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Food, Agriculture & Environment Vol.2 (2) : 157-165. 2004
Investigation of acrylamide levels in selected fried and baked foods in Jordan Hani M. Al-Dmoor1*, Mohammad A. Humeid 2 and Mahmoud A. Alawi
3
1
Dr. of Food Science,Al-Balqa Applied University, Faculty of Agriculture, Department of Nutrition and Food Technology, Al-Salt, Jordan. e.mail:
[email protected]. 2 Department of Food Chemistry. e-mail:
[email protected]. 3 DEpartment of Analytical Chemistry & Environmental Analysis, University of Jordan, Jordan. *e-mail:
[email protected]
Received 11 February 2004, accepted 25 April 2004.
Abstract Acrylamide is a potential health hazardous compound, occurring in baked and fried food as a result of excessive dry heating during preparation and/ or processing of foods. This study aimed at investigating the acrylamide content in selected fried and baked foodstuffs commonly consumed in Jordan and comparing the effect of different processing factors on its level. Seventeen food items representing four groups of food (breads, fine bakery products, Arabic sweets and fried foodstuffs) were chosen and purchased from the local markets after collecting data on their formulation and preparation or processing conditions. The selected foods are produced under different conditions (ingredients, method of preparing, pH, heating time and temperature). Furthermore, a controlled study was conducted on four types of foods (falafel, fried kobbeh, potato and karabeej Halab). Acrylamide content was extracted with 2–butanone and analyzed via GC/MS/EI. Acrylamide was found in 15 out of 17 of the tested food samples at varying levels as a result of the differences in formulation and preparation or processing conditions. Acrylamide content of yeast fermented Arabic bread that is usually baked at high temperature and short time (35-45 sec. at 450-500ºC for thin Lebanese type and 60-90 sec. at 300-350ºC for thick kmaaj type), boiled potatoes and unheated dough of falafel, fried kobbeh and karabeej Halab were below the detectable level (180 µg kg-1) of acrylamide. Whereas the Arabic bread leavened by yeast and sodium bicarbonate (mashrooh) or solely by sodium bicarbonate (shrak) and baked under comparable conditions of Arabic bread were found to contain acrylamide at levels of 1200 and 1800µg kg-1 respectively. This demonstrates the effect of increasing pH, due to the addition of sodium bicarbonate as a leavening agent. In comparison to the rapidly baked Arabic bread, hamam bread that is a loaf type, baked at 280ºC for 18 min. and having a well developed brown crust showed an acrylamide average of 3300µg kg-1. Fine (cracker type) bakery products, Arabic sweets and fried foods, their pH values ranging between 6.2 to 8 showed relatively high concentrations of acrylamide (2400-5200 µg kg-1). Kirshaleh baladieh that is leavened by both bacterial flora and sodium bicarbonate was found to have the highest value (5200µg kg-1) among cracker types which contain 4300, 4700 and 4700 µg kg-1 or fermented kirshaleh, sweet kirshaleh and improved ka´ak respectively. Bormah crust which is thoroughly browned showed the highest level (4600 µg kg-1) of acrylamide among the Arabic sweet samples of kunafah crust, awamah, haresah and karabeej Halab, that contained 2900, 4000, 4200 and 4200 µg kg-1, respectively. Fried potato fingers (French fries), that represent simple starchy food contained high level of acrylamide (4100 µg kg-1) compared with falafel (3500µg kg-1) and fried kobbeh (3600 µg kg-1) that represent composed foods rich in protein. Extending the time of frying of falafel, karabeej Halab and fried kobbeh caused a significant increase in the acrylamide content. The excessive use of frying oil caused a significant increase in the acrylamide content (33%) in falafel compared with that fried in fresh oil. Key words: Arabic bread and sweets, fine bakery products, fried foodstuffs, processing.
Introduction Acrylamide is a highly water-soluble compound with a low molecular weight. It could be easily polymerized to give polyacrylamide. It is used as a papermaking aid, a soil-conditioning agent, a separation gel in analytical biochemistry work and as a clarifying agent in sewage and wastewater treatment as well as in drinking water 2. In April 2002, a group of Swedish scientists presented results on detected trace levels of acrylamide in baked, roasted and fried food 26. In June 2002, World Health Organization (WHO) and the Food and Agriculture Organization (FAO) convened an expert consultation on acrylamide 29. The meeting concluded that the presence of acrylamide in food is a major concern, since it is a probable human carcinogen, and recommended more research on acrylamide occurrence, mechanisms of formation and toxicity 29. The awareness about acrylamide as a hazard which is currently under review by several world health authorities and scientists has focused the attention on the occurrence and formation of acrylamide in food. The WHO/FAO consultation advised that food should not be cooked excessively, for too long time or at too Food, Agriculture & Environment, Vol.2 (2), April 2004
high temperature, but also advised that it is important to cook food thoroughly (particularly meat and meat products) to destroy foodborne pathogens that might be present 29. Analysis conducted on various food samples collected in the United Kingdom, Norway, Switzerland and the United States of America have verified the results of the Swedish researchers, who found that acrylamide is formed primarily as a result of the preparation of carbohydrate-rich foods at high temperatures (>120°C). Relatively high levels of acrylamide were found in potato chips (French fries), potato crisps and other fried, deep-fried or oven-baked potato products, together with some crisp bread, biscuits, crackers and breakfast cereals 9. The presence of high level of acrylamide (more than 10000 µg kg-1) in overcooked fried chips demonstrates the influence of cooking temperature and cooking duration 24. No acrylamide was detected in raw and boiled foodstuffs. The levels of acrylamide found in the prepared carbohydrate-rich foods were much higher than that specified for drinking water (0.5 µg L-1) 27. Other foodstuffs that undergo similar processing may also contain acrylamide; however, no data are 157
available for many commodities of food 29. Other expected source of acrylamide may be due to the contact between food and food packaging materials 2. The migration of acrylamide into food should not exceed 0.01 mg kg-1 of food 7. Recent studies 3, 16, 17, 19, 21, 30 show that the pathways of acrylamide formation probably involves Strecker degradation of amino acids, especially asparagine in the presence of dicarbonyl products from the Maillard reaction. The production of acrylamide is indissolubly linked to the formation of colors and flavors in food preparation. Acrylamide avidly participates in nucleophilic addition reactions with nucleophile (electron-rich) compounds which include protein functional groups such the SH group of cysteine, N-terminal NH2 groups, e-NH2 groups of lysine and the NH group of the imidazole ring of histidine 16. Acrylamide is very rapidly absorbed through the intestinal tract and fairly evenly distributed throughout the body organs. However, with the exception of testes, it does not accumulate in any particular tissue. Once exposure ceases, it is rapidly excreted from the body. Among the acrylamide metabolites glycidamide is considered the most likely candidate for causing genetic damage. Glycidamide has been found in mice, rats and human who were exposed to acrylamide 25. High doses of acrylamide (>203 mg kg-1) caused adverse developmental and reproductive effects in neonatal rodents 11. For example, nerve degeneration and abnormal changes in intestinal enzymes have been observed. Abnormal sperm, reduced fertility and spontaneous abortions have been elevated in treated rodents. Acrylamide is considered to be genotoxic in vivo and carcinogenic in experimental animals 2, 13. A joint epidemiological study by researchers from the Swedish Karolinska Institute and the American Harvard University 18 was based on food-frequency questionnaire data from a 1999 population-based Swedish study of diet and cancer along with the food administration’s data on the acrylamide content of foods. The study showed no correlation between the high intake of acrylamide in food and intestinal, bladder or kidney cancer. Also in another study 6 an evidence was found for the lack of an important association between consumption of fried/baked potatoes and cancer risk. The acute oral LD50 for acrylamide in rats and mice is 107-203 mg kg-1 of body weight and the acute dermal LD50 for rats was reported to be 400 mg kg-1 of body weight 16. The no observed effect level (NOEL) for acrylamide was reported to be up to 2000 µg kg-1 rat body weight per day 29. Jordanians consume considerable amounts of baked and fried traditional foodstuffs, which are expected to contain acrylamide. No studies have been carried out on the acrylamide content of these foods. The main objectives of this study were to evaluate the levels of acrylamide in selected fried and baked foodstuffs in Jordan, and to compare the effect of different processing factors on these levels. Materials and Methods Food items: Seventeen samples of locally produced food that represent four groups of food were collected from different production sites in Amman. The ingredients and preparation conditions of the products were recorded using a questionnaire. Heating temperatures and time were measured during sample collection using a portable digital thermometer (Multi– Thermometer, LCD, USA) and a stopwatch. pH of dough samples and raw potatoes was measured by mixing 10 g of sample in 100 ml 158
water as described in the American Association of Cereal Chemists1 method No. 52-2 using a pH meter (Biosan, Spain). The food groups were bread, fine bakery products, Arabic sweets and fried food. Group 1 bread: Arabic bread (thin Lebanese type); white, flat, very thin (~ 3 mm), pocket forming, produced from straight flour (extraction rate 78%), leavened by yeast fermentation, short baking time, (35-45 sec. at 450-500ºC). Arabic bread (thick Kmaaj type); white, flat, thickness (8-10 mm), pocket forming, produced from straight flour (extraction rate 78%), leavened by yeast fermentation, relatively short baking time, (60-90 sec. at 300-350ºC). Mashrooh; white bread with some bran and brown spots on the surface, flat, relatively thin (4-6 mm), not pocket forming, produced from straight flour (extraction rate 78%), leavened by yeast fermentation in addition to sodium bicarbonate >1800 ppm, relatively short baking time, (~ 60 sec. at 300-350ºC) 8. Shrak; White bread with some bran and brown spots on the surface, flat, very thin (1-2 mm), not pocket forming, produced from straight flour (extraction rate 78%), leavened by addition sodium bicarbonate >1800 ppm, short baking time (~ 30 sec. at 360-370ºC) on a hot plate. Hamam; Small loaf white bread, form a browned crust, long baking time (15–20 min at 280–300ºC), produced from zero flour (extraction rate 72%) with addition of milk, oil, improvers (α-amylase, emulsifying agent and ascorbic acid) and leavened by yeast fermentation. Group 2 Fine bakery products: Fermented kirshaleh; A cracker type with substantial brown crust, composed of white flour (zero type, extraction rate 72%), milk, oil/fat, sesame seeds, water and leavened by yeast fermentation, long baking time (20–25 min. at 280–300ºC). Sweet kirshaleh; A cracker type with substantial brown crust, composed of white flour (zero type, extraction rate 72%), milk, sugar, oil/fat, water and leavened by ammonium bicarbonate, long baking time (20–25 min. at 280–300ºC). Improved ka´ak; A cracker type with substantial brown crust, composed of white flour (zero type, extraction rate 72%), milk, sugar, oil/fat, improvers (α-amylase, emulsifying agent and ascorbic acid), water and leavened by ammonium bicarbonate, long baking time (20–25 min. at 280–300 ºC). Kirshaleh baladieh; A cracker type with substantial brown crust, composed of white flour (zero type, extraction rate 72%), milk, sugar, oil/fat and leavened by chickpea steep liquor (a fermented liquid, containing active bacterial flora, prepared by pouring hot water (90ºC) on coarsely broken chickpeas followed by incubation at 40ºC for 48-72 hours) and by sodium bicarbonate, long baking time (20–25 min. at 280–300ºC)14. Group 3 Arabic sweets: Kunafah; A crust made of vermicelli like threads prepared from a white flour batter, baked in a metal plate on flame heater after dying and adding shortening and filling with desalted white cheese, sweeten after baking with a heavy sugar syrup, baked at 180–200ºC/contact surface for ~10 min., analysis was carried out on the crust without filling, crust was 40% of the whole sweet. Awamah; Fried small dough balls resembling doughnuts type sweet, substantial browning crust, deep fried at 160-180ºC for 6-8 min. in vegetable oil, leavened by yeast, sweeten by dipping after Food, Agriculture & Environment, Vol.2 (2), April 2004
frying in a heavy sugar syrup. Haresah; A crust made of coarse semolina, milk, sugar and small quantity of oil, leavened by sodium bicarbonate, spread on greased metal plate baked in an oven at 200-250ºC for 25 min., sweeten after baking with heavy sugar syrup. Karabeej Halab; Fried longitudinally extruded dough resembling doughnuts type sweet, substantial browning crust, deep fried at 160-180ºC for 6-8 min. in vegetable oil, composed of flour, milk, eggs and leavened by yeast, sweeten by dipping after frying in a heavy sugar syrup. Bormah; A crust made of coarse vermicelli like threads prepared from a white flour batter, stuffed with pistachio, deep fried at 160180ºC for ~ 15 min. in vegetable oil, through browning, analysis was carried out on the crust without stuffing, crust was 50% of the whole sweet. Group 4 Fried food: Falafel; A dough made of a mixture of soaked ground chickpeas, leafy vegetable, onions, spices and leavened by sodium bicarbonate, formed to patties just before deep-frying in vegetable oil for 6-8 min. at 160–180ºC, gritty crust and thoroughly browned. Fried Kobbeh; A dough made of a ground bulghur mixed with minced beef meat formed as balls stuffed with cooked ground meat, onion, nuts and spices, deep fried at 160-180ºC for 8- 10 min. in vegetable oil, gritty crust and thoroughly browned. Analyses were carried out on the crust without stuffing; crust was 70% of the whole piece. Fried potato; Fingers (6-8 mm) thickness, deep-fried at 160-180ºC for 6-8 min. in vegetable oil. Sample preparation and extraction of acrylamide: The food samples were prepared and analyzed using method of Biedermann et al. 4 with some modifications to optimize the extraction and recovery of acrylamide from food samples. The fresh food samples were disintegrated, homogenized and a representative sample of a 20 g was accurately weighed and allowed to swell by soaking in 200 ml of hot distilled water (60±1.0°C) in a 600 ml beaker for 20 min. The swelled food sample was homogenized by a mixer (Rotel, Aarburg, Switzerland) for 2 min. Forty ml of the homogenate was transferred into a 50 ml screw capped centrifuge tube and centrifuged at 4000 rpm for 30 min. using (Centaur 2 centrifuge, MSE, UK). Twenty five ml of the supernatant was transferred into 100 ml glass centrifuge tube. Twenty g of ammonium sulfate (BDH Laboratory, England) and 25 ml 2-butanone (Fluka Buchs, Switzerland) were added, shaken for 20 min. using a mechanical shaker (Memmert, Germany) and centrifuged for 15 min. at 4000 rpm. Twenty ml of the organic phase layer (butanone) was transferred into a 25 ml test tube. The organic solvent was evaporated with the aid of a stream of nitrogen which was directed over the liquid in the test tube that was fitted in a thermostatically controlled aluminum heating block adjusted at 40±1.0°C, especially constructed for this purpose by the researching group. The dried residue was redissolved in 800 µl of acetonitrile (GC grade, Lab-Scan, Dublin, England) and 2 ml of hexane (HPLC grade, Lab-Scan, Dublin, England), and then mixed by vortex (V1 plus Biosan, Spain) for 30 sec. The fat in the extracted sample was removed by mixing the acetonitrile layer two times with 2 ml of hexane and the hexane layer was always sucked with a glass Pasteur pipette and discarded. The fat free acetonitrile (800 µL) was placed in a 1.5 ml GC-glass vial, mixed with 200 µL of 5 mg L-1 acrylamide Food, Agriculture & Environment, Vol.2 (2), April 2004
(acrylamide 99.9% SIGMA, O5632, USA) as a standard addition techniques and placed in the GC autosampler for GC/MS analyses. GC/MS analysis: The prepared samples were analyzed using Shimadzue GC/MS (Model QP5050A; Shimadzu Inc., Koyoto, Japan) operated on the EI (electron impact ionization) as shown in Appendix 2. 1 µL of the final extract was autoinjected on the splitless mode onto a 100 cm x 0.32 mm i.d. precolumn deactivated with diphenyl tetramethyl disilazane (BGB Analytik AG, Anwil, Switzerland), connected to a 30 m x 0.32 mm i.d. analytical column coated with a 0.25 µm film of FFAP (Free Fatty Acid Phase) (BGB Analytik AG, Anwil, Switzerland). The carrier gas was helium (99.999%), inlet pressure 75 kPa and the flow rate 50 ml min-1. The initial oven temperature was 110°C and programmed as follows: 110-230°C (10°/min), 230-250°C (25°/min) and 250°C (1 min.). The injector temperature was 250°C. Mass spectrometry involved positive ion electron impact ionization (EI) with full scan monitoring (m/z 42-100) and the ion source was at 230°C. Calibration curve of acrylamide: The calibration curve was prepared for acrylamide using different concentrations and the respective peak areas. Acrylamide (99.9 %, SIGMA, O5632, USA) was dissolved in acetonitrile (GC grade, Lab-Scan, Dublin, England) and the concentrations of 0.25, 0.5, 1.0, 2.0 and 2.5 mg L1 were prepared and measured using the GC/MS under the above mentioned working conditions. Determination of % recovery: Two quantitative recoveries at two concentrations (0.5 and 3.6 µg mL-1) of acrylamide were determined by complete analyses of three replicates using 20 g of dough sample precisely spiked with 550 µL of 10 µg mL-1 acrylamide to have a final concentration of 0.5 µg mL-1 and 400µL of 100 µg mL1 acrylamide to have a final concentration of 3.6 µg mL-1. The extracts were injected three times onto the GC/MS column at three separate days. Recoveries, standard deviations and coefficient of variations were calculated using acrylamide calibration curve . Method reproducibility: Analysis of 1 µg mL-1 of the acrylamide standard solution in triplicate at seven separate days was used to calculate the reproducibility for the method given as the coefficient of variations is about 5.1%. Standard addition technique: An addition of acrylamide standard solution (200 µL of 5 mg L-1) to the final extracts of every food sample was performed in order to confirm the retention time (6.2±0.05 min.) and to increase the recovery of minute amounts of acrylamide in some food samples. Detection limits (DL): The detection limit of the method was calculated from the reproducibility of 1 mg L-1 using the following mathematical equation 31: DL = t 0.99, (n - 1) . STD , DL = 3.707 x 0.05 = 180 µg L-1. where STD = standard deviations of analysis of 1 mg L-1 of the acrylamide as shown in Table 3, n = number of replicates (7), t (tabulated) = 3.707 at 99% confidence limit 23. Controlled study: A controlled study was carried out on four products to investigate the effect of frying time, effect of excessive use of frying oil and effect of heating methods as shown in Table 1. 159
Table 1. Fried food items were subjected to different treatments. Food item
Heat treatment
Exposed time
Falafel Falafel Falafel Falafel Fried Kobbeh Fried Kobbeh Fried Kobbeh Karabeej Halab Karabeej Halab Karabeej Halab Boiled potato Fried potato
Unheated Frying Frying Frying Unheated Frying Frying Un heated Frying Frying Boiling Frying
0 4 min 6 min 6 min 0 5 min 10 min 0 4 min 6 min 30 min 8 min
1
Temperature ºC 0 170 170 170 0 170 170 0 170 170 100 170
States of oil used Old1 Old Fresh2 Old Old Old Old Old
Intermittently used for 48 h, 2 freshly filled
Statistical analysis of experimental data: The data obtained were analyzed for significance using the general linear model (GLM) procedure of the SAS institute Inc., Cary, NC, and USA 1998 version seven software. Duncan’s multiple range tests was applied to determine significance between different treatments 20. Results and Discussion Acrylamide content in selected types of bread: Table 2 presents the pH and acrylamide concentrations (µg kg-1) of the selected types of bread. An example of subtracted GC/MS chromatogram of acrylamide in hamam bread (as an example of this group) is shown in Fig. 1. It is obvious from the results that Arabic bread, regardless of its thickness, does not contain acrylamide (less than the detection limit DL of the used method which was found to be
