INTRODUCTION

• Dietary lipids, naturally occurring in raw food

materials or added during food processing, play an
important role in food nutrition and flavor
• Lipid oxidation is a major cause of food quality
deterioration
– challenge for manufacturers and food scientists.
Lipids are susceptible to oxidative processes in the presence of
light, heat, enzymes, metals, metalloproteins, and micro-
organisms,
• off-flavors and
• loss of essential amino acids,
• Loss of fat-soluble vitamins, and
• other bioactives.
Lipids may undergo
• autoxidation,
• photo-oxidation,
• thermal oxidation, and
• enzymatic oxidation
• Autoxidation is the most common process leading to oxidative
deterioration
– spontaneous reaction of atmospheric oxygen with lipids
• Accelerated at higher temperatures, such as in deep-fat frying,
thermal oxidation,
• Increases in free fatty acid and polar matter contents, foaming,
color, and viscosity.
• Unsaturated fatty acids are generally the reactants affected by such
reactions, free fatty acids, triacylglycerols (as well as DAG or MAG),
or phospholipids
• Both autoxidation and thermal oxidation of unsaturated fatty acids
occurs via a free radical chain reaction
– 3 steps of initiation, propagation, and termination.
Mechanism of autooxidation
• As oxidation normally proceeds very slowly at the initial
stage,
– the time to reach a sudden increase in oxidation rate is referred to
as the induction period.
• Lipid hydroperoxides have been identified as primary
products of autoxidation;
• Decomposition of hydroperoxides yields aldehydes, ketones,
alcohols, hydrocarbons, volatile organic acids, and epoxy
compounds, secondary oxidation products.
• These compounds, together with free radicals, constitute
the bases for measurement of oxidative deterioration of
food lipids
 METHODS FOR MEASURING LIPID OXIDATION
• Numerous analytical methods are routinely used for measuring lipid
oxidation in foods
• No uniform and standard method to detect all oxidative changes in all
food systems
• It is necessary to select a proper and adequate method for a particular
application
• The available methods to monitor lipid oxidation in foods
– absorption of oxygen,
– loss of initial substrates,
– formation of free radicals,
– formation of primary and secondary oxidation products
A number of physical and chemical tests, including
instrumental analyses for lipid oxidation
parameters.
• weight-gain and headspace oxygen uptake
method for oxygen absorption;
• chromatographic analysis for changes in reactants;
Mechanisms
• MEASUREMENT OF OXYGEN ABSORPTION
• MEASUREMENT OF REACTANT CHANGE
• MEASUREMENT OF PRIMARY PRODUCTS OF
OXIDATION
 I. MEASUREMENT OF PRIMARY PRODUCTS OF
OXIDATION
Peroxide Value (PV)
• Formation of hydroperoxides as primary oxidation
products
• The primary product may break down to a variety of
nonvolatile and volatile secondary products.
• The formation rate of hydroperoxides outweighs
their rate of decomposition during the initial stage
of oxidation
– This becomes reversed at later stages.
• Therefore, the peroxide value (PV) is an indicator of the
initial stages of oxidative change.
• Assesses whether lipid is in the growth or decay portion of
the hydroperoxide concentration by monitoring the
amount of hydroperoxides as a function of time
• The PV, the total hydroperoxide content (quality indicators)
– production and storage.
• A number of methods have been developed for
determination of PV, among which the
– iodometric titration,
– ferric ion complex measurement spectrophotometry,
– infrared spectroscopy
 1. Iodometric Titration Method
• Iodometric titration assay is based on the oxidation of the
iodide ion (I-) by hydroperoxides (ROOH), standard methods
for determination of PV.
– A saturated solution of potassium iodide is added to oil samples
to react with hydroperoxides.
– The liberated iodine (I2) is then titrated with a standardized
solution of sodium thiosulfate and starch as an endpoint
indicator.
• The PV is obtained by calculation and reported as
milliequivalents of oxygen per kilogram of sample
(meq/kg).
 Chemical reactions

Iodometric titration as a method for measurement

of PV, suffers from several disadvantages
The procedure is time-consuming and labor
intensive .
• The assay includes six steps:
– accurate weighing of the sample,
– dissolution of lipids in chloroform,
– acidification with acetic acid,
– addition of potassium iodide,
– incubation for exactly 5 minutes, and
– titration with sodium thiosulfate.
• Drawbacks of the method
– Absorption of iodine across unsaturated bonds and
– Oxidation of iodide by dissolved oxygen

•Limitations
• lack of sensitivity,
• possible interferences, and
• difficulties in determining the titration endpoint
Modification of the classical iodometric assay.
colorimetric determination at 560 nm,
potentiometric endpoint determination
spectrophotometric determination of the I3-1 chromophore at 290
nm or 360 nm
 2. Thiobarbituric Acid (TBA) Test
Oxidative deterioration of fat-containing foods
• During lipid oxidation, malonaldehyde (MA), a
minor component of FAs with 3 or more double
bonds, degradation of PUFAs
• An indicator of the lipid oxidation process, both for
the early appearance as oxidation occurs and for
the sensitivity of the analytical method
In this assay, the MA is reacted with thiobarbituric acid (TBA)
to form a pink MA-TBA complex that is measured
spectrophotometrically at its absorption maximum at 530–
535 nm
The extent of oxidation is reported as the TBA value
Expressed as milligrams of MA equivalents per kilogram
sample/as micromoles of MA equivalents per gram of
sample
 3. p -Anisidine Value (p -AnV)
• The p-anisidine value (p-AnV) method measures the
content of aldehydes (principally 2-alkenals and 2,4-
alkadienals) generated during the decomposition of
hydroperoxides.
• It is based on the color reaction of p-methoxyaniline
(anisidine) and the aldehydic compounds
• The reaction of p-anisidine reagent with aldehydes
under acidic conditions gives yellowish products
that absorb at 350 nm
• The color is quantified and converted to p-AnV.
• The p-AnV is defined as the absorbance of a
solution resulting from the reaction of 1 g of fat in
isooctane solution (100 ml) with p-anisidine
(0.25% in glacial acetic acid)
More sensitive to unsaturated aldehydes because the
colored products from unsaturated aldehydes absorb
more strongly at this wavelength
 4. Oil Stability Index (OSI)

• During lipid oxidation, volatile organic acids, mainly

formic acid and acetic acid, as secondary volatile
oxidation products at high temperatures,
simultaneously with hydroperoxides
• In addition, other secondary products, including
alcohols and carbonyl compounds, can be further
oxidized to carboxylic acids
• The oil stability index (OSI) measures the volatile
acids by monitoring the change in electrical
conductivity when effluent from oxidizing oils is
passed through water
• The OSI value is defined as the point of maximal
change of the rate of oxidation, attributed to the
increase of conductivity by the formation of
volatile organic acids during oxidation
• This method requires a higher level of oxidation (PV >
100) to obtain measurable results than other methods
in which hydroperoxides are the most important
products formed and detected
– To determine oil stability, oxidation is accelerated by
elevated temperatures and excess amount of air/oxygen
• Using a flow of air and high temperatures to accelerate oxidation
• The OSI test measures the changes in conductivity
caused by ionic volatile acids
• Rancimat is employed for determining the OSI value.
In the Rancimat assay,
• A flow of air is bubbled through a heated oil, usually
at 100C or above.
• Volatile compounds formed during accelerated
oxidation are collected in distilled water, increasing
the water conductivity.
• The change of conductivity is plotted automatically
and
• The induction period of the oil or the time taken to
reach a fixed level of conductivity is recorded
 5. Sensory Evaluation
• For the food industry, the detection of oxidative off-
flavors by taste or smell is the main method of
deciding when a lipid-containing food is no longer
fit for consumption
• In the edible oil industry, the AOCS (American Oil
Chemists’ Society) Flavor Quality Scale with
separate grading and flavor intensity has been
employed for describing lipid oxidation
• The descriptive analysis, including the detection and
the description of both the qualitative and
quantitative sensory aspects of a product, is
performed by a trained panel
• Sensory induction period can be determined.
 MEASUREMENT OF FRYING FAT DETERIORATION
• Deep-fat frying is a popular method for food
preparation, as a heat-exchange medium, but also
contribute to the quality of fried products
• Lipid oxidation easily occurs at relatively high
temperatures, producing a complex compounds
that exerts undesirable effects on flavor and quality
• The measurement of lipid oxidation is essential to
determine its effect on food and oil quality, as well
as the useful life of fats or oils subjected to frying.
The oxidative changes in frying fats are characterized
by a decrease in the total unsaturation of the fat
with increases in the
• free fatty acid content,
• foaming,
• color, and
• viscosity as well as the
• content of polar compounds and polymeric
material
Physical methods estimate oxidative degradation by
monitoring changes in physical properties of frying fats
• Molecular weight,
• specific gravity,
• smoke point,
• refractive index,
• chromatic parameter,
• viscosity,
• surface tension, and
• dielectric constant
Chemical methods include the
• iodine value,
• saponification value,
• free fatty acid content,
• peroxide value,
• TBA value, or
• p-anisidine value
PV is less useful because hydroperoxides decompose
at about 150C, and no accumulation of peroxides
Cyclic cpds from frying
 METHODS FOR MEASURING ANTIOXID. ACTIVITY
• A variety of natural and synthetic antioxidants are used in fat-
containing foods in order to inhibit lipid oxidation with a wide
range of efficiencies
– depending on their properties, concentrations, and processing
conditions.
• The essential features of any test are
– a suitable substrate,
– an oxidation initiator, and
– an appropriate measure of endpoint
• Certain aspects,
– the model food system used for the test, and
– the means by which oxidation is accelerated and monitored
• Most assessments of antioxidant activity are performed in
oil/other model systems, sensible prediction for the
activity
– oil or water-in-oil emulsions
– oil-in-water emulsions
• Stripping of oils may be necessary in such evaluations
because the endogenous antioxidats enhance the
oxidative stability
• In addition to oils and fats, lipid substrates used for testing
antioxidant activity could be fatty acids, fatty acid ethyl
esters or triacylglycerols and beta-carotene
• Approaches proposed for testing antioxidant
activity
– measuring of the current state of oil samples
– radical scavenging assays
• Radical scavenging methods measure
– the relative abilities of antioxidants to scavenge synthetic
radicals or natural in comparison with the antioxidant
potency of a standard antioxidant
• Trolox (6-hydroxy-2,5,7,8-tetramethylchroma-2-
carboxylic acid), ascorbic acid, and quercetin are
among the standard antioxidants
The most common synthetic radicals are
– DPPH (2,2-diphenyl-1- picrylhydrazyl) and
– ABTS (3-ethylbenzthiazoline-sulfonic acid) radicals
• DPPH test and ABTS assay are simple, rapid, and
involve no substrate
• However, artificial substrate-free methods do not
always adequately mimic the processes in food
systems,
– less valuable for predicting the effectiveness of the
antioxidant in foods