Exercise 8: Lipid Oxidation In this lab we are concerned with the chemical properties of food lipids in particular the volatiles that can form by fragmenting a triglyceride into its constituent fatty acids or by the autoxidation of polyunsaturated fatty acids. Lipid Molecules. Lipids are usually defined as those components that are soluble in organic solvents (such as ether, hexane or chloroform), but are insoluble in water. This group of substances includes triacylglycercols, diacylglycercols, monoacylglycercols, free fatty acids, phospholipids, sterols, caretonoids and vitamins A and D. The lipid fraction of a fatty food therefore contains a complex mixture of different types of molecule. Even so, triacylglycercols (triglycerides) are the major component of most foods, typically making up more than 95 to 99% of the total lipids present. Triacylglycerols are esters of three fatty acids and a glycerol molecule. The fatty acids normally found in foods vary in chain length, degree of unsaturation and position on the glycerol molecule. Consequently, the triacylglycerol fraction itself consists of a complex mixture of different types of molecules. Each type of fat has a different profile of lipids present which determines the precise nature of its nutritional and physiochemical properties. The terms fat, oil and lipid are often used interchangeably by food scientists. Although sometimes the term fat is used to describe those lipids that are solid at the specified temperature, whereas the term oil is used to describe those lipids that are liquid at the specified temperature. Glycerol Fatty Acids Triacylglycerol (triglyceride) The ester link between glycerol and its fatty acids can be broken down by lipase enzyme. Triglycerides are too large to be volatile (and hence have aroma) but free fatty acids, particularly the small ones, can be volatile enough to have characteristic smells. lipase H + H
Lipid Oxidation. Unsaturated (particularly polyunsaturated) fatty acids are prone to oxidation. The significant end products of lipid oxidation are rancid-smelling flavor aldehydes derived from the fatty acids (either free or as part of the triglyceride). Each fatty acid can form several flavor volatiles and the large range of fatty acids in a real oil mean a wide range of flavors can be generated. Lipid oxidation follows a free radical mechanism that starts with the abstraction of a hydrogen atom (not an ion) from the fatty acid. The methylene interrupter group between two double bonds (-CH = CH-CH 2 -CH =CH-) is particular prone to loosing a hydrogen. The lipid radical form (R ) rapidly reacts with oxygen to form a peroxy radical via a free radical chain reaction. The peroxy radical (ROO ) can gain a hydrogen atom to form a lipid hydroperoxide (ROOH) which is relatively stable and exists in significant quantities in many natural fats. The lipid hydroperoxide has no off-flavor but rapidly break down (particularly in the presence of heat and a metal catalyst) to form rancid flavors. -CH = CH-CH 2 -CH =CH- -CH = CH-CH-CH =CH- + O 2 -CH = CH-CH-CH =CH- -CH = CH-CH-CH =CH- -OOH Hydroxyperoxide (ROOH) The hydroperoxide breakdown starts with the loss of a hydroxy radical ( OH) to form a lipid alkoxy radical (RO ). The alkoxy radical rearranges and breaks the molecule into two parts including a volatile and rancid-smelling aldehyde. The aldehydes formed are often so odor active that even a few ppm can lead to a food being unacceptable.
Lipid oxidation can be inhibited by rigorously excluding oxygen, but this is practically difficult in many foods and even if it could be done, pre-existing lipid hydroperoxides can breakdown to rancid flavors in the absence of additional oxygen. Instead antioxidants can be added to limit the rate of the reaction. Metal chelators (e.g. EDTA and citric acid) can bind the metal ions that catalyze the breakdown of lipid hydroperoxides or the generation of radicals. Chain breaking antioxidants (e.g. BHA and BHT) react with radical intermediates and break the chain reaction before rancid flavors are generated. Each time a chain breaking antioxidant reacts with a radical it is itself oxidized and no longer effective so this class of antioxidant will only serve to delay oxidation until they are all consumed. Measurement of Lipid Oxidation. There are many intermediates and products of the lipid oxidation reaction. Even a single fatty acid will break down to form over a dozen significant products and real, complex food oil will produce many hundreds. This poses an analytical problem as there is no single product to measure and there may be immeasurably small losses of starting material before the product is obviously rancid. Instead, many analytical procedures have been developed to measure classes of compounds formed in the reaction. The selection of a test is somewhat complex as some products (e.g. volatile aldehydes) will form relatively late in the reaction and will only be detected after a lot of oxidation has occurred while others (e.g. conjugated dienes) may be formed in large numbers early in the reaction but breakdown to immeasurable levels later in the process.
Chromatography. Chromatography is the most powerful method of monitoring lipid oxidation. A detailed profile of fatty acids and other molecules present in lipids can be detected and monitored using chromatography methods. Iodine Value. Iodine value is a measure of the unsaturated linkages in fat and is expressed in terms of percentage if iodine absorbed. The decline in iodine value can be used to monitor lipid oxidation. Peroxide Value. Peroxides are primary reaction products of lipid oxidation. Peroxides can be measured based on their ability to liberate iodine from potassium iodide, or to oxidize iron ions (from ferrous to ferric ions). The peroxide value is applicable for the early stages of lipid oxidation. During the course of oxidation, peroxide values reach a peak and then decline. Conjugated Dienes. Almost immediately after peroxides are formed, the nonconjugated double bonds that are present in natural unsaturated lipids are converted to conjugated double bonds. Conjugated double bonds (conjugated dienes) absorb ultraviolet light strongly at 233nm. In the later stages of lipid oxidation the conjugated dienes (primary products) are broken down into secondary products (which do not absorb UV-visible light strongly) which leads to a decrease in absorbance. Thiobarbituric Acid (TBA). TBA is the most widely used test for measuring the extent of lipid oxidation. This test is preferred due to its simplicity and because its results correlate highly with sensory scores. In this experiment we use a TBA test to measure the extent of the reaction. TBA (thiobarbituric acid) reacts with two molecules of malonaldehyde (formed as a byproduct of lipid oxidation) to form a pink color. The color of the malonaldehyde-tba complex can be quanititated spectrophotometrically (530nm). Unfortunately other aldehydes (esp. reducing sugars) can also react with TBA and give a false positive result.
Procedures Oxidative Rancidity in Potato Chips 1. Wrap a jar with aluminum foil. Tape the foil in place so that no light can enter the container. 2. Place fresh potato chips in the foil-wrapped jar and in a clear jar without foil around it. 3. Taste the fresh potato chips and rate their flavor on a 5-point scale: 1 = extremely dislike, 2 = slightly dislike, 3 = neither like nor dislike, 4 = slightly like, and 5 = extremely like. 4. Place the two jars on a window sill where they will be exposed to sunlight. 5. Describe the aroma of the potato chips after 1 week. 6. Taste potato chips from each after 1 week and rate their flavor. Aromas 1. Describe the aromas of all oil samples provided. Samples include heated oils used for the TBA test (treatment 1) and two old commercial oils. 2. Compare the aroma of the oil samples with the aroma of the potato chips in the clear jar (after 1 week). TBA Test of Food Oils/Fats Sample A: Corn oil Sample B: Olive oil Sample C: Shortening Each group will test only one oil sample (Group 1+2 = corn oil, group 3+4 = olive oil, group 5+6 = shortening). These samples have been prepared for you in advance. All samples have been heated at 60ºC for two weeks and four different treatments have been applied to the samples: 1. Oil 2. Oil + BHT 3. Oil + Copper 4. Oil + Copper + EDTA
Perform the following procedure in triplicates: 1. Carefully pipet (oil is viscous!) 1ml oil into a screw cap test tube. 2. Add 1ml hexane to the samples and close the tubes. THIS HAS TO BE DONE IN THE FUME HOOD! 3. Add 10ml TBA-reagent and mix. 4. Loosen the caps on your test tubes and place them in a boiling waterbath for 40 min. 5. Remove the tubes from the waterbath; carefully remove the top layer of liquid (into provided waste containers). 6. Measure the absorbance of the remaining liquid at 540nm. Data Presentation: 1. Table 1 shows the flavor ranking (5-point scale) and the aromas of the potato chips for fresh samples and samples stored in the different jars for 1 week. 2. Table 2 is a description of the aroma for the various oil samples compared to the potato chips. 3. Figure 1 shows the absorbencies measured for the different oil treatments (bar graph). 4. Table 3 contains a ranking of oxidation state of the oil samples (4 different treatments) based on the results of the TBA tests. Discussion: 1. Why did wrapping the potato chip jars in aluminum foil affect the flavor quality of the chips? How are potato chips packaged in the store and why? 2. Compare the aromas of the oil samples and the potato chips. What factors may influenced the different oxidation levels of the samples? 3. Briefly describe the function of BHT, copper and EDTA with regard to lipid oxidation. Do your results agree with what you expected?
Exercise 8 Pre-Lab Questions 1) What is the structure of BHT? (2 points) 2) What does TBA stand for? (2 points) 3) In addition to the TBA test, how can lipid oxidation be monitored (2 examples)? (2 points) 4) Fish oil is particularly prone to lipid oxidation. What is it about fish lipids that makes them so prone to rancidity? (4 points)