INSTRUCTION Oils and fat quality. Lipid oxidation. Determination of peroxide value in fats and oil.

It is arguable that the two most important chemical reactions that occur in food systems are lipid oxidation and non-enzymatic browning. This lab exercise focuses attention on the former reaction. Lipid oxidation, which is also called auto-oxidation, occurs in lipid material by way of a free-radical mechanism. After an induction period, hydrogen peroxides, or primary products, are formed. Ultimately these peroxides break down, and secondary products, e.g., aldehydes, ketones, organic acids, and hydrocarbons, are formed. The peroxide value (PV) test, which is one of the most common tests used to evaluate the extent of lipid oxidation, is based on measuring peroxides. Objective: To measure the PV or a number of food samples, and to evaluate the meaning of the results. Reagents: Acetic acid (glacial) Chloroform (CCl4) 15% Potassium iodide (KI) 0.01 N (0.01M) sodium thiosulfate (Na2S2O3) Starch indicator 0.5 % concentrated hydrochloric acid HCl 0.01 N (0.00167M) potassium dichromate K2Cr2O7 (fix.) Procedure Determination of the titre of the sodium thiosulfate solution Measure off 10 ml of 0.01N K2Cr2O7 solution to a 200 ml conical flask. Add 0.5 ml concentrated HCl and 1.0 ml 15% KI solution. Mixed exactly 1 minute and leave for 5 minutes in a dark place. Add 0.5 ml starch solution, 20 ml distilled water. Mix and titrate with sodium thiosufate solution. Calculate the exact normality of Na2S2O3 knowing that in this chemical reaction 1 gramequivalent of K2Cr2O7 react with 1 gram-equivalent of Na2S2O3 (1 mole K2Cr2O7 react with 6 moles Na2S2O3). Determination of peroxide value. Weigh 3.00 g oil (with precision of 0.001 g) into a 250 ml Erlenmeyer flask. Add 10 ml chloroform and swirl to dissolve oil. Add 15 ml acetic acid, 1.0 ml KI solution, mix and leave for 5 minutes (exactly !) in a dark place. Add 30 ml distilled water and 1 ml starch inducator. Solution titrate with sodium thiosulfate until blue colour disappears. Do a blank determination (10 ml chloroform + 15 ml acetic acid + 1.0 ml KI + 30 ml H2O). Add starch indicator (1 ml) before titrating and titrate dropwise. Repeat the titration procedure at least 3 times. Individual results shouldnt vary more than 0,3 ml. Calculation Calculate the peroxide value PV for all samples from the following formula:

PV = (V1 V0 ) x T x 1000 / m

[miliequivalent available oxygen/kgsample ]

[meq. / kg]

where: V1 volume of thiosulfate solution required to titrate the sample [ml]; V0 volume of thiosulfate solution required to titrate the blank determination [ml]; T - titre of the sodium thiosulfate solution [normality]; m mass of sample [g] REPORT CONTENTS: - filled up analysis report; - description of the aim of the exercise; - shown calculations of individual results; - discussion of results and errors; - comparison of the experimental data with references.

ANALYSIS REPORT Oils and fat quality. Lipid oxidation. Determination of peroxide value in fats and oil.
Name: Date:

Product:

1. Determination of the titre of the sodium thiosulphate solution. Normality of K2Cr2O7 Volume of Na2S2O3 V [ml] Average V The titre of Na2S2O3 T [N]

Determination of peroxide value. A. Blank determination Volume of Na2S2O3 Vo [ml] Average Vo [ml]

B. PV determination m mass of sample Volume of Na2S2O3 [g] V 1 [ml]

PV [meq. / kg sample ]

Average PV [meq. / kg sample ]

Fats and oils play an important role in the flavor, aroma, texture, and nutritional quality of foods, pet foods, and feeds. Fats and oils may be added during manufacturing or they may be inherent to the product or ingredient. The product may be pure oil or it may be part of a complex mixture with proteins, carbohydrates, minerals, and vitamins. The product may contain almost no fat or it may contain a considerable amount. Regardless of the source of fat, the amount of fat, or the product composition, predicting and monitoring fat and oil quality is an important component of developing and manufacturing high quality products. As soon as a food, feed, or ingredient is manufactured, it begins to undergo a variety of chemical and physical changes. Oxidation of lipids is one common and frequently undesirable chemical change that may impact flavor, aroma, nutritional quality, and, in some cases, even the texture of a product. The chemicals produced from oxidation of lipids are responsible for rancid flavors and aromas. Vitamins and other nutrients may be partially or entirely destroyed by highly reactive intermediates in the lipid oxidation process. Oxidized fats can interact with proteins and carbohydrates causing changes in texture. Of course, not all lipid oxidation is undesirable. Enzymes, for example, promote oxidation of lipid membranes during ripening of fruit. For most products, though, predicting and understanding oxidation of lipids is necessary to minimize objectionable flavors and aromas arising from fat rancidity. Lipid oxidation processes.
It is now well admitted that lipids are non-enzymatically oxidized through two types of reaction: - autoxidation - photo-oxygenation. Besides these mechanisms, fatty acids may also be oxidized via enzyme activities: - enzymatic peroxidation

Autoxidation The radical-chain processes are generally considered to occur in three phases: - an initiation or induction phase, - a propagation phase, and - a termination phase. In complex systems, the products of each of these phases will increase and decrease over time, making it difficult to quantitatively measure lipid oxidation. During the initiation phase, in a peroxide-free lipid system, in the present of the initiation - to
abstract a hydrogen atom from a methylene group (- CH2-). These hydrogen having very high mobility. This attack generates easily free radicals from polyunsaturated fatty acids.

RH = initiators= R + H For this phase to occur at any meaningful rate, some type of oxidative initiators must also be present, such as chemical oxidizers, transition metals (i.e., iron or copper), or enzymes (i.e., lipoxygenases). Heat and light also increase the rate of this and other phases of lipid oxidation. The reactive products of this initiation phase will, in turn, react with additional lipid molecules to form other reactive chemical species. During the propagation phase, molecular oxygen combines with unsaturated fatty acids to produce hydroperoxides and free radicals, both of which are very reactive.

R + O2 = ROO ROO + RH = R + ROOH ROOH = RO + OH

As a peroxyl radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in the presence of metals such as copper or iron, thus causing an autocatalytic chain reaction. The peroxyl radical combines with H to give a lipid hydroperoxide (or peroxide). This reaction characterizes the propagation stage.

The propagation of further oxidation by lipid oxidation products gives rise to the term autooxidation that is often used to refer to this process. In the final, termination phase of lipid oxidation, relatively unreactive compounds are formed including hydrocarbons, aldehydes, and ketones. R + ROO = ROOR R + R = RR ROO + ROO = ROOR + O2 RO + R = ROR Any kind of alkyl radicals (lipid free radicals) R. can react with a lipid peroxide ROO. to give non-initiating and non-propagating species such as the relatively stable dimers ROOR or two peroxide molecules combining to form hydroxylated derivatives (ROH). Some bonds between lipid peroxides and membrane proteins are also possible. Lipid oxidation moves through each of these three phases as a product ages and a number of factors can influence the rate of lipid oxidation in a product. These include: the initial quality of the fat or oil used for manufacturing the product, conditions used to manufacture the product, storage conditions (heat, light, packaging), surface area exposed to atmospheric oxygen, presence of transition metals, concentration of active lipoxygenases, application of appropriate of synthetic or natural preservatives, or presence of chemical oxidizers. Early in the lipid oxidation process, peroxides and hydroperoxides are the predominate reaction products. These reaction products continue to increase until a) storage conditions change, b) one or more initiators is depleted, c) available oxygen is consumed, or d) the lipid substrate is exhausted. Increased peroxide and hydroperoxide concentrations will initiate a series of reactions that eventually lead to increasing concentrations of aldehydes, ketones, hydrocarbons, and other termination phase products. Because many compounds produced during the termination phase are volatile, their concentration in the product may also begin to decrease over time. The rate of decrease varies

with storage conditions, packaging, and fat content. The consequence of all of these changing concentrations is that any attempt to evaluate the rancidity of a product will likely be taking aim at a moving target. Peroxide values could be low because minimal oxidation has occurred or because peroxide concentrations have begun to decrease. Low aldehyde concentrations may be the result of limited oxidation or the aldehydes may have volatilized. It is generally not possible to predict the best indicator of lipid oxidation and any attempt to characterize rancidity of a product will likely require multiple tests. Appropriate control samples (freshly manufactured or other nonrancid product) are also helpful when historical values are unavailable. And finally, while a variety of chemical tests can objectively quantify various lipid oxidation products, subjective sensory evaluations may be the key to understanding the data. Ultimately, correlation to sensory testing is the basis for determining which chemical tests are appropriate for measuring lipid oxidation in any product. Peroxide value. One of the most widely used tests for oxidative rancidity, peroxide value is a measure of the concentration of peroxides and hydroperoxides formed in the initial stages of lipid oxidation. Milliequivalents of peroxide per kg of fat are measured by titration with iodide ion. Peroxide values are not static and care must be taken in handling and testing samples. It is difficult to provide a specific guideline relating peroxide value to rancidity. High peroxide values are a definite indication of a rancid fat, but moderate values may be the result of depletion of peroxides after reaching high concentrations. Photo-oxidation As singlet oxygen (1O2) is highly electrophilic, it can react rapidly with unsaturated lipids but by a different mechanism than free radical autoxidation. In the presence of sensitizers (chlorophyll, porphyrins, myoglobin, riboflavin, bilirubin, erythrosine, rose bengal, methylene blue...), a double bond interacts with singlet oxygen produced from O2 by light. Oxygen is added at either end carbon of a double bond which takes the trans configuration. Thus, one possible reaction of singlet O2 with a double bond between C12 and C13 of one fatty acid is to produce 12- and 13-hydroperoxides. The lifetime of singlet O2 in the hydrophobic cell membrane is greater than in aqueous solution. Furthermore, photo-oxidation is a quicker reaction than autoxidation since it was demonstrated that photo-oxidation of oleic acid can be 30 000 times quicker than autoxidation and for polyenes photo-oxidation can be 1000-1500 times quicker. Similar effects have been described in liposomes and in intact membranes. The inhibition of photosensitized oxidation is efficiently inhibited by carotenoids, the main protective role played by these compounds in green plants. The inhibitory mechanism is thought to be through an interference with the formation of singlet oxygen from the oxygen molecule. In contrast, tocopherols inhibit this oxidation by quenching the previously formed singlet oxygen, this forms stable addition products. Unexpectedly, It was shown that carotenes are efficient inhibitors in vegetal oils only if tocopherols are also present to protect the former.
Literature: www.cyberlipid.org www.ralstonanalytical.com www.ag.auburn.edu