Autoxidation of Unsaturated Lipids in Food Emulsion Fariha Irshad

Food emulsion  Mixture of two or more liquids that are normally immiscible (unmixable or unblendable) Continuous and dispersed phase Oil-in-water emulsion Water-in-oil emulsion

Lipid oxidation Oxidative deterioration of lipids containing any number of carbon-carbon double bonds Produces various primary and secondary by- products Undesirable flavors Toxic products during storage Quality deterioration Change in nutrition, taste, texture, appearance Shorten the shelf life of product Limit the use as beneficial lipid in functional foods Oxidized foods can cause oxidative stress in biological systems and cause diseases

Enzyme -catalysed oxidation Oxidation of unsaturated lipids Oxidation of unsaturated fatty acids in their structures Photoxidation oxidation caused by the action of light Singlet oxygen is involved Require sensitizers Autoxidation Oxidation that occurs in open air or in presence of oxygen (and sometimes UV radiation) and forms peroxides and hydroperoxides Free Radical Chain-reaction Enzyme -catalysed oxidation An oxidative enzyme is catalyses oxidation reaction. peroxidases, which use hydrogen peroxide, and oxidases, which use molecular oxygen Cyclooxygenase and lipoxygenase catalyze the reactions between oxygen and polyunsaturated fatty acids

Mechanism of lipid oxidation The overall mechanism of lipid oxidation consists of three phases Initiation Fatty acid radical is produced. Initiators are reactive oxygen species (ROS), e.g OH· Forms H2O + fatty acid radical Propagation The fatty acid radical is unstable It reacts readily with O2 & creates a peroxyl-fatty acid radical. This is unstable ∴ reacts with another FFA A different fatty acid radical is produced Cycle continues Termination Radical reaction stops when two radicals react and produce a non-radical species. This happens when radical concentration is high

Initiation: RH + O2 -->R· + ·OH R· + O2 --> · + ROO· Propagation:   ROO· + RH --> R· + ROOH ROOH--> RO· + HO·     Termination: R· + R· --> RR R· + ROO·--> ROOR ROO· + ROO· --> ROOR + O2

RH is any unsaturated fatty acid; R· is a free radical formed by removing a labile hydrogen from a carbon atom adjacent to a double bond; ROOH is a hydroperoxide, one of the major initial oxidation products that decompose to form compounds responsible for off-flavors and odors. Such secondary products include hexanal, pentanal, and malonaldehyde.

Unsaturated lipids include fatty acids and their derivatives (including tri-, di-, and monoglycerides, and phospholipids), as well as other compounds such as sphingolipids, sterol lipids, and saccharolipids The important lipids involved in oxidation are the unsaturated fatty acid moieties, oleic, linoleic, and linolenic The rate of oxidation of these fatty acids increases with the degree of unsaturation. Oleic – 1 times rate Linoleic – 10 times Linolenic – 100 times

Factors affecting unsaturated lipid oxidation in food emulsions Chemical structure of lipids Oxygen concentration Antioxidants Temperature Surface area Water content

Interfacial material between lipid material and aqueous phase Oil in interior of emulsion droplet Interfacial material between lipid material and aqueous phase Composed of proteins, small emulsifiers or mixture of number of these compounds Aqueous phase Contain ions,macromolecules, salts, amino acids Exert stabilizing and destabilizing effect Behavior of oil in water emulsion

Interfacial membrane Interfacial layers depands on: Quantities of material absorbed Composition and structure of that material Interfacial layer formed by emulsifier or protein or combination of both, present b/w lipid substrate enhance Stability of emulsion Provide physical barrier against pro-oxidant penetration and diffusion Higher the interfacial membrane thickening, higher the physical barrier and slower the lipid oxidation

Emulsifier type Water-in-oil emulsion the oxidative rate of lipid is similar to that in the bulk oil phase Oxidation of lipid in oil-in-water emulsion is different from the bulk oil phase Many unabsorptive emulsifier molecules exist in aqueous solution form micelles and increase the emulsion stability by dissolving The lipid, Antioxidant, and Prooxidant Oxidative stability of oil-in-water emulsions containing different emulsifiers increased in the order: Whey protein isolate >lecithin >mono-/ diacylglycerols> Tween 20>sucrose fatty acid ester.

Droplet characteristic Oil conc. Total oxidation, Conc. Of volatiles (Jo and Ahn, 1999) Oil conc. Rate of initiation, Rate of propagation (Coupland et al., 1996) For fixed droplet concentration Droplet surface Mean diameter, Lipid oxidation

Diffusion of oxygen Rate-limiting step is the diffusion of oxygen in water phase when the oxygen concentration is low When oxygen concentration is high, the diffusion rate of oxygen is faster than the oxidation rate (Marcuse and Fredriksson, 1968; L¨ u et al., 2008). Decreasing oxygen concentration is effective method to retard oxidation

Determination of autoxidation Primary oxidation Secondary oxidation Hydroperoxide major initial lipid peroxidation reaction product Undergo both enzymatic and non enzymatic degradation Produce secondary products The assay for hydroperoxides is the most direct measure of lipid peroxidation

Primary product determination tests Peroxide value test Peroxide value directly measures the concentration of hydroperoxides formed in the initial stage Sodium thiosulfate titrate iodine produced from a reaction of lipid peroxide and potassium iodine. Thus the content of peroxide can be calculated directly by titration of iodine produced ROOH+2H++2KI−→ROH+I2+2K++H2O Drawbacks Susceptibility toward background reactions Absorption of iodine by unsaturated fatty acids Underestimation of the peroxide value Oxygen error leads to high PV levels

Iron Thiocyanate Method Spectrophotometric method Ability of peroxides to oxidize ferrous ions to ferric ion Fe2++2H++O→Fe3++H2O Simple and easy method to master Drawback Sensitive to oxygen in the solution, which will disturb the measure

Conjugated dienes value Conjugated dienes are produced during the refining process of oil or autoxidation Strong characteristic UV absorption at 234 nm Indicators of free radical production Study pure lipid autoxidation only Drawback Unsuitable for determination of lipid containing high proportion of saturated fatty acid Reflect only the extent of lipid autoxidation at an early stage

Active oxygen method AOM value Number of hours required for the peroxide concentration to reach 100 meq/kg of lipid. The more stable the lipid, the longer it will take to reach that level Predicts the stability of lipid by bubbling air through a solution of the lipid Drawbacks Costly Time-consuming Stable lipid may require 48 h or more before reaching the required peroxide concentration

High Performance Liquid Chromatograph (HPLC) Separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation Hydroperoxide with different volatility, different molecular weight, or different polarity Determine lipid peroxide present

Determination of Secondary Products Thiobarbituric Acid Reactive Substance (TBARS) Test Two molecular TBAs react with malondialdehyde (MDA) originated from oxidation of polyunsaturated fatty acids result in red color which can be measured using a spectrophotometer The solution takes on yellow when it reacts with the other aldehyde under acid condition. TBA can form an adduct not only with MDA but also with other oxidized products, the method is also called thioharhituric acid reactive substances (TRARS) test Drawback Only fatty acid containing three or more double bonds can produce enough substrate to react with TBA

Anisidine Value (AnV) Test Nonvolatile reaction product can be measured by reaction with anisidine. High anisidine values may be a hint that lipid has oxidized The technique is convenient when applied to oils and edible fats Drawback When lipids are extracted from a product, a correction for the product’s own absorbance may be a source of error in AnV determination

Other methods Oxidative Stability Index (OSI) Method Rancimat Method Acid value Fluorescence Spectroscopy Method Gas Chromatograph Method

Method to control lipid oxidation Processing characteristics Using antioxidants Ingredients low in pro-oxidants, transition metals & hydroperoxides Proper storage temperature Protection from light, aeration and moisture Vacuum packaging Inactivation of enzymes

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