ANTIOXIDANT

ASSAYS IN
FOOD
SYSTEMS

NAME- VEDANT SAWANT

ROLL NO.- 19FET107
SUBJECT- PRINCIPLE OF FOOD
ANALYSIS
  Antioxidant assays are based on a concept called total antioxidant capacity
(TAC).
 TAC is measured as the amount of free radicals quenched by a test solution
used to determine the AO capacity of a biological sample.
 Used to evaluate the antioxidant activity of food ingredients, additives, and
products.
WHAT ARE
ANTIOXIDANT  Major applications

ASSAYS? 1. Screening potential food additives for their antioxidant activity.

2. Determining the optimal concentration of antioxidants.
3. Comparing the antioxidant activity of different food ingredients.
4. Studying the effects of processing, storage, and packaging on the
antioxidant activity of food products.
5. Evaluating the antioxidant activity of dietary supplements and functional
foods.
TYPES
Hydrogen atom transfer (HAT) reaction based
Electron transfer (ET) reaction based assays
assays
 Ferric ion reducing antioxidant power (FRAP) assay  Oxygen radical absorbance capacity (ORAC) assay

 Potassium ferricyanide reducing power (PFRAP)
assay
 Cupric ion reducing antioxidant capacity (CUPRAC)
assay
 Folin-Ciocalteu reagent (FC) assay
 Thiobarbituric acid reactive substances (TBARS)
assay
 DPPH free radical scavenging assay
 ABTS radical scavenging method
 Total radical-trapping antioxidant parameter (TRAP)
assay
 Hydroxyl radical antioxidant capacity (HORAC)
assay

Analytical techniques available to measure the antioxidant property of samples-

spectrometry, electrochemical technique, and chromatography
  These assays measure the reducing capacity of the
ELECTRON antioxidant compounds.
TRANSFER (ET)  It is based on the simple redox reaction, where
antioxidant compounds reduce the free radicals and get
REACTION themselves oxidized.
BASED ASSAYS  Reduction by antioxidant compounds results in the
color change of the reagent, which correlates with the
antioxidant capacity, which is measured by the change
in absorbance.
FRAP (FERRIC
REDUCING/ANTIOXIDANT
POWER) ASSAY

 The FRAP assay relies on the

reduction of Fe3+–TPTZ (2,4,6-
tri(2-pyridyl)-1,3,5-triazine) to
produce Fe2+–TPTZ by the
antioxidants.
 The binding of Fe2+ to the ligand
creates a very intense navy blue
color .
 The absorbance at 593 nm can be
measured to test the amount of
iron reduced and can be
correlated with the amount of
antioxidant.
  An absorbance increase can be correlated to the reducing ability of
antioxidants/antioxidant extracts.
 The compounds with antioxidant capacity react with potassium
ferricyanide (K3[Fe(CN)6]) to form potassium ferrocyanide
PFRAP (K4[Fe(CN)6]).
(POTASSIUM  The latter reacts with ferric trichloride, yielding ferric ferrocyanide, a
FERRICYANIDE blue-colored complex, with a maximum absorbance at 700 nm.
REDUCING
POWER) ASSAY
 CUPRAC (CUPRIC
REDUCING
ANTIOXIDANT
CAPACITY) ASSAY
 In CUPRAC assay, Cu (II) was reduced
to Cu (I) through the action of electron-
donating antioxidants.
 Neocuproine (Nc; 2,9-dimethyl-1,10-
phenanthroline) is the ligand- form a
copper–ligand complex to facilitate
absorbance measurement.
 After 30 min incubation, the absorbance
was recorded at 450 nm.
 Results are expressed in milligrams of
Trolox per liter of sample.
 Trolox equivalent= standard antioxidant
  During FC assay, the reaction between FC reagent and phenolic
compounds occurs at alkaline medium (~pH 10), which is reached by
FC (FOLIN- adding sodium carbonate (Na2CO3).
CIOCALTEAU REAGENT)  Under this basic condition, dissociation of a phenol leads to the
ASSAY formation of phenolate ion, which is responsible to reduce the FC
reagent.
 Upon reduction, the intense yellow colour of FC reagent turns into a
blue colour and maximum absorbance at 765 nm.
TBA
(THIOBARBITURIC
ACID) ASSAY
 TBA assay used 2-thiobarbituric
acid to interact with the lipid
oxidation product malonaldehyde
to form a pink compound, with a
maximum absorption value at
532 nm.
 The test result is usually
expressed as the product of the
absorbance at 532 nm and the
absorption coefficient of
malonaldehyde, which is
commonly referred to as the TBA
value.
Antioxidant Assay Principle Absorption maximum, End Product
colour Determination
FRAP Antioxidant reaction with a 593 nm, navy blue Colorimetry
Fe (III) complex
PFRAP Potassium ferricyanide 700 nm, blue Colorimetry
reduction by antioxidants
and subsequent reaction of
potassium ferrocyanide
with Fe3+
CUPRAC Cu (II) reduction to Cu (I) 450 nm, yellow-orange Colorimetry
by antioxidants
FC Reduction of FC reagent 765 nm, blue Colorimetry
TBA thiobarbituric acid to 532 nm, pink Colorimetry
interact with the lipid
oxidation product
malonaldehyde
HYDROGEN  These assays measures/quantify the hydrogen atom
donating ability of the antioxidant compounds by a
ATOM proton-coupled ET reaction, where it measures the
TRANSFER chain breaking antioxidant capacity.
 These assays based on the reaction between synthetic
(HAT) free radical generator, oxidisable molecular probe, and
REACTION antioxidant where reaction kinetics is derived from the
kinetic curve.
BASED ASSAYS
 ABTS (2,2′-AZINO-BIS(3-
ETHYLBENZOTHIAZOLINE-
6-SULFONIC ACID) ASSAY

 ABTS can be oxidized by potassium

persulfate or manganese dioxide to
ABTS radical (ABTS•+).
 the test measures the antioxidants’
capacity to neutralise (ABTS•+) stable
radical , a blue-green chromophore of
maximum absorption at 743 nm.
 In the presence of Trolox (or of
another hydrogen donating
antioxidant), the nitrogen atom
of ABTS•+ quenched the hydrogen
atom, yielding the solution
decolorization (pale blue), and the
absorbance at 743 nm was decreased.
 DPPH (2,2-DIPHENYL-
1-PICRYL-
HYDRAZYL) ASSAY
 The DPPH free radical is a long-lived
organic nitrogen radical with a deep
purple color.
 When a DPPH solution is mixed with an
antioxidant, its color turns from purple to
yellow of the corresponding hydrazine.
 The reducing ability of antioxidants
toward DPPH can be evaluated by
monitoring the decrease of its absorbance
at 515–528 nm.
 The results are expressed as IC50 or as %
scavenging of DPPH• at a fixed
antioxidant concentration for all the
samples.
IC50 - Antioxidant concentration required to inhibit 50% free radicals.

% scavenging= absorbance control- absorbance sample/absorbance control

  The ORAC assay measures the antioxidant scavenging capacity
against peroxyl radical generated by thermal decomposition of AAPH
(2,2′-azobis-2-amidino-propane) at 37°C.
 Fluorescein was used as the fluorescent probe. The loss of fluorescence
ORAC (OXYGEN
of fluorescein is an indication of the extent of damage from its reaction
RADICAL with the peroxyl radical.
ABSORBANCE  This method uses the area-under-curve technique in the presence and
CAPACITY) ASSAY in absence of the antioxidant.
 The protective effect of an antioxidant is measured by assessing the
area under the fluorescence decay curve (AUC) relative to that of a
blank in which no antioxidant was present.
 Antioxidant capacity measured in Trolox equivalent.
  This technique relies on the measurement of the metal-chelating
activity of antioxidants, under the conditions of Fenton-like reactions

HORAC In Fenton reactions, a highly reactive hydroxyl radicals (OH* ) is
produced during the reaction of H2O2 with iron (II) .
(HYDROXYL
 The method uses a Co(II) complex and hence evaluates the protecting
RADICAL
ability against the formation of hydroxyl radical.
ANTIOXIDANT
CAPACITY) ASSAY  Fluorescein is incubated with the sample to be analyzed, then the
Fenton mixture (generating hydroxyl radicals) was added.
 The initial fluorescence is measured, after which the readings were
taken every minute after shaking. Gallic acid solutions were used for
building the standard curve.
TRAP (TOTAL
 The luminol-enhanced chemiluminescence (CL) was exploited to
PEROXYL monitor the reactions involving the peroxyl radical.
RADICAL-  The CL signal was driven by the production of luminol-derived
TRAPPING radicals, resulted from the thermal decomposition of AAPH (2,2′-
ANTIOXIDANT azobis-2-amidino-propane).
PARAMETER ) A  The TRAP value was determined from the duration of the time period
SSAY (induction period) during which the sample quench the
chemiluminescence signal, due to the presence of antioxidants.
Antioxidant Assay Principle End Product Determination
ABTS Antioxidant reaction with an Colorimetry
organic radical
DPPH Antioxidant reaction with Colorimetry
an organic radical
ORAC Antioxidant reaction with peroxyl Loss of fluorescence of fluorescein
radicals, induced by AAPH
HORAC Antioxidant capacity to quench Loss of fluorescence of fluorescein
OH radicals generated by a Co (II)
based Fenton-like system
TRAP Antioxidant capacity to scavenge Photo chemiluminescence
luminol-derived radicals, generated quenching
from AAPH decomposition
CHROMATOGRAPHY

Antioxidant Assay Principle End Product Determination

GC Separation of the compounds in a mixture is based on Flame ionization or thermal
the repartition between a liquid stationary phase and a conductivity detection
gas mobile phase
HPLC Separation of compounds in a mixture is based on the UV–vis detection, fluorescence, mass
repartition between a solid stationary phase and a spectrometry or electrochemical
liquid mobile phase with different polarities, at high detection
flow rate and pressure of the mobile phase
TLC Separation of compounds is based on the repartition UV–vis detection
between a solid stationary phase (silica gel) and a
liquid mobile phase (mixture of acetate, formic acid
and water)
 ELECTROCHEMICAL TECHNIQUES

Antioxidant Assay Principle End Product Determination

Cyclic voltammetry The potential of a working electrode is
linearly varied from an initial value to a final
value and back, and the respective current
intensity is recorded
Amperometry The potential of the working electrode is set
at a fixed value with respect to a reference
electrode
Measurement of the intensity of the
cathodic/ anodic peak
Measurement of the intensity of the
current generated by the
oxidation/reduction of an electroactive
analyte
ANTIOXIDANT ASSAYS – SELECTION PARAMETERS

 PH- There are tests operating in acidic (FRAP), neutral (CUPRAC) or alkaline (Folin−Ciocalteu) conditions.

 hydrophilic and lipophilic antioxidants- While the ABTS and CUPRAC tests can measure both hydrophilic, and
lipophilic antioxidants, some methods only measure hydrophilic antioxidants (FRAP and Folin−Ciocalteu), and others
only apply to hydrophobic systems (DPPH).
 background colour in the food matrix- may trigger absorbance modifications, which have more significant adverse
effects in the case of discolouration reactions (ABTS, DPPH), as compared to colour-formation reactions (FRAP,
CUPRAC).
REFERENCES

 https://innovareacademics.in/journals/index.php/ajpcr/article/view/13092

 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464350/

 https://ijaem.net/issue_dcp/Antioxidant%20detection,%20estimation,%20and%20evaluation%20in%20food%20systems.
pdf
 https://onlinelibrary.wiley.com/doi/full/10.1002/fft2.10