(0.15 mM) solutions were prepared fresh in methanol at room tem-
perature (22 ± 2 °C). The equivalent concentration of the antioxidants
in ppm has also been calculated and shown in
Table S1 Supplementary
Data
. 2 mL of DPPH solution was then added into 2 mL of antioxidant
solutions and mixed vigorously using a vortex mixer. The mixture was
left at room temperature in the dark for 30 min and absorbance read at
517 nm using a UV/visible spectrometer (Beckman DU 530). A solution
containing 2 mL of methanol and 2 mL antioxidants solution was used
as the blank, whereas a solution containing 2 mL of DPPH with 2 mL of
methanol used as the control. The DPPH scavenging capacity was
measured using the following equation
=
−
∗
DPPH Scavenging Capacity
A
A
A
%
100
c
s
c
(1)
where,
A
C
is the absorbance of the control, and
A
S
is the absorbance of
the test solution.
2.3.2. Metal chelating ability test
Metal chelating ability of the antioxidant was tested by measuring
its chelating activity towards Fe
2+
using a method described by
Maqsood and Benjakul (2010)
with slight modi
fi
cations. In brief,
5
–
400 µM solutions of the tannic acid, the ca
ff
eic acid and the
L
-as-
corbic acid were prepared in distilled water. The pH of the solutions
was then increased to 8
–
9 using 1 M NaOH and kept at that pH for
30 min to improve the solubility of the antioxidants and then it was re-
adjusted to 7.3 using 1 N HCl. All other antioxidant solutions were
prepared in ethanol. Approximately, 40 µL of freshly prepared FeCl
2
(2 mM) solution was added to 3.5 mL of antioxidant solution, and after
5 min, 80 µL of ferrozine (5 mM) solution was added into the mixture
for excess iron quanti
fi
cation and mixed vigorously. The mixture was
left at room temperature in dark for 20 min for the reaction to happen
and afterwards the absorbance of the ferrozine-iron complex was
measured at 562 nm. A control for each antioxidant was prepared in the
same way, using all the other reagents in either water/ethanol without
adding any antioxidant. The metal chelating activity was calculated
using the following formula,
=
−
∗
Metal Chelating Ability
A
A
A
%
100
c
s
c
(2)
where
A
C
is the absorbance of the control,
A
S
is the absorbance of the
sample.
2.4. Oxidative stability of FSO
For oxidative stability of FSO natural antioxidants were selected
based on their highest antioxidant capacities (See Results and
Discussion section) within each of the hydrophilic (tannic acid), hy-
drophobic (alpha tocopherol) and intermediate polarity (ascorbyl pal-
mitate) categories. Synthetic antioxidant TBHQ was also used as a
control. Di
ff
erent concentrations (0, 50, 100, 200 and 400 ppm) of
antioxidant were added in methanol at room temperature and the re-
quired amount of antioxidant solutions were added into the oil.
Solutions were prepared in such a way that the amount of methanol
needed was 50 µL per 10 g of oil in all cases to minimize the e
ff
ect of
any excess methanol. The blends were mixed vigorously using a vortex
mixer for 5 min and the methanol was removed by passing dry nitrogen
through the sample for 10 min at 4 °C to avoid any oxidation. The same
amount of methanol is also added to the control oil (
fl
axseed oil without
antioxidants) and treated the same way as its mixtures with anti-
oxidants. Di
ff
erent aliquots of mixtures were then taken for the de-
termination of peroxide value (PV),
p
-anisidine value (AV) and the
accelerated oxidation using a rancimat. The FSO as well as its mixtures
with antioxidants were stored at 25 °C, 40 °C, and 60 °C in dark for
30 days, and the PV and the AV values were determined on the same
day (0 d) after an hour of storage, and after 1, 10, 20 and 30 days of
storage. The accelerated oxidative stability of the samples was done at
110 °C using a rancimat to rapidly determine the high temperature
stability of the oil mixtures.
2.4.1. Accelerated oxidative stability
Accelerated oxidative stability of the samples was determined using
a Metrohm rancimat model 743 (Herisau, Switzerland) according to
Frega, Mozzon, and Lercker (1999)
. Approximately, 3 g of FSO or its
mixtures with di
ff
erent concentrations of antioxidants were taken in a
disposable glass test tube and heated at 110 °C at a gas
fl
ow of rate
20 L/h. The volatile oxidized compounds coming out of the sample
during heating were transported to a reaction vessel containing 60 mL
of Millipore (deionized) water, and the change in conductivity was
measured as a function of time. The time at which the volatile com-
pounds were detected in the measuring vessel, known as the oxidative
stability index (OSI), was indicated by a sharp increase in conductivity
of water as noted by the in
fl
ection point of the conductivity vs time
graph recorded by the rancimat.
2.4.2. Peroxide value
Peroxide value (PV) of the FSO and FSO-antioxidant mixtures were
evaluated using International Dairy Federation (IDF) method (
Shantha
& Decker, 1994
). In brief, approximately 150 mg of each sample was
dissolved in a 10 mL mixture of 7:3 chloroform/methanol. Approxi-
mately, 50 µL of ammonium thiocyanate solution (30%) was then
added into the mixture and mixed using a vortex mixer for 10
–
20 s.
Then
∼
50 µL of freshly prepared iron (II) solution (
∼
20 mM) was
added and mixed again using the vortex mixer. The resulting mixture
was kept for 5 min at room temperature in the dark and absorbance
read at 500 nm against a blank that contained all reagents except the oil
sample using a UV/Visible spectrometer (Beckman DU 530). Note, the
iron (II) chloride (FeCl
2
) solution was prepared every day by adding a
50 mL of iron (II) sulphate (0.5 g FeSO
4
·7H
2
O dissolved in 50 mL dis-
tilled water) into a 50 mL of barium chloride solution (0.4 g of barium
chloride dihydrate in 50 mL of distilled water), and adding 2 mL of 10 N
HCl into the resulting solution. The barium sulphate precipitate was
fi
ltered out to obtain a clear iron (II) chloride solution. Ammonium
thiocyanate solution was prepared by adding 3 g of ammonium thio-
cyanate into 10 mL of water every day. A standard curve for iron (III)
were prepared using FeCl
3
solution. A working solution of 2.5 mg/mL of
iron (III) chloride solution was prepared in distilled water and a set of
solutions of increasing iron (III) concentration in the range 0
–
50 µg of
iron (III) in 10 mL 7:3 chloroform/methanol were prepared by diluting
the working solution. The standard curve was obtained by plotting
absorbance versus iron (III) concentration. The peroxide value, ex-
pressed as mille-equivalents of peroxide per kilograms of sample was
calculated using the following equation:
=
−
∗ ∗
∗
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