 °C until analysis.
2.3. Analysis of Acrylamide
Acrylamide in potato and onion samples were analyzed after the xanthydrol derivatization by GC-MS14). The stir-fried potato or onion sample was pulverized and the 10 g portion was used for analysis. The pulverized sample mixed with 100 mL water, 2 µg acrylamide-d3 as an internal standard, and 20 mL n-hexane, was homogenized, shaken for 10 min, and centrifuged. The aqueous phase (20 mL) was loaded onto an EXtrelut® NT column (Merck Millipore Co., Darmstadt, Germany), stood for 30 min at room temperature, and eluted with 150 mL of ethyl acetate. To this eluate, 0.1 mL of 10% diethylene glycol solution in methanol was added. The sample was dried in vacuo, dissolved in 40 mL of water, and refined using a jointed cartridges of Sep-Pak C18 (Waters Co., Milford, MA, USA) and Sep-Pak AC-2 (Waters Co.). Acrylamide was eluted from Sep-Pak AC-2 with 5 mL of methanol. To this eluate, 0.1 mL of 10% diethylene glycol solution in methanol was added. The sample was dried in vacuo and dissolved in 1 mL methanol. Acrylamide in the sample was derivatized with xanthydrol, and analyzed by GC–MS (6890N/5973intert, Agilent Technologies Inc., Santa Clara, CA, USA) with a DB-5 MS column (0.25 mm I.D. 30 m, 0.25 μm film thickness; Agilent Technologies Inc.) as described by Yamazaki et al.14) The molecular ion peak of N-xanthyl acrylamide at m/z 251, its fragment ion peak at m/z 234, and the molecular ion peak of N-xanthyl acrylamide-d3 at m/z 254 were monitored using the selected ion monitoring (SIM) mode. The acrylamide concentrations in the food samples were calculated from the peak height ratio of m/z 251 to m/z 254.
3. Results and Discussion
Total 257 persons are participated as questionees. Among the questionees cooking curry, stew and/or Niku-jaga at home, 67% of them stir-fry potato prior to boiling for curry, 62% for stew, and 59% for Niku-jaga (Table 1). As for onion, 90% of them stir-fry for curry, 83% for stew, and 73% for Nikujaga (Table 1). Thus, in total, 63% and 82% stir-fry potato and onion, respectively, for these popular simmered dishes in Japan.
Table 1. Result of questionnaire about stir-frying of potato and onion at cooking of curry, stew and Niku-jaga.
| Curry | Stew | Niku-jaga | ||||
|---|---|---|---|---|---|---|
| Potato | Onion | Potato | Onion | Potato | Onion | |
| Stir-fry | 160 | 207 | 144 | 184 | 135 | 143 |
| Not stir-fry | 78 | 24 | 88 | 39 | 94 | 52 |
| Not use | 12 | 3 | 5 | 0 | 0 | 0 |
| Not cook at home | 5 | 5 | 17 | 17 | 22 | 22 |
| No answer | 2 | 18 | 3 | 17 | 6 | 40 |
| Total | 257 | 257 | 257 | 257 | 257 | 257 |
In the next step, 88 questionees were selected as candidates of cooperators of the survey cooking. The relative frequency of eating of curry, stew, and Niku-jaga in Japan was estimated to be about 3:1:1 ratios from background data of the National Health and Nutrition Survey12). Considering the rate of these cookings, we asked 54 volunteers to offer stir-frying intending to cook curry, 17 volunteers intending to cook stew, and 17 volunteers intending to cook Niku-jaga. Among them, 61 people sent us the stir-fried samples, of whom 38 set out cooking of curry, 11 of stew, and 12 of Niku-jaga. The 61 cooperators included 10 men and 51 women. Majority of the cooperators were women because we asked that the samples were to be cooked by family members who mainly cook at home. The ages of the cooperators were, 2 teens, 14 twenties, 11 thirties, 14 forties, 16 fifties, 3 sixties, and 1 eighties. Locality of their origin covered Tokyo Metropolitan and 21 different prefectures in Japan.
Total 58 potato samples and 60 onion samples were obtained in this survey. Of these samples 5 potato samples were excluded for analysis. Three of them were not stir-fried, one of them was stir-fried with curry powder, which might include acrylamide, and the rest one was mixed with water after stir-frying, which might release parts of acrylamide to the water. Of the onion samples obtained, one of them was stir-fried with curry powder, and one of them was mixed with water after stir-frying. These samples were excluded for analysis. As the result, 53 stir-fried potato samples and 58 stir-fried onion samples were used for the analysis.
Accuracy of acrylamide analysis was confirmed by recovery test using stir-fried potato and onion samples. The results were shown in Table 2, and satisfactory results were obtained for both potato and onion. Limit of detection (LOD) and limit of quantification (LOQ) were calculated based on standard deviation of seven repeated analyses of a sample with acrylamide concentration about 2 ng/g using following formulae, where n means number of analysis, t(n-1, 0.05) means t value of one-side-test with 5% significance level, and s means standard deviation of the analytical values.
Table 2. Result of recovery test of acrylamide.
| Added acrylamide /sample |
Potato | Onion | ||||
|---|---|---|---|---|---|---|
| Recovery (%) | Average (%) | Relative standard deviation (%) | Recovery (%) | Average (%) | Relative standard deviation (%) | |
| 0.2 µg / 10 g | 113.3 | 119.3 | ||||
| 113.6 | 113.3 | 0.3 | 112.5 | 116.4 | 3.0 | |
| 113.0 | 117.3 | |||||
| 1 µg / 10 g | 98.0 | 101.6 | ||||
| 97.9 | 97.5 | 1.0 | 95.4 | 98.3 | 3.2 | |
| 96.3 | 97.8 | |||||
| LOD = 2 × t(n-1, 0.05) × s |
| LOQ = 10 × s |
LOD and LOQ of potato sample were 4 ng/g and 10 ng/g, respectively, and those of onion sample were 3 and 8 ng/g, respectively.
The results of acrylamide analyses of potato and onion samples are summarized in Table 3, and the frequency distributions of acrylamide concentration are shown in Fig. 1. In cases of acrylamide concentration was less than LOD and less than LOQ, one-half of LOD and average of LOD and LOQ were adopted, respectively, to calculate the median and average. Median and average values of acrylamide in stir-fried potato were 2.0 ng/g and 11 ng/g, respectively. This median was markedly lower than those of fried potato (180 ng/g) and potato snacks including potato chips (550 ng/g) reported as result of monitoring in 2013 fiscal year by MAFF Japan5). For simmered dishes, potato are not necessary to be fully cooked during stir-frying. The incomplete cooking is likely to reduce formation of acrylamide. Among 53 stir-fried potato samples, acrylamide concentrations of 27 samples (51%) were less than LOD, and those of 13 samples (25%) were less than LOQ. Number of potato samples decreased with increase of acrylamide concentration (Fig. 1). Appearance of stir-fried potato samples are shown in Fig. 2. A sample with acrylamide less than LOD is whitish without dark brown part (Fig. 2A). On the other hand, the sample with maximum acrylamide has burnt parts especially at the edge (Fig. 2B).
Table 3. Summary of acrylamide analysis.
| Stir-fried sample | Median (ng/g) | Average (ng/g) | Maximum (ng/g) |
|---|---|---|---|
| Potato, n =53 | 2 | 11 | 120 |
| Onion, n =58 | 14 | 36 | 420 |
Fig. 1.
Frequency distribution of acrylamide content in stir-fried potato and onion samples.
Fig. 2.
Pictures of stir-fried potato and onion samples. A, potato with acrylamide < 4 ng/g; B, potato with acrylamide 120 ng/g; C, onion with acrylamide < 3 ng/g; D, onion with acrylamide 420 ng/g.
Among 58 stir-fried onion samples, acrylamide concentration of only one sample (2%) was less than LOD, and those of 15 samples (26%) were less than LOQ. Median and average values of acrylamide concentration in the stir-fried onion were 14 ng/g and 36 ng/g, respectively. These values are comparable to those reported for stir-fried onion by MAFF Japan (median 19 ng/g, average 25 ng/g)15). But the maximum value of stir-fried onion 420 ng/g in the present study is much higher than the reported maximum value of stir-fried onion (70 ng/g)15). Frequency distribution of the stir-fried onion samples shifted to higher concentration than that of the stir-fried potato (Fig. 1). A frequency peak was found at around 10 ng/kg. Besides number of sample was more at acrylamide concentration 31–40 ng/g than that at acrylamide concentration 21–30 ng/g, and sample with acrylamide concentration > 41 ng/g was 8. These onion samples with high acrylamide concentration may be due to a custom to stir-fry onion to brown as shown in Fig. 2D for flavoring. This suggests that onion stir-fried brown for flavoring of various dishes will be one of the intake sources of acrylamide from daily meal.
The volunteers of home cooking submitted information about storage temperature of potato and onion, weight of used vegetables and meat, size of their pieces, heat strength (low, medium or high) and heating time during stir-frying (Supplementary material 2 and 3). Size of cooking pans and type of material of the pans, including whether coated with Teflon or not, were also reported if possible. None of these factors, however, showed statistically significant relationship with acrylamide concentration in stir-fried potato and onion. Although the exact reason is obscure, this may suggest the involvement of multiple cooking factors in acrylamide formation. The dominating factor for the acrylamide formation may vary on a case-by-case basis, and none of these cooking parameters alone thus may be used to predict acrylamide concentration in stir-fried potato and onion. Color of stir-fried potato and onion was inhomogeneous within a piece (Fig. 2). Small burnt part, for example at the edges of the potato pieces in Fig. 2B and a part of the potato surface, may be a major source of acrylamide in spite of large light color area in the rest part. Estimation of acrylamide concentrations in a sample composed of big thick potato pieces by color analysis of a picture is, therefore, difficult while L* (lightness) of homogeneously crushed potato chips correlated with acrylamide content16).
Analysis of samples prepared by volunteering cooperators at home under the conditions of their accustumed home cooking afforded actual profiles of acrylamide concentration in stir-fried potato and onion for simmered dish cooking. Compilation of analytical data of individual food components like market basket total diet study is not applicable to risk assessment of hazards formed in cooking during mixing and heating of food stuffs. Moreover, cooking conditions at home varies depend on cooking customs, which reflect age and locality of origin of cook and family members. It is thus almost impossible to specify a set of conditions for a unified model cooking representing a certain type of home-cooked dish. Analyses of samples collected from survey cooking through general citizens are necessary to know actual situation of the dish. This study is a good example of survey of such a carcinogenic hazard, acrylamide, formed by heating, and will contribute to of acrylamide intake from home cooking in Japan.
Acknowledgments
We express thanks to volunteer cooperators who prepared stir-fried potato and onion samples. This study was supported by a grand from the Food Safety Commission, Cabinet Office, Government of Japan (Research Program for Risk Assessment Study on Food Safety, No. 1507).
Abbreviations: JECFA, Joint FAO/WHO Expert Committee on Food Additives; LOD, limit of detection; LOQ, limit of quantification; MAFF, Ministry of Agriculture, Forestry and Fisheries; MHLW, Ministry of Health, Labour and Welfare; SIM, selected ion monitoring
Footnotes
Conflict of interest statement: The authors had no conflict of interest to declare in this article.
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