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. 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. 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. Lipid oxidation follows a free radical mechanism that starts with the abstraction of a hydrogen atom from the fatty acid. 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
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 eliminating oxygen, but this is practically difficult 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. For example, metal chelators (e.g. EDTA and citric acid) can bind the metal ions that catalyze the breakdown of lipid hydroperoxides while BHA and BHT are chain breaking antioxidants that react with radical intermediates and break the chain reaction before rancid flavors are generated. There are many intermediates and products of the lipid oxidation 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. ● Acid Value. Acid value is defined as the number of milligrams of potassium hydroxide required to neutralize the free fatty acids present in one gram of fat. It is a relative measure of rancidity as free fatty acids are normally formed during decomposition of triglycerides. The value is also expressed as percent of free fatty acids calculated as oleic acid. ● 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. ● 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 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.