4.1.1 Measuring lipid oxidation rates
The rate at which lipid oxidation occurs will define the time that a lipid oxidation prone product can be stored before it is no longer satisfactory for consumption. The focus of all experiments must be on the rates of lipid oxidation and how the rates of lipid oxidation change when changes are made to the oxidation system. It is, therefore, important to develop a method to track lipid oxidation reactions in such a way as to fully describe the oxidation rates in both bulk oils and oil-in-water emulsions.
4.1.2 Tracking initial versus final products of lipid oxidation
As has been discussed in the literature review, there are a number of different methods that can be used to describe the state of a lipid. Each method essentially measures the concentrations of reactants or products of lipid oxidation. Some methods track the initial products of lipid oxidation while others track the changes in secondary or final oxidation products such as ketones and aldehydes. The value of each of these measures depends on the stage of oxidation that is most important to the experiment being conducted. For example, gas chromatography (GC) analysis of volatiles such as hexanal tend to correlate well with sensory analysis which suggests that it would be a useful way of identifying when a product is rancid. Conversely, peroxide values (PV) are an indicative measure of the concentration of lipid hydroperoxides present in a sample.
As suggested in the literature review, lipid hydroperoxides are the only semi-stable product formed during the initial stages of lipid oxidation. PVs are, therefore, good at determining the initial rates of lipid oxidation which tend to account for most of the time required to produce a rancid product. Furthermore, lipid hydroperoxides are always formed in lipid oxidation while individual tertiary products (hexanal, propanal etc.) can be formed in different ratios according to the conditions in which oxidation is taking place.
PVs have been used for a number of years by a vast number of different researchers and industry groups. The methods have been well established and are published as official methods by the American Oil Chemists Society. Even though PVs are an indicative and empirical measure, they are capable of explaining changes in lipid hydroperoxide concentrations without the need for expensive and complicated high performance liquid chromatography (HPLC) techniques. Furthermore, their ability to track changes in primary oxidation products makes them ideal for explaining the effects of the slow initiation reactions and induction phase in the lipid oxidation pathway. Additionally, since the lipid oxidation pathway must include the formation of lipid hydroperoxides (unlike tertiary products which may or may not be formed depending on the reaction system), PVs should always give good estimates of lipid
oxidation rates irrespective of the oxidation system. For these reasons, PVs will be used extensively during future investigations.
4.1.3 Tracking common reactants versus reaction products
While PVs are a good universal measure of the net rates of lipid hydroperoxide formation, they are not capable of describing the entire lipid oxidation process. To do so using conventional techniques would require an array of measures targeting secondary and tertiary products so that any changes in reaction pathway and mechanism could be accounted for. The results of such analysis would be hard to understand and would yield questionable results purely because the researcher would have to make assumptions about the reaction pathway and know which reactions are or are not taking place. Such measures would not be practical as they would require highly skilled persons and an excessive amount of time. Furthermore, the exercise would have to be repeated every time a new product or lipid were being analysed.
Rather than measuring the array of potential secondary and tertiary products, some researchers have turned to measuring the concentrations of fatty acids and/or lipids in a sample. Since the lipids are the initial reactants, it makes sense that a drop in the number of double bonds (by oxidation of unsaturated fatty acids) would correlate with lipid oxidation rates. This is indeed the case during initial reactions but does not account for the reaction of radical species to form the tertiary products. Like the measures described earlier, measuring fatty acid concentrations requires extensive prior knowledge and expensive equipment that most industries would struggle to justify.
Unlike fatty acids and lipids, oxygen consumption rates can be easily measured and require far more cost effective equipment. Like fatty acids/lipids, oxygen is consumed during lipid oxidation. Unlike lipids, oxygen is consumed during the vast majority of the lipid oxidation pathway from the early initiation reactions to the tertiary reactions. Lipid oxidation, as has been discussed earlier, occurs far more slowly, if at all, in the absence of oxygen. Such dependence, coupled with the ease of measurement and low cost of equipment makes oxygen an excellent measure of lipid oxidation rates.
Coupled with PVs, oxygen consumption rates should be able to explain the changes in lipid oxidation rates in any system and under any conditions.
4.1.4 Method requirements
As discussed in Sections 2.3.8 and 2.3.9 of Chapter 2, both the Oxidograph and Rancimat methods require the use of high temperatures to accelerate the rates of lipid oxidation. Without the use of high temperature, both methods would likely require weeks or months to gain useful oxygen consumption data. Unfortunately, lipid oxidation is the combination of many reactions. It is, therefore, risky to assume that all the reactions would be affected by temperature changes equally. That is, the rates of all the lipid oxidation reactions occurring at 25ºC are unlikely to be the same at 100ºC. Furthermore, Rancimat and Oxidograph tests are reliant on pressure and conductivity measurements and, as a result, are sensitive to the addition of other compounds (i.e. BHT or BHA antioxidants) and the evolution of volatile products.
Due to their sensitivity to additives and volatile production as well as the need for high temperatures, the Rancimat and Oxidograph methods are unlikely to be able to measure oxidation rates in real food systems. To be able to measure oxygen consumption rates at lower temperatures (~35ºC), without being sensitive to the effects of additives and within a reasonable timeframe a new method must be developed. The method must:
1) Accurately and reliably measure oxygen consumption rates.
2) Measure oxygen concentrations directly and not rely on pressure or conductivity measurement.
3) Be sensitive enough to describe the effects of system changes. 4) Yield useful information in a short period of time (<24hr).