Research: Effects of Frying Oil on Acrylamide Formation in Potatoes

Abstract
In 2002, Swedish scientists discovered acrylamide formation in some starchy foods prepared at high temperatures (1). This has since become a major concern in the food industry due to the potential negative health effects of acrylamide.  Different cooking oils have varying fatty acid contents, which are likely to affect acrylamide formation.  In order to investigate this difference, potatoes were fried in corn, soybean, and sunflower oil.  A standard of acrylamide-spiked Pringle chips, for which the acrylamide content was known, was used.  The acrylamide was removed from each sample utilizing the Soxhlet extraction method, using first pentane and secondly methanol as solvents.  The resulting extracts were evaporated, resuspended in methanol, and analyzed via GC-MS.  It was found that the potatoes fried in sunflower oil had the highest level of acrylamide formation.

Introduction

The compound 2-propenamide, commonly known as acrylamide, is an α-β unsaturated conjugate molecule with the structure H2C=CH—CONH2 (Figure 1).  Acrylamide’s vinylic structure makes it a convenient tool in biochemical research to selectively modify thiol groups in compounds (2).   However, this reactivity is likewise effective on biological molecules, and therefore poses a danger upon human exposure.  This study investigates exposure to acrylamide from a common food source—fried potatoes—and how the conditions under which the source is prepared affects the formation of acrylamide.

Consistent evidence from recent studies has shown that acrylamide is formed in foods with a high content of the free amino acid asparagine and of reducing sugars.  Taubert, Harlfinger, et al. discovered that when these conditions are met under high temperatures (120-230°C), acrylamide formation is possible by the Malliard browning reaction (3).  The optimum conditions for such formation in food occur in potato chips, since potatoes contain very high concentrations of asparagine per mass (93.9 mg/100 g) (2).   When exposed to heat, the α-NH2 group of asparagine reacts with the aldehyde of the D-glucose sugar, also present in high concentration in potatoes, undergoing a nucleophilic addition to form a Schiff base.  The Schiff base then rearranges to the asparagines derivative N-glycoside, an intermediate which can undergo decarboxylation and loss of the asparagine α-NH2 to form acrylamide.  This heat induced reaction between an amino acid and reducing sugar is the oxidative Malliard browning reaction (2).  Other amino acids such as methionine, arginine, threonine, and valine can also form acrylamide through this reaction, but these play a minor role in comparison with asparagine. (2)

Once formed, acrylamide from external sources can be easily absorbed by inhalation, ingestion, or skin absorption, and reacts with proteins to form its metabolite glycidamide, an epoxide, which can then interact with DNA.  The electrophilic double bond of acrylamide also allows it to interact with other active hydrogen-containing functional groups in the body, especially –SH and α-NH2  groups of free amino acids, and the NH group on histidine (2). The effect of these reactions is the formation of hemoglobin adducts and neurotoxins, as indicated by the work of Calleman, Stern, et al. (4) . These adducts and toxins have led to serious health effects such as protein malfunction and muscle control problems.

Discovery of acrylamide in potatoes and other food sources has sparked study of the various conditions in which acrylamide can form during cooking.  Although extensive studies have been done on the temperature dependence of acrylamide formation, less time has been devoted to investigating the effects of the type of cooking oil used for frying.  This study looks at three common cooking oils—corn, sunflower, and soybean oil—and how their properties influence acrylamide formation via the Malliard reaction.  Upon heating, variation in triglyceride and fatty acid composition among the three oils causes differing rates of degradation and contaminant formation, which may or may not influence acrylamide formation.  Among the differences in post-frying oil composition were a far higher branched-chain and steryl ester content in corn oil than either of the other two, and greater “flavor stability” in corn oil fried products (5). Likewise, triglyceride composition becomes altered upon heating in all three oils, where production of polar contaminants is proportional to the amount of unsaturated fatty acids present (6). These characteristics also cause varied rates of temperature increase and thus may impact Malliard reaction conditions.

Materials and Methods
The first stage of the experiment involved verifying the current methods of acrylamide extraction and detection.  For this verification, one container of Pringles® Original was obtained and ground to a fine powder using a mortar and pestle.  This powder was divided into four samples of approximately 40 grams each and placed in cellulose thimbles for the Soxhlet extractors labeled Samples 1-4.  The mass of each sample, including powder and thimble, was determined and recorded (Table 1).

Samples 1 and 2 were spiked with 630 µL of 0.01791 M acrylamide in methanol (8.0 x 10-4 g). Each sample was then placed in a Soxhlet extractor, and 200 mL of pentane was added to the extractor.  The Soxhlet extractors were used to extract the acrylamide from the fried potatoes.  Soxhlet extraction is known to be extremely effective in extractions of acrylamide (7, 8). The samples were then heated and refluxed in the Soxhlet extractor.  Samples 1 and 3 were refluxed in pentane for two days, while the pentane in samples 2 and 4 was replaced after 24 hours with 200 mL acetone.  Extra pentane solvent was added to Samples 1 and 3 to maintain a sufficient volume for refluxing.  After the fat present in the samples was sufficiently solvated, the thimbles containing defatted sample were removed from the extractor, dried in an oven to evaporate the pentane, and changes in mass were determined and recorded.  The solution of pentane in the Soxhlet extractor was then replaced with approximately 200 mL methanol, to solvate acrylamide, and the sample was heated and refluxed for seven days.

Following the extraction of Samples 1-4, the cellulose thimbles were removed from the Soxhlet extractors, dried to evaporate residual methanol, and the new masses were then determined and recorded.  The methanol containing the acrylamide in solution was collected from the Soxhlet extractor.  The samples were evaporated on a  rotary evaporator down to a small, measured volume for each sample, between 0-3 mL.  This volume was centrifuged to remove any residual particles as a “pellet,” and a 1 µL aliquot was run on a Gas Chromatograph/Mass Spectrometer (GC/MS) to determine compound identity and concentration (Table 2). The GC-MS technique was chosen for this experiment because this technique has worked successfully in the past for similar experiments.

A calibration curve was generated by injecting 1 µL samples of acrylamide in methane into the GC/MS at the calculated concentrations of 173 ppm, 108 ppm, 43.25 ppm, and 0.16 ppm (this curve originally also incorporated 161 ppm and 16 ppm samples, but these were later rejected due to inconsistency).  These concentrations were calculated by dividing the number of moles of acrylamide added by the 100 mL volume of the methane solvent.

Once the calibration curve had been created from the standard solutions generated in the laboratory and acrylamide concentration values were obtained from Samples 1-4, the test samples were prepared. Two potatoes were obtained for each of three sample oils, plus one potato for a control.  These potatoes were sliced into small pieces approximately 1 cm3 in size and then fried for 15 minutes in approximately 700-1000 mL of oil preheated to 160°C.  Some variation after addition of potatoes was noted (see Discussion).

After frying was complete, the samples were dried to remove excess oil, and ground into a course powder using a mortar and pestle.  This powder was then divided into 2 to 3 samples for each oil type, placed into cellulose thimbles, and the mass was determined for each sample and recorded.  The samples included Soybean Oil #1, #2 and #3, Sunflower Oil #1, #2 and #3, and Corn Oil #1 and #2.  Finally, the procedure above for extraction and detection of acrylamide was repeated with several changes to the procedure.

First, a refrigeration system was used to maintain the Soxhlet condenser at 10° C.  Also, no additional acetone solvent was added to the samples after 24 hours, as this was determined to solvate only acrylamide added, but no acrylamide contained naturally in the potato chips (see Discussion).  Lastly, additional centrifuging was necessary to remove all residual particles from the samples before GC/MS injection.

Results and Discussion
Acrylamide formation in fried foods is emerging as a major concern in the food industry given its potential adverse health effects.  As acrylamide formation predominantly occurs during the cooking process, an investigation of the effects of different cooking oil types was done.  Since corn, soybean, and sunflower oil vary in triglyceride and fatty acid composition, their rates of degradation and contaminant formation are likely to vary as well.  Thus, there should be a difference in acrylamide formation depending on which type of oil is used in cooking.

In this study, a standard sample with a known concentration of acrylamide was used to validate the experimental techniques used in the study.  The standard chosen was Pringles Original Baked potato chips.  Although the preparation method for these differed from the frying technique commonly used to make potato chips, these were specifically chosen because they contained the highest acrylamide amount of any such product on the market at 1200 ppb (9). The standardization included two control samples, as well as two samples spiked with additional acrylamide.  One spiked sample and one control sample was dissolved in acetone while the other  spiked sample and the other control sample was dissolved in methanol in order to compare the most effective method.  However, when the extraction was complete, the acetone had solvated no acrylamide from the un-spiked Pringles sample, and therefore only the samples solvated in methanol samples were used.  Upon analysis of the samples post-Soxhlet processing, the difference between spiked and un-spiked samples was used to calculate the percent of acrylamide extracted.  The calculation included using the total mg of acrylamide in the spiked sample and subtracting this value from the amount that should have been removed from the potato, found from the un-spiked control trial.  This difference gives the acrylamide yielded from spiked, which is divided to give a 94 percent extraction. This meant results gathered could be taken as the data from a valid technique.

The three trials of sunflower and soybean oil, and two trials of corn oil yielded different concentrations of acrylamide.  The average for each oil is shown in Figure 8, indicating sunflower oil to have the highest acrylamide concentration, followed by corn oil, and lastly soybean oil.  Figure 9 shows the corrected values with respect to percentages extracted based on the internal methyl acrylamide standards.  These adjusted values were calculated by taking the parts per million present in each sample vial multiplied by the solvent and its density, dividing by 1000 to give the mg amounts.  To calculate the ppm (mg per kg) value, this value is divided by total mass.  The numbers calculated using these corrected values, however, cannot be taken individually as “parts per million” figures; rather, they must only be considered in terms of their relative amounts in comparison to the other oils to show that the acrylamide formation trend is sunflower, corn, then soybean.  This is because these values are found using the internal standard, methyl acrylamide, of which the affinity of extraction, as compared to acrylamide, is unknown.  While acrylamide was shown to be extracted at 94 percent as per the experimental method, methyl acrylamide extraction varied from 38 percent to 80 percent.  Without correction, however, measurements of the three oils can be considered as numerical parts per million values, calculated from acrylamide extracted after concentrating each sample with a rotary evaporator from approximately 35 grams to 2 grams of acrylamide in methanol solution.

Several factors may have swayed experimental results that must be accounted for in terms of human error.  Soxhlet extraction is a notably accurate method of extraction, and it is not expected that any acrylamide was lost in the solvation process.  No acrylamide should have been lost in the pentane solvent, as acrylamide is insoluble in pentane, however, the pentane was not checked for the presence of acrylamide, which may have been an oversight.

Some variation in slicing causes each piece of potato to fry at a slightly different consistency in the oil.  An attempted to account for these size discrepancies was done by taking a random sample of twelve pieces and calculating the average surface area to volume ratio for all pieces.  Along with these incongruities in sample preparation, some oversights in frying must also be accounted for.  Because of the different heat drop and recovery times when the potatoes were added to the oil, the volumes of oils used in frying differed.  Only about 800 mL were used to fry the soybean samples, while 1000 mL were used to fry the sunflower and corn samples.  Because soybean oil takes much longer to recover it heat, a smaller volume was used in an effort to keep temperature of oil versus time consistent with the other two oils.  However, this changed the oil to potato ratio, and better consistency in this ratio would have reduced the error.

The key conclusion to be made based on the results of this study, shown by both the adjusted and unadjusted values of acrylamide found after frying and extraction, is that a difference in acrylamide formation can be seen amongst the three oils.  Sunflower oil generates the largest concentration of acrylamide, while soybean oil generates the least.  Interestingly, sunflower oil has the lowest specific heat value of the three oils tested, while soybean had the highest.  However, an attempt to correlate acrylamide formation with specific heat values cannot be accepted as legitimate.  Experiments displayed drops in temperature followed by varied rates of recovery for each oil during the frying process, but the net temperature difference of each oil is the same.  That is, while sunflower oil, with its low specific heat dropped the most and the quickest in temperature, its temperature also increased the fastest to reach the 160°C frying temperature.

Although acrylamide formation may be temperature dependent, this temperature dependency is not altered by the specific heats of the oils.  This means a likely possibility for influencing the formation of acrylamide may lie in the inherent compositions of the oils themselves before and after heating.  Because the composition of each oil differs in triglyceride and contaminant content, it is possible the presence of certain molecules in the oils, whether from the original plant source or added or created during the refinement process could inhibit acrylamide formation.  In fact, recent studies by Danisco et al. have found biomolecules in seaweed with the ability to oxidize the reducing sugar before it has a chance to react with asparagine in the Maillard reaction (10).  Further experiments must be done to determine if such molecules could be found in specific cooking oils, as this could make a significant impact on food preparation techniques.

Acknowledgements
The authors would like to thank professors Gordon Gribble and Siobhan Milde for their guidance. The research was conducted as part of the Chem. 63 course.

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