Chapter 2
Downloaded via BETHEL UNIV on April 2, 2019 at 15:09:16 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Acrylamide Content of Vegetable Chips J. Stephen Elmore,* Fei Xu, Anisa Maveddat, Rea Kapetanou, Heming Qi, and Maria-Jose Oruna-Concha Department of Food and Nutritional Sciences, University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom *E-mail:
[email protected] Vegetable chips—beets, sweet potatoes, carrots, and parsnips fried like potato chips and sold separately or as a mixture—are an increasingly popular snack. Little data currently exists on acrylamide levels in these products, and there is sparse information on the free amino acid composition of these four vegetables, with asparagine, a precursor of acrylamide, being of interest. Acrylamide concentrations in 35 vegetable chip samples and free amino acids in two to three samples of each of the four vegetables were measured. Of the chip samples, nearly 70% (24 samples) contained levels of acrylamide above the European Commission’s benchmark value of 750 µg/kg for potato chips. In comparison, from 2012 to 2016, approximately 10% of potato chip samples exceeded this value. The food industry has reduced the acrylamide content of potato chips by using cultivars developed specifically for chip manufacturing. Because the vegetable chip market is still significantly smaller than the potato chip market, equivalent cultivar development has not yet occurred. Hence, it is more likely for vegetable chips to be high in acrylamide than potato chips.
© 2019 American Chemical Society Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Introduction Potato chips have often been highlighted as a major source of the potential carcinogen acrylamide. As a result, ongoing work by the food industry has resulted in a reduction in the acrylamide content of potato chips (1, 2). On January 10, 2011, based on European Food Safety Authority (EFSA) monitoring data from 2007–2008, the European Commission set indicative values for eight food product categories known to contain acrylamide. Investigations by the member states’ competent authorities were recommended if the acrylamide level found in a specific foodstuff exceeded the indicative value. An indicative value of 1000 µg/kg for acrylamide concentrations in potato chips was introduced (3). On July 19, 2017, member states’ representatives voted in favor of the European Commission’s proposal to reduce acrylamide in food. European Commission Regulation (EU) 2017/2158 summarizes mitigation measures and establishes benchmark levels for the reduction of acrylamide in food. The benchmark level for acrylamide in potato chips was set at 750 µg/kg. When benchmark levels are exceeded, food business operators shall review the mitigation measures applied and adjust processes and controls with the aim of achieving levels of acrylamide as low as reasonable achievable (ALARA principle). These regulations have been in place in European Union member states since April 11, 2018 (4). Vegetable chips are becoming an increasingly popular snack. Beets, sweet potatoes, carrots, and parsnips are fried in the same manner as potato chips and are sold separately or, more commonly, as a chip mixture. Few values have been reported for acrylamide concentrations in vegetable chips (5–9). Existing values are collated in Table 1. It is clear that more than half of these values are greater than the benchmark level and at least 11 of the 30 samples exceed the indicative value for acrylamide in potato chips. However, vegetable chips are not included in the current list of foods for which benchmark values exist (4). Acrylamide concentrations in vegetable chips should be investigated further, and this chapter provides new information on acrylamide levels in commercially available products. In addition, this chapter includes data on free asparagine levels in samples of the types of vegetables used for vegetable chip manufacturing. As asparagine is a key precursor of acrylamide, its levels in the vegetables used for chip manufacturing are also of interest, but, again, there is little published information on free amino acid levels in these types of vegetables. Although reducing sugar levels are also key in indicating the amount of acrylamide formed in chip frying, we did not analyze the vegetables for reducing sugars because samples were purchased from supermarkets in July and it was unknown whether they had been recently harvested or had undergone a period of storage (10). The parsnips were almost certainly stored as they are usually harvested during winter, but it was less certain for the other vegetables. Increased concentrations of sucrose and reducing sugars have been reported in cold-stored parsnips (11) and sweet potatoes (12). Asparagine changes in root vegetables other than potatoes during storage have not been reported. However, asparagine shows relatively small changes in potatoes during storage (13). 16 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 1. Published Data on Acrylamide Concentrations in Vegetable Chips Sample
Acrylamide Content (µg/kg)
2002–2006 Food and Drug Administration (5) Snyder’s of Hanover Veggie Crisps
832
Snyder’s of Hanover Veggie Crisps
1340
Terra Exotic Vegetable Chips
828
Trader Joe’s Vegetable Chip Mix with Salt
73
Blue Mesa Grill Sweet Potato Chips
4080
2014 Food Standards Agency (6)
17
Tyrrells Veg Crisps
962
ASDA Extra Special Mixed Vegetable Crisps with Sea Salt
2795
Waitrose Vegetable Crisps
2652
Kettle Vegetable Chips—Golden Parsnip, Sweet Potato & Beetroot Chips
1508
2016 Food Standards Agency (7) Tyrrells Veg Crisps
1324
Marks & Spencer Vegetable Crisps
360
Waitrose Vegetable Crisps
716
Kettle Vegetable Chips—Golden Parsnip, Sweet Potato & Beetroot Chips
812 Continued on next page.
Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 1. (Continued). Published Data on Acrylamide Concentrations in Vegetable Chips Sample
Acrylamide Content (µg/kg)
2017 Changing Markets (8) Tyrrells Sweet Potato Lightly Sea Salted Crisps
2484
Co-Operative Smoked Paprika Sweet Potato Hand Cooked Crisps
1681
2017 Ernährung und Kosmetik, Stiftung Warentest (9) 15 vegetable crisp samples
3 samples >1000 µg/kg 3 samples ~1000 µg/kg
18 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Experimental Materials Vegetable chips were purchased from various supermarkets in Reading, U.K. in June 2016. Components included orange sweet potato, purple sweet potato, parsnip, beet, golden beet, and carrot. Three products were orange sweet potato alone while the other eleven were vegetable mixtures. Mixtures of chips were separated into their components prior to analysis. All samples were ground into a fine powder in a coffee grinder before extraction. Vegetable samples were purchased from farmers’ markets and various supermarkets in Reading during July 2016. Vegetable samples included purple beets, carrots, and parsnips grown in the United Kingdom and sweet potatoes grown in the United States. Because vegetable chips are often prepared from unpeeled vegetables, those studied were sliced without peeling and then frozen for 1 hour in a blast freezer at –18°C. They were then freeze-dried for 96 hours. The freeze-dried samples were ground into powder using a coffee grinder. Acrylamide (>99%) was purchased from Sigma-Aldrich (St Louis, MO). 1,2,3-13C3-Acrylamide (1000 mg/L in methanol) was purchased from LGC Standards (Teddington, U.K.); 1 mL was diluted to 1 L in deionized water to provide a 1 mg/L stock solution.
Acrylamide Analysis The acrylamide extraction method was adapted from Halford et al. (13). Ground chip samples (1.00 ± 0.01 g) were weighed into 50-mL Falcon tubes. Samples were extracted with water (40 mL, containing 2 mL of 1 mg/L 1,2,3-13C3acrylamide) at room temperature. After shaking for 20 min using a mechanical shaker, tube and contents were centrifuged for 10 min at 9000 rpm at 15°C. Two milliliters were removed from the aqueous layer and passed through a 0.22-µm syringe into a 2-mL vial. All samples were extracted and analyzed in triplicate. The three replicate analyses were all from the same packet. Chip samples were analyzed by liquid chromatography–mass spectrometry/ mass spectrometry using a 1200 high-performance liquid chromatography system connected to a 6410 triple quadrupole mass spectrometer with electrospray ion source in positive ion mode (both Agilent Technologies, Santa Clara, CA). The mobile phase was 0.1% aqueous formic acid at a flow rate of 0.3 mL/min. Injection volume was 5 µL. An isocratic separation was carried out at room temperature using a 100 × 3.0 mm Hypercarb column with a 10 × 3.0 mm Hypercarb pre-column (both 5 µm particle size; Thermo Fisher Scientific, Waltham, MA). The transitions m/z 72→55 and m/z 72→27 were measured for acrylamide and the transition m/z 75→58 was measured for 13C3-acrylamide. Concentrations of acrylamide in chips were expressed as μg/kg fresh weight (13), calculated from a calibration curve prepared from solutions in water containing 1, 5, 20, 100, 500, and 1000 μg/L acrylamide alongside 50 μg/L 13C3-acrylamide. 19 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Free Amino Acids in Freeze-Dried Vegetables Free amino acids were extracted from powdered freeze-dried vegetables (0.100 ± 0.01 g) using 10 mL of 0.01 M hydrochloric acid. Samples were stirred at room temperature for 15 min and allowed to settle for an additional 15 min. One milliliter of the supernatant was loaded into a 1.5-mL Eppendorf centrifuge tube and then centrifuged at 7200 g for 10 min at room temperature. Samples were derivatized using EZ:faast (Phenomenex, Torrance, CA) and then analyzed by gas chromatography–mass spectrometry using a 6890 gas chromatograph with 5975 mass spectrometer (both Agilent Technologies). One microliter of the derivatized amino acid solution was injected at 250°C in split mode (20:1) onto a 10 m × 0.25 mm Zebron ZB-AAA capillary column (Phenomenex). The oven temperature was 110°C for 1 min, then increased at 30°C/min to 320°C, and held at 320°C for 2 min. The transfer line was maintained at 320°C, and the helium carrier gas flow rate was kept constant throughout the run at 1.1 mL/min. The ion source was maintained at 220°C in electron impact mode at 70 eV. Amino acid concentrations were expressed as mmol/kg per dry weight, calculated from a 3-point calibration curve, prepared using amino acid standards (13). Statistical Analysis Means and standard deviations for all data were calculated using Microsoft Excel for Office 365 (Microsoft Corporation, Redmond, WA).
Results and Discussion Acrylamide Contents of Vegetable Chips Acrylamide concentrations were measured in 35 vegetable chip samples: 9 orange sweet potato, 9 parsnip, 9 purple beet, 4 carrot, 2 purple sweet potato, and 2 orange beet. The values, summarized in Table 2, covered a similar range to those in Table 1, with a maximum value of 4457 μg/kg in an orange sweet potato chip and a minimum value of 135 μg/kg in a purple sweet potato chip. The largest variation within a product was for parsnip, with a maximum acrylamide concentration 8 times higher than the minimum concentration. There was a 5-fold variation between the highest and lowest values for orange sweet potato, which increased to 30-fold when purple and orange sweet potato data were combined. The limited number of data suggested that a shift from orange to purple sweet potato could result in chips lower in acrylamide, although, as only two purple sweet potato chip samples were studied, this could not be assessed statistically. Conversely, golden beets appeared to create chips lower in acrylamide than purple beets but again this observation could not be statistically confirmed. Of the 35 samples studied, 24 exceeded the European Commission benchmark level of 750 µg/kg for acrylamide in potato chips (4), while 18 exceeded the indicative value of 1000 µg/kg for acrylamide concentrations in potato chips (3). In particular, levels of acrylamide in all 9 orange sweet potato samples were above 20 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
750 µg/kg, with levels in 7 samples being above 1000 µg/kg. For purple beet chips, 7 exceeded the benchmark level and 4 the indicative value, while in parsnip chips the numbers were 5 and 4, and in carrot chips the numbers were 3 and 3. None of the purple sweet potato or golden beet samples contained acrylamide at a concentration above the benchmark level. It can be calculated that acrylamide concentrations in 68.6% of the samples exceeded the benchmark level while concentrations in 51.4% of the samples exceeded the indicative value of 1000 µg/kg. Powers et al. (2) pooled and analyzed published data on acrylamide levels in potato chip samples from 2002 to 2016. In 2002, the first year that moves were made to reduce acrylamide in food products, 42 potato chip samples were analyzed, with a mean value of 763 µg/kg and 23.8% of the samples exceeding the indicative value. In 2011, 12,213 samples were analyzed and only 3.2% exceeded the indicative value, with a mean acrylamide concentration of 358 µg/kg. From 2012 to 2016, acrylamide concentrations in potato chips have leveled off, with 3.7–6.2% of samples in each year being above the indicative value. In addition, approximately 10% of samples exceeded the benchmark value (based on 90% quantile data for each year (2)). Comparison of the vegetable chip acrylamide data presented in this chapter with the potato chip data from the 2002 study reveals that the mean value for acrylamide content across all the vegetable chip samples is 1214 µg/kg, around 1.5 times higher than that for the potato chips.
Table 2. Experimental Values for Acrylamide Concentrations in Vegetable Chips
Vegetable
Number of Samples
Mean (µg/kg)
Standard Error
Minimum (µg/kg)
Maximum (µg/kg)
Orange sweet potato
9
1713
202
868
4457
Purple sweet potato
2
171
16
135
207
Parsnip
9
1133
148
386
3019
Purple beet
9
1143
107
494
2231
Golden beet
2
592
20
552
632
Carrot
4
1266
145
566
1799
Free Amino Acids in Sweet Potato, Beet, Carrot, and Parsnip There is little information on free amino acids in root vegetables other than potatoes. Takahata et al. (14) analyzed eight varieties of sweet potato and found asparagine was the major free amino acid in all of them. Nandula et al. (15) reported arginine as the major free amino acid in carrots, followed by unresolved asparagine and aspartic acid. Kugler et al. (16) analyzed purple and golden beets 21 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
and found that in both types glutamine was the major free amino acid, followed by glutamic acid, then asparagine. There appears to be no published free amino acid data for parsnips. In this chapter, free amino acid data are presented for three different samples of orange sweet potato, three of purple beet, three of parsnip, and two of carrot (Table 3). The EZ:faast technique is not suitable for arginine, so values for this amino acid are not presented. In addition, histidine and ornithine levels were too low to quantify. Of the three orange sweet potato samples analyzed, asparagine was the amino acid present at the highest concentration, while in the other sample, aspartic acid was at significantly higher concentrations than asparagine (p < 0.05). Compared to the other vegetables studied, the relative molar concentration of asparagine was by far the highest in orange sweet potato, from 22% to 57%, which may explain, to some extent, why acrylamide levels were highest on average in orange sweet potato chips. Of the two carrot samples studied, alanine was the major amino acid in one and serine in the other. Arginine was reported as the major amino acid in carrots by Nandula et al. (15) but could not be detected using the EZ;faast technique. Relative molar concentrations of asparagine were low at 6-7%. In the three beet samples, levels of glutamine were very high; the one sample, bought from a farmers’ market, gave a value of 1.85 mol/kg dry weight. Although this value is well outside the calibration range used for this work and should be treated with caution, comparable values for glutamine in red and golden beets were reported by Kugler et al. (16). Because of these very high glutamine values, relative molar amounts of asparagine were very small, from 1 to 5%. Apart from glutamine and asparagine, serine, alanine, glutamic acid, and aspartic acid were present at relatively high concentrations in all three beet samples. Like carrot, parsnip was high in serine and alanine and, overall, carrots and parsnips were relatively similar in composition, when compared to orange sweet potatoes and purple beets. On average, asparagine was the third most abundant amino acid measured in all three parsnip samples, although it was only the fifth most abundant in parsnip sample 1. The relative molar amounts of asparagine varied from 7 to 18%. Parsnips and carrots are both members of the Apiaceae family. In comparison, relative amounts of asparagine in 20 varieties of potatoes, grown at the same location and analyzed using the same technique, varied from 13 to 31% (17), while relative amounts of the same varieties grown at a second site varied from 25 to 52% (18). These data, alongside reducing sugar and acrylamide data, showed the relative importance of asparagine in acrylamide formation when reducing sugar levels were low (18). These potatoes covered all types of food use, from chips to French fries and household use.
22 Granvogl and MacMahon; Food-Borne Toxicants: Formation, Analysis, and Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Table 3. Free Amino Acid Compositions of Purple Beet, Carrot, Parsnip, and Orange Sweet Potatoa Purple Beet Amino Acid
Carrot
Parsnip
Orange Sweet Potato
23
1
2
3
1
2
1
2
3
1
2
3
Asparagine
12.2
6.9
21.7
5.1
4.5
3.8
6.4
12.9
105.2
69.3
28.7
Alanine
21.6
11.0
44.2
17.1
14.3
8.1
14.1
18.2
7.3
9.4
6.4
Aspartic acid
20.0
15.7
15.0
10.0
10.4
4.3
2.5
6.1
25.2
10.4
38.0
Glutamic acid
12.0
13.2
16.8
3.9
5.5
tr
5.9
tr
3.7
11.9
6.8
Glutamine
89.2
89.1
1850
nd
nd
nd
nd
nd
nd
1.5
1.3
Glycine
2.9
3.9
8.7
1.9
2.7
1.5
1.4
1.6
6.1
6.0
2.9
Isoleucine
4.2
4.2
13.4
2.8
4.0
2.5
2.6
1.5
2.7
4.0
2.8
Leucine
4.0
3.9
17.4
3.9
4.8
3.9
3.0
2.4
4.5
6.1
4.9
Lysine
2.0
tr
9.5
nd
nd
nd
2.0
nd
nd
2.0
5.3
tr
tr
tr
tr
tr
tr
tr
tr
nd
1.1
0.8
Phenylalanine
0.6
tr
5.5
0.9
0.9
0.9
5.0
1.7
2.2
5.0
2.4
Proline
1.4
5
3.6
1.6
1.1
1.7
5.2
1.4
1.3
1.4
1.5
Serine
36.3
26.5
65.3
15.0
21.1
17.2
31.4
19.5
11.6
11.7
9.8
Threonine
7.2
3.8
7.7
2.3
2.4
2.4
3.0
1.7
9.7
7.0
7.2
Tryptophan
1.0
nd
1.9
nd
nd
nd
tr
nd
nd
nd
0.8
Tyrosine
0.8
nd
6.3
nd
nd
nd
1.2
nd
nd
1.1
1.9
Valine
6.6
3.0
15.6
4.4
4.4
3.6
3.9
2.8
5.0
6.4
4.7
Methionine
tr: trace (0.2–0.5 mmol/kg); nd:
