Determination of the Aldehyde Levels in Frying Oil Using p-Anisidine Test Lee C.Y.1, Zhou W.B.

2 and Bansal G.3

Department of Food Science and Technology, Faculty of Science, National University of Singapore 10 Kent Ridge Road, Singapore 117546 ABSTRACT During frying, extensive oxidation of the lipids take place, producing aldehydes as secondary oxidation products. The levels of aldehydes in sunflower oil, palm-olein and cooking oil were determined using the p-anisidine test and AlkalSafeTM test. Both tests agreed that sunflower oil contained the highest amount of aldehydes, followed by palm-olein and cooking oil. This confirmed that the initial level of unsaturated fatty acids was one of the factors that determined the levels of aldehydes. The correlation between the two tests was found to be fair though, the results from both tests appeared to have different trends for the three types of oil. INTRODUCTION Deep fried food is very popular nowadays and many health issues associated with the quality of the oil used for frying have been raised. Oxidation of the unsaturated fatty acids starts via a free-radical reaction, producing hydroperoxides (RO2H), which are the primary oxidation products. The hydroperoxides are very unstable and quickly decompose via C-C bond scission to form secondary oxidation products, such as aldehydes, alcohols or hydrocarbons. The objective of this project is to evaluate the levels of aldehydes in cooking oil, sunflower oil and palm olein that has been used for frying chicken nuggets using the p-anisidine test and AlkalSafeTM test kit. The p-anisidine (para-anisidine) test, the AOCS official method for determining the levels of aldehydes, was used to determine the level of non-volatile aldehydes, principally 2,4-dienals and 2-alkenals (Tompkins and Perkins, 1999). The samples were also tested with the AlkalSafeTM kit, which measures the concentration of alkenals (Osawa et al., 2007) in the samples in nanomoles per milliliters.

1 2

Student Supervising lecturer 3 Co-supervisor

MATERIALS AND METHODS Sample Five litres of sunflower oil (SO), palm-olein (PO) and cooking oil (CO) were used to fry chicken nuggets. The oil was maintained at about 180oC to 185oC for an hour before the frying and also throughout the frying. Frying was carried out for approximately 100g of chicken nuggets at 30 minutes intervals. Samples were collected at every 5 fry intervals, such that samples ranging from 0 fry to 45 fry were obtained. Determination of p-Anisidine value p-Anisidine was dissolved in glacial acetic acid to make up a 0.25g/100ml solution. Isooctane was used as a solvent for the oil samples. The test was conducted in triplicates for all samples. 0.5g to 0.7g of the oil samples were accurately weighed in 25ml volumetric flasks, noting down the mass of oil sample used (m). It was then diluted to volume with isooctane. The absorbance (Ab) of the resulting solutions at 350nm was measured using isooctane as the reagent blank. Glass cuvettes were used for all absorbance measurements. 5ml of the solution was then pipetted into a test tube and 5ml of isooctane into another test tube. 1ml of the p-anisidine solution was added to both and the solutions were mixed. After 10 minutes, the absorbance (As) of the sample solutions was read with the isooctane as blank. The p-Anisidine values were calculated using the following equation: (1) Determination of aldehyde concentration using AlkalSafeTM kit AlkalSafeTM kits were purchased from Safety Associates, Inc. The preparation reagent (99% propanol) was used to dilute the oil samples and AlkalSafeTM reagents A (92% propanol) and B (99% propanol) were also added to the oil samples. Calibrators of aldehyde concentration of 8nmol/ml, 16nmol/ml, 32nmol/ml and 64nmol/ml were used to plot a standard curve of absorbance against concentration. 500l of the oil samples were diluted in 1.5ml of the preparation reagent. These solutions were vortexed for about 5s and 150l of the solutions were then pipetted into a new tube. Reagents A and B were added to the tubes and the resulting solution was vortexed at high speed for 30s and left to stand for 20 minutes. Any sample that appeared to be cloudy was placed in a heat block for 5 minutes. At the end of which, the samples were inserted into the analyzer and the aldehyde concentrations of the samples were automatically calculated. Triplicates were carried out for all samples and repeated until a standard deviation of less than 10% was obtained.

RESULTS AND DISCUSSION

Figure 1. p-Anisidine test results

Figure 2. AlkalSafeTM test results

p-Anisidine value, therefore the level of aldehydes, increased with increasing number of frying for all three oils. Sunflower oil had the highest p-anisidine values, followed by palm-olein and lastly cooking oil. This was because oleic acid and linoleic acid, the main types of unsaturated fatty acids that were oxidized, were present in the highest amount in sunflower oil (88%) but was comparable in palm-olein (59.2%) and cooking oil (59%). Although palm-olein and cooking oil had similar amounts of the two fatty acids, palm-olein had significantly higher values than cooking oil (F > Fcrit at P= 0.05). Further investigations into the production of aldehydes in cooking oil and palm-olein could be undertaken to determine the reasons for the difference in the aldehyde levels despite similar initial fatty acid content. Methyl linoleate reacts much faster than methyl oleate during secondary oxidation and has a shorter induction period (Allen and Hamilton, 1989). Since sunflower oil contained the highest amount of linoleate acid (67.8%) as compared to palm-olein (40.5%) and cooking oil (40.3%), the initial rate of increase in the amount of aldehydes was the fastest for sunflower oil, followed by palm-olein and cooking oil. The results of the AlkalSafeTM test confirmed that the trends for the aldehyde levels in all three types of oil were increasing. The level of correlation between the both tests were found to be fair (sunflower oil: R2= 0.955, palm-olein: R2= 0.759, cooking oil: R2= 0.834). Though both tests produced rates that were of the same trend, the differences between the rates of the three oils were significantly different in figure 1 but similar in figure 2. The p-anisidine test showed that there was a significant difference between the aldehyde levels in the three types of oils at a 95% confidence interval but the results of AlkalSafeTM test showed that there were no significant differences. The

reliability of the AlkalSafeTM test and whether it was more accurate than the p-anisidine values could not be determined since the reaction of the AlkalSafeTM reagents and aldehydes were unknown. Based on the findings of this study, some further studies could be suggested. Firstly, the results from this studies could only be applied to oil that has been used to fry chicken since the composition of the food, such as lipid and water levels, affect the rate of oxidation in the oil and therefore the levels of aldehydes produced. Investigations into how the aldehyde levels change when frying other food materials should be determined to find out if similar trends exists for the three oils. Further studies can also be conducted to determine the reasons for the difference in the aldehyde levels of cooking oil and palm-olein despite similar initial fatty acid content. Lastly, the reasons for the difference in the results from p-anisidine test and AlkalSafeTM test will also have to be determined. One important point to note is that the aldehyde level is not the only parameter that indicates the extent of oxidation of the oil. The levels of various other compounds, such as ketones and malondialdehyde also have to be taken into account. CONCLUSION Of the three types of oil used in this study, sunflower oil had the highest amount of aldehydes after being used to fry chicken nuggets, since it contained the highest levels of unsaturated fatty acids initially. The p-anisidine test had significantly different results from the AlkalSafeTM test and a fair level of correlation. ACKNOWLEDGEMENTS I would like to thank Professor Zhou Wei Biao for his invaluable guidance as a supervisor and also Geeta for all the help that she has provided throughout the whole course of the project. I would also like to express my gratitude to Chooi Lan and Hwee Lee for helping me with the apparatus and the machines. Lastly, I would like to thank all those who have also helped me in one way or the other throughout the project. REFERENCES Allen J.C. and Hamilton R.J. (1989) Rancidity in Foods 2nd ed. Pub. Elsevier Science, England Tompkins C. and Perkins E.G. (1999) The Evaluation of Frying Oils with the p-Anisidine value. Journal of American Oil Chemists Society 76 945-947 Swee Y.F., Cuppett S. and Schlegel V. (2006) Evaluation of SafTestTM Methods for Monitoring Frying Oil Quality. Journal of American Oil Chemists Society 83 15-20