International Journal of Food Properties

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Fortification of refined soybean oil by hull oil of

two Iranian wild pistachios: Improving thermal
stability during frying process

Javad Tavakoli & Nazanin Sorbi

To cite this article: Javad Tavakoli & Nazanin Sorbi (2017) Fortification of refined soybean oil by
hull oil of two Iranian wild pistachios: Improving thermal stability during frying process, International
Journal of Food Properties, 20:sup3, S2990-S3003, DOI: 10.1080/10942912.2017.1396339

To link to this article: https://doi.org/10.1080/10942912.2017.1396339

© Javad Tavakoli and Nazanin Sorbi

Published online: 09 Jan 2018.

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INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. S3, S2990–S3003
https://doi.org/10.1080/10942912.2017.1396339

Fortification of refined soybean oil by hull oil of two Iranian wild

pistachios: Improving thermal stability during frying process
Javad Tavakoli and Nazanin Sorbi
Faculty of Agriculture, Department of Food Science and Technology, Jahrom University, Jahrom, Fars, Iran

ABSTRACT ARTICLE HISTORY

In this research, various concentrations of Kolkhoung (Pistacia khinjuk) hull oil Received 8 June 2017
(KHO), Beneh (Pistacia atlantica) hull oil (BHO), and Sesame (Sesamum indicume Accepted 20 October 2017
L. cv. Dezful) seed oil (1%, 2%, 4%, and 6%) as natural antioxidants were added KEYWORDS
to refined soybean oil (RSO) and antioxidant effect of these compounds was Kolkhoung hull oil; Beneh
compared with that of tert-butyl hydroquinone (TBHQ) during 32 h frying hull oil; Oxidative stability;
process at 180°C. For this purpose, variation of acid value (AV), conjugated Tocotrienols; Sterols; Sesame
diene value (CDV), carbonyl value (CV), total polar compounds, and total tocol seed oil
(TT) and total sterol contents of RSO was monitored during frying process.
Results of oxidative stability assays revealed that 4%KHO had the best
antioxidant effect which was fairly equal to that of TBHQ, followed by 4%
BHO. It should be mentioned that in contrast to TBHQ, KHO and BHO are not
pure antioxidant. The remarkable antioxidant effect of KHO and BHO during
thermal process was due to high tocotrienol content and presence of suitable
amount of Δ-7-avenasterol and Δ-5-avenasterol, respectively [TT content of
KHO (2041 mg/kg) much higher than that of ordinary edible oils and 92.7% of
TT content in KHO is formed by tocotrienols]. Antioxidant activity of
Δ-7-avenasterol and Δ-5-avenasterol and tocotrienols during frying process
was attributed to formation of stabilized tertiary alkyl radicals. These radicals
cut down radical chains formed during frying process.

Introduction
Soybean oil is one of the most common oils across the world and has suitable nutritional value due to
high content of linoleic acid, linolenic acid, and antioxidant compounds. Since this oil has high amount
of linolenic acid, its oxidative stability, especially at high temperatures, is low and thus is not suitable for
frying.[1] Oxidative stability of this oil has been improved through development of low linolenic acid
soybean varieties. Moreover, in oil-producing factories, oxidative stability of the soybean oil is enhanced
by hydrogenation. However, use of hydrogenated oil causes various health-related problems such as the
risk of cancer and cardiovascular diseases due to formation saturated and trans fatty acids during
hydrogenation process.[2] Addition of antioxidants to edible oils improves their oxidative stability. Use
of some synthetic antioxidants in food and oils has been reduced in developed countries because they are
suspected as causing cancer in the human body.[3] In contrast, application of natural antioxidants has
increased due to their positive effect on human health. Application of natural antioxidants also results in
some problems. Most of these antioxidants have low oxidative stability at high temperature (frying).
Moreover, they are usually found in small amount and their extraction is costly. Thus, researchers seek
for suitable natural antioxidants with high thermal stability and low extraction cost that are available in
sufficient amount. There are rich sources of natural antioxidants in Iran that have been investigated by
many authors.[4–7] Fruits of two wild Pistacia species called Beneh (Pistacia atlantica) and Kolkhoung

CONTACT Javad Tavakoli ja_tavakoli@yahoo.com; javadtavakoli@jahromu.ac.ir Faculty of Agriculture, Department of

Food Science and Technology, Jahrom University, Jahrom, Fars, Iran.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljfp.
© Javad Tavakoli and Nazanin Sorbi
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2991

(Pistacia Khinjuk) represent a source of natural antioxidants that are available in high amount in Zagros
forests in West and Southwest Iran.[4,5,8,9] Zagros forests cover an area of 5 million ha where Beneh,
Kolkhoung, oak, and wild almond are found. Tavakoli et al. showed that Kolkhoung hull oil (KHO) is an
oil containing tocopherols with unique oxidative stability during thermal process. Oxidative indices such
as carbonyl value (CV), acid value (AV), conjugated diene value (CDV), and polar components of this oil
show low increase. Moreover, it has been revealed in rancimat test that the effect of 100 ppm KHO on
oxidative stability of olive oil was equal to that of 100 ppm tert-butyl hydroquinone (TBHQ).[8] In
another study, it was revealed that Beneh hull oil (BHO) is an oil with high oxidative stability during
thermal process at 170°C.[9] Also, Pazhouhanmehr et al. showed that addition of this oil increased
oxidative stability of fish oil during thermal process.[10] Based on previous researches, the present study
was designed to evaluate the antioxidant effect of the addition of KHO into refined soybean oil (RSO)
during the frying process, and then further measurement and comparison of the oxidative stability
indexes with of the BHO and Sesame (Sesamum indicume L. cv. Dezful) seed oil (SSO) (as two oils
containing high antioxidants) and TBHQ (as a typical synthetic antioxidant) added RSO.

Materials and methods

Materials
Kolkhoung and Beneh fruits (10 kg) were collected from forest located in Meimand, Iran (summer,
2016). Sesame seeds (S. indicume L. cv Dezful) were gathered from a farmland in Dezul, Khoozestan
Province (summer, 2016). RSO (30 kg) without added antioxidants was supplied by the Narges Company
(Shiraz, Iran). All samples were stored at 4°C until the initiation of the experiments (maximum 4 weeks).
All chemicals and solvents used in this experiment had analytical reagent grade and were purchased from
Merck and Sigma-Aldrich Chemical Companies (Darmstadt, Germany).

Oil extraction
The soft green hull of the Kolkhoung and Beneh fruits was removed using a dehuller device and,
together with sesame seeds, was powdered in a mill (Mullinex Depose-Brevete S.G.C.G., France). The
prepared powders were mixed with normal hexane (1:4) and placed on a shaker for 48 h in the dark
condition. The mixtures were then placed under vacuum oven (OV11, Jeiotech, Korea) at 40°C for
6–12 h to remove the remained solvent.[8,9,11]

Extraction of unsaponifiable matters

The saponification of the oils was carried out as following: 5 g of oil was mixed with 50 mL 1 N ethanolic
KOH and subsequently heated at 95°C for 1 h. The mixture was cooled at ambient temperature and then
100 mL distilled water was added and completely mixed. The resulting solution was extracted twice using
100 mL diethyl ether in a decanter funnel. In each extraction step, the upper organic layer was collected
and washed twice with 75 mL distilled water, once with 100 mL of 0.5 N ethanolic KOH and finally
neutralized with 100 mL distilled water. Then, the organic layers were separated and dried using Na2SO4.
The solution was filtered and evaporated to dryness in a vacuum oven (OV11, Jeiotech, Korea) at 45°C.
For further purification of unsaponifiable compounds, they were dissolved in chloroform, filtered and
finally evaporated at 45°C under vacuum conditions.[4,6,12]

Preparation of various samples of RSO

To perform frying, different RSO samples containing 1%, 2%, 4%, and 6% KHO, BHO, SSO, and
100 ppm TBHQ were prepared to be compared with RSO without antioxidant as control.
S2992 J. TAVAKOLI AND N. SORBI

Frying process
Potatoes were peeled and cut into 6 cm × 1 cm × 1 cm slices and submerged in water until needed and
dried with a cotton cloth. Frying was performed using a conventional fryer (Tefal model 1250, Paris,
France) equipped with a thermostat and stainless steel grid basket (in each replication, 2.5 L oil sample
was used). Potato pieces were fried in 20-g batches at constant frying temperature (180°C) and were
subjected to frying for 5 min. The rest time between frying periods was 15 min. The frying process was
conducted for four consecutive days, 8 h per day. In 4 h intervals, about 10 g of oil was taken from the
fryer, cooled at room temperature, and, after nitrogen injection, kept at −18°C until experiment time
(maximum 1 week). The token volume of oil was not replenished during the frying process.[5,6,13]

Fatty acid composition

Fatty acid composition of the oil samples was determined by a gas–liquid chromatography and reported
in relative area percentages. Fatty acids were transesterified into their corresponding FAMEs (fatty acid
methyl ester) by vigorous shaking of a solution of the oil in hexane (0.3 g in 7 mL) with 2 mL of 7 M
methanolic potassium hydroxide at 50°C for 10 min. The FAMEs were identified using an HP-5890 gas
chromatography (Agilent, Palo Alto, CA) equipped with a CP-FIL 88 (Supelco Inc., Bellefonte, PA)
capillary fused silica column (60 m × 0.22 mm i.d., 0.2 mm film thickness) and the flame ionization
detector. Nitrogen was used as a carrier gas at flow rate of 0.75 mL/min. The oven temperature was fixed
at 198°C and that of the injector and the detector at 250°C.[4,5]

Gas chromatography analysis for sterol compounds of KHO, BHO, and SSO
To quantify sterol compounds of KHO, BHO, and SSO, a gas chromatography system using Betulin as
a standard was applied. The compounds were separated by an SE 54 CB column (Macherey-Nagel,
Duren, Germany; 50 m long, 0.25 mm ID, 0.25 µm film thickness). Further used parameters can be
summarized as follows: hydrogen as a carrier gas, split ratio 1:20, injection and detection temperature
adjusted to 320°C, temperature program, 240–255°C at 4°C/min.[7,8,14]

HPLC analysis for tocol compounds of KHO, BHO, and SSO

Tocol compounds in the KHO were determined using a HPLC system (Waters ACQUITY UPLC®
System, Milford, Massachusetts, USA) with a Spherisorb column (25 cm × 4 mm i.d., WATERS, USA)
packed with silica (5 µm particle size) and a fluorescence detector operating at an excitation wavelength
of 290 nm and an emission wavelength of 330 nm were utilized. The used mobile phase consisted of
acetonitrile and water (9:1, v/v) at a flow rate of 0.5 mL/min. Tocopherols in test samples were verified by
the comparison of retention time with reference standards.[15]

Total sterol and total tocol contents of RSO samples

Total sterol (TS) content of RSO samples was quantified according to the Lieberman–Burchard color
reaction.[16] Total tocol (TT) content of RSO was also determined according to the colorimetric
method described by Wong et al.[4,7,17]

Acid value, conjugated diene value, and carbonyl value

AV was determined according to the AOCS Official Method Cd 3d-63.[4,7,18] CDV was measured
using the described procedure by Saguy et al.[4,7,19] In this method, oil sample was mixed with HPLC
grade hexane in 1:600 ratio and the absorption was determined spectroscopically (UV/Visible Philips
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2993

Cambridge, UK) at 234 nm. CV of the oil samples was measured according to the developed method
by Endo et al.[4,7,20]

Total polar compounds content

The total polar compounds (TPC) content was determined according to the economical micro
method described by Schulte with a slight modification on removing the solvent from the eluate.[7,21]
The solvent was evaporated in a vacuum oven at 40°C for 25 min.

Statistical analysis
Initially, the data obtained from the experiments of chemical properties in the present investigation were
analyzed in triplicate based on a randomized complete design (RCD). The subsequent tests of frying
process of these oils were developed as factorial RCD with three replications (the variable in these
experiments was oil samples and time of frying time). All data were analyzed using ANOVA. MStatC and
SlideWrite software were used for ANOVA and regression analysis, respectively. The mean comparison
was performed by Duncan multi-step test to determine significant differences (P < 0.05).

Result and discussion

Chemical properties
Chemical properties of KHO, BHO, and SSO are represented in Table 1. As seen, the dominant fatty
acids of KHO and BHO and SSO were oleic acid (52–52.1%) and linoleic acid (47.1%), respectively.
SFA value in KHO, BHO, and SSO was 25.8%, 25.6%, and 16.8%, indicating no significant difference
(P > 0.05) between BHO and KHO. The dominant saturated fatty acid in the three oils was palmitic
acid (10.2–23.4%), followed by stearic acid (2.4–5.7%). The content of linolenic acid as a highly
oxidation-sensitive oil in KHO, BHO, and SSO was 1.5%, 1.2%, and 5%, suggesting that there is
significant difference (P > 0.05) among the oils. Considering fatty acid composition of KHO, BHO,
and SSO (ignoring other factors affecting oxidation stability of edible oils), it can be predicted that
BHO is probably more stable than KHO and SSO.

Table 1. Chemical characteristics of KHO, BHO, and SSO.*

Parameter KHO BHO SSO
Fatty acid (%)
C16:0 23.4 ± 0.24a 22.55 ± 0.07a 10.2 ± 0.32b
C16:1 7.74 ± 0.07b 14.05 ± 0.22a 0.28 ± 0.1c
C18:0 2.39 ± 0.05c 3 ± 0.14b 5.71 ± 0.22a
C18:1 52.03 ± 0.16a 52.12 ± 0.17a 30.01 ± 0.42b
C18:2 12.02 ± 0.2b 5.35 ± 1.02c 47.1 ± 0.69a
C18:3 1.5 ± 0.19b 1.16 ± 0.12b 5.01 ± 0.45a
C20:1 0.25 ± 0.05b 0.76 ± 0.06a 0.44 ± 0.11b
C22:0 – – 0.59 ± 0.22
SFA 25.79 ± 0.21a 25.55 ± 0.9a 16.79 ± 0.51b
MUFA 60.02 ± 0.3b 66.93 ± 0.7a 30.73 ± 0.58c
PUFA 13.52 ± .19b 6.51 ± .11c 52.11 ± 0.52a
USM content (% of oil) 0.64 ± 0.06a 0.72 ± 0.04a 0.78 ± 0.09a
TT content (mg/kg oil) 2041 ± 19a 703 ± 12c 994 ± 17b
TP content (mg gallic acid/kg oil) 100 ± 7b 82 ± 10c 646 ± 14a
TS content (mg/kg oil) 1601 ± 13c 1649 ± 12b 5401 ± 16a
*Mean ± SD within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; KHO: kolkhoung hull oil; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty
acid; SFA: saturated fatty acids; SSO: Seasame seed oil; TP: total phenolics; TT: total tocols; TS: total sterols;
USM: unsaponifiable matter.
S2994 J. TAVAKOLI AND N. SORBI

USM values of the studied oils are presented in Table 1. As can be observed, there is no significant
difference (P > 0.05) among KHO, BHO, and SSO regarding USM value (0.64–0.78%). USM compounds
can delay deterioration of food. The main components of USM compounds include hydrocarbons,
terpene alcohols, esterols, and tocopherols. USM of edible oils varies between 0.5% and 2.5% and
sometimes reaches 5–6%. The content of these compounds is highly affected by purification; thus,
their value is extensively used as an index of purified oils’ quality.[22] Sharayei et al. used USM of Beneh
kernel oil as an antioxidant additive in canola oil and, based on frying process results, concluded that
these compounds can be used as a stable natural antioxidant in edible oil.[7]
Tocopherols are compounds with antioxidant and vitamin activities. Presence of these compounds is
effective in improvement of oxidative stability of edible oils. TT content of KHO (2014 mg/kg) was
significantly (P > 0.05) higher than that of other oils, followed by SSO (994 mg/kg) and BHO (703 mg/kg)
(Table 1). TT content of KHO was apparently higher than that found in common plant oils.[23] Tavakoli
et al. reported that TT content of kernel oil from Kolkhoung was 619.4 mg/kg which is much lower than
that measured in KHO.[4] Also, amount of TT content of Beneh kernel oil was measured as 818.6 mg/
kg.[24]
Phytosterols as the one of the most important minor compounds of plant oils could play a critical
role in food grading and are considered as an index for recognizing the fake and original edible oils.
Most of plant oils have 1000–5000 ppm of esterol compounds.[25,26] As is evident in Table 1, the
highest TS content was found in SSO (5041 mg/kg), followed by BHO and KHO (1649 and
1601 mg/kg).
As natural antioxidants, phenolic compounds represent a major factor for evaluation of edible
oils’ quality. There is direct relation between oxidative stability and organoleptic properties of edible
oils and their phenolics content. Moreover, phenolics play biological role in human body that
prevents from diseases caused by formation of free radicals via improving antioxidation defense
capacity of the body.[27,28] As can be seen from Table 1, there was significant difference (P > 0.05)
among the three oils regarding TP value. The highest content of TP was found in SSO (994 mg/kg),
followed by KHO (100 mg/kg) and BHO (82 mg/kg). Presence of large amount of antioxidant
compounds in edible oils is a positive property; however, possessing high thermal stability especially
during frying process is more important.

Thermal stability during frying process

CDV measurement as primary oxidation index is a suitable test for evaluating thermal stability of
edible oils with high content of linoleic and linolenic acids including soybean oil. During oxidation of
polyunsaturated fatty acids, diene and conjugated triene bonds are formed. By increase in the number
of conjugated diene bonds, optical absorbance in 234 nm is enhanced which is used as an index of
oxidation and reported as increase in oxygen absorption and formation hydroperoxides during first
steps of oxidation.[23,29,30] CDV variation in RSO treated with various concentrations of KHO, BHO,
and SSO and 100 ppm TBHQ during 32 h frying process at 180°C is presented in Table 2. CDV
increase for pure RSO and RSO samples containing 1%, 2%, 4%, and 6% KHO, BHO, and SSO and
100 ppm TBHQ was 885%, 867%, 725%, 769%, 793%, 862%, 721%, 779%, 869%, 846%, 822%, 780%,
and 616%. As can be seen in Table 1, after 32 h of frying, the lowest CDV variation was observed in the
sample containing 100 ppm TBHQ, followed by those containing 2% KHO, 4%BHO, 4% and 6% KHO,
1% and 6% BHO and 6%SSO, 4%SSO, 2%BHO and SSO, 1%KHO, 4%BHO, and pure RSO. KHO in
level of 2% and BHO in level of 4% had antioxidant activity equal to 80% and 85% of TBHQ,
respectively. Considering impurity of these oils compared to TBHQ, it is a remarkable result because
some synthetic antioxidants cause cancer and cardiovascular diseases. 2%KHO was determined as the
best sample because it is half of 4%BHO.
Moreover, this assay showed that by increase of KHO, BHO, and SSO in RSO, CDV increase was
not regularly changed. For example, 2%KHO and 4%BHO were the best sample and promoted RSO
stability against CDV variation. This may be attributed to impurity of these oils. Besides
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2995

Table 2. Changes in CDV (mmol/L) of the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process at 180°C.
RSO contain
Time (h) RSO 1%KHO 2%KHO 4%KHO 6%KHO 1%BHO 2%BHO
0 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a
4 30.8 ± 1.2b 33.3 ± 0.2a 32 ± 0.5b 23.5 ± 1.2d 21.7 ± 0.1 27.1 ± 0.9c 26.4 ± 0.3c
8 47.1 ± 2.1bcd 49.3 ± 0.8b 46.6 ± 0.3d 37.3 ± 0.3g 36.2 ± 0.4h 42.9 ± 2ef 42.6 ± 0.8e
12 67.1 ± 2.5abc 67.6 ± 0.1a 67.7 ± 0.3a 56.4 ± 1. 57.4 ± 4.5 60.8 ± 0.1e 60.6 ± 0.2e
16 80.4 ± 1.2a 76.4 ± 0.8c 78.8 ± 0.4b 52.4 ± 3.7k 65 ± 0.1h 70.8 ± 0.5f 71.1 ± 0.1e
20 92.9 ± 1.1a 90.2 ± 1.5b 90 ± 0.3b 79.8 ± 0.3f 77.6 ± 0.5g 83.5 ± 0.3d 83.4 ± 0.4d
24 101 ± 1.4a 99.9 ± 1.1b 103.3 ± 1.9a 88.4 ± 1.4e 86.1 ± 0.5f 91.4 ± 1.1d 91.9 ± 0.4d
28 107.9 ± 0.1a 97.8 ± 0.3e 99 ± 0.5e 105.9 ± 0.5b 104.9 ± 0.1c 104.2 ± 0.2c 100.8 ± 0.3d
32 116.8 ± 3.7a 114.7 ± 0.5a 97.9 ± 0.4d 103.1 ± 1c 103.3 ± 0.5c 106 ± 2.7c 114.1 ± 3.1ab
RSO contain
4%BHO 6%BHO 1%SSO 2%SSO 4%SSO 6%SSO 100 ppm TBHQ
0 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a 11.9 ± 0.3a
4 20.5 ± 0.1e 26.6 ± .25c 27.2 ± 0.8c 26.4 ± 0.4c 22.5 ± 0.3d 25.5 ± 2.5cd 19.1 ± 0.2f
8 33.2 ± 0.2j 40.3 ± 0.6f 53.1 ± 1.4a 47.5 ± 0.5c 34.4 ± 0.4i 40.3 ± 0.9f 27.1 ± 0.1k
12 50.4 ± 0.4 63.8 ± 1.1d 66.8 ± 1b 65.3 ± 0.6c 52.4 ± 0.2 55 ± 3.1 36 ± 0.6
16 59.9 ± 0.4j 69.2 ± 0.4g 79.9 ± 1.1a 77.1 ± 0.4c 62.9 ± 0.5i 72 ± 0.6d 43 ± 0.6l
20 72.2 ± 0.3h 80.8 ± 0.4e 91.5 ± 0.5ab 89.4 ± 0.3c 72.2 ± 0.8h 80.9 ± 0.7e 54 ± 0.5i
24 82.9 ± 1.7g 87.5 ± 0.4ef 102.9 ± 0.8a 94 ± 0.6c 83 ± 2.5g 94.1 ± 1c 64.1 ± 0.6h
28 96.7 ± 0.9f 95.3 ± 0.8g 104.2 ± 1.8bc 100.8 ± 0.4d 96.2 ± 1.8f 98.1 ± 0.8e 75.1 ± 0.6h
32 97.4 ± 2d 104.3 ± 0.6c 114.9 ± 1.7a 112.2 ± 2.9ab 109.4 ± 2.7b 104.4 ± 1.3c 85 ± 0.5e
Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; CDV: conjugated diene value; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: seasame seed oil;
TBHQ: tertiary butyl hydroquinone.

antioxidants, there are also other compounds in KHO, BHO, and SSO that may cause unpredictable
reactions in RSO. When concentration of these oils in RSO exceeds a threshold value, antioxidative
state is replaced by prooxidative one. Such condition occurs when antioxidants’ concentration is
increased in edible oils. Similar results have been reported by Sharayei et al. By increase in Beneh
kernel oil content in canola oil, resistance against CDV variation during thermal process was
improved and then reduced. Moreover, it was observed that 1% Beneh kernel oil had antioxidant
activity equal to 65% of TBHQ.[4] In another study, it was found that Beneh kernel oil at 1000 ppm
concentration was not significantly different (P > 0.05) from TBHQ (100 ppm) in prevention of
CDV increase in canola oil blended with palm olein and virgin olive oils.[6]
Carbonyl compounds such as aldehydes and ketones are products of lipid secondary oxidation
formed by decomposition of hydroperoxides and cause bad taste, deterioration, and reduced nutri-
tional value of fried foods. They are more stable than peroxides during frying; thus, CV is a good
index of oxidation variation of edible oils during thermal process.[20] CV variations of different RSO
samples during 32 h of frying process at 180°C are presented in Table 3. The highest increase of CV
was observed in pure RSO (2051%), followed by 4%SSO (1445%), 6%SSO (1338%), 1%SSO (1263%),
1%KHO (1248%), 2%SSO (1177%), 6%KHO (1089%), 2%KHO (1078%), 2%BHO (995%), 1%BHO
(929%), 4%BHO (867%), 100 ppm TBHQ (826%), and 4%KHO (580%). Based on these results,
antioxidant effect of 1%, 2%, 4%, and 6% of KHO, BHO, and SSO was determined 66%, 77%, 142%,
76%, 89%, 83%, 95%, 86%, 65%, 70%, 57%, and 61% of that of TBHQ. Among various samples, 4%
KHO had the best antioxidant activity in preventing from formation of carbonyl compounds in RSO
which is even higher than that of TBHQ. The results of this assay showed that KHO and BHO can be
used as substitutes for TBHQ to prevent the problems caused by this synthetic antioxidant. As
observed in CDV test, by increase in KHO, BHO, and SSO in RSO, CV increase was not regularly
changed. Evaluation of CV variation of canola oil blended with different concentrations of Beneh
kernel oil indicated that by increase of this oil in canola oil, CV variation was not regularly changed
so that among canola oils containing 0.05%, 0.1%, 0.2%, and 0.4% of Beneh kernel oil, the sample
with 0.2% of this oil had the lowest antioxidant effect in preventing formation of carbonyl com-
pounds, followed by samples containing 0.1%, 0.05%, and 0.4% of this oil. Moreover, it was observed
S2996 J. TAVAKOLI AND N. SORBI

Table 3. Changes in CV (µmol/g) of the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process at 180°C.
RSO contain
Time (h) RSO 1%KHO 2%KHO 4%KHO 6%KHO 1%BHO 2%BHO
0 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a
4 26.6 ± 0.3a 19 ± 0.1e 24.7 ± 1.7b 15.3 ± 0.4f 15.2 ± 0.3f 27.1 ± 0.9a 20.3 ± 0.7d
8 40.7 ± 0.4a 31 ± 1c 39 ± 0.6b 27 ± 2.6d 25.4 ± 0.8d 42.9 ± 2a 32.3 ± 0.7c
12 65.9 ± 0.5a 43.6 ± 0.9e 57.4 ± 1.9c 44.3 ± 1.2e 34.5 ± 2.2hi 60.8 ± 0.1b 47.6 ± 1.4d
16 65.6 ± 0.3b 59 ± 1.9c 57 ± 0.9c 48.8 ± 3.7d 43.7 ± 1.5e 70.8 ± 0.5a 57.7 ± 2c
20 84.1 ± 0.9ab 79.6 ± 2.2c 86.5 ± 2a 54 ± 1h 60.1 ± 0.1g 83.5 ± 0.3b 72.9 ± 1.4d
24 108.3 ± 1.4a 97.4 ± 3b 87.7 ± 3.3cd 52.7 ± 0.4j 73.9 ± 0.9g 91.4 ± 1.1c 77.6 ± 2.2ef
28 119.8 ± 0.9b 131 ± 3.7a 91.5 ± 2gh 68.4 ± 0.8k 116.6 ± 4.4cd 104.2 ± 0.2e 101.9 ± 1.6f
32 219.4 ± 2.2a 138.8 ± 4.1d 122.5 ± 4.5f 70 ± 1.2j 123.7 ± 3.5f 106 ± 5.7g 111.7 ± 0.2g
RSO contain
4%BHO 6%BHO 1%SSO 2%SSO 4%SSO 6%SSO 100 ppm TBHQ
0 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a 10.3 ± 1.4a
4 21.4 ± 0.4c 18.8 ± 0.7e 10.3 ± 1.4g 10.3 ± 1.4g 17.9 ± 0.6e 14.6 ± 0.9f 18.1 ± 0.2e
8 32.7 ± 3.1c 26.3 ± 0.7d 17.2 ± 0.2e 15.6 ± 0.5f 31.2 ± 0.9c 27.8 ± 1.1d 25.5 ± 0.7d
12 42 ± 0.1f 46.1 ± 1.3d 36.4 ± 0.4h 33.2 ± 1i 34.5 ± 0.5i 46.5 ± 1.6d 38.3 ± 0.6g
16 47.3 ± 1.3d 51.4 ± 2.7d 49.5 ± 1.6d 44.5 ± 1e 58.7 ± 2.7c 35.5 ± 2.3f 48.8 ± 1.9d
20 60.8 ± 4.4fg 67.2 ± 1.1ef 67.8 ± 2.4ef 63.6 ± 3.2f 70.1 ± 1.9de 66.4 ± 1.6ef 55.2 ± 2.4dh
24 70.5 ± 2.4h 80.5 ± 2.5e 75.7 ± 2.2fg 78.8 ± 0.6ef 85.7 ± 2.4d 77 ± 3.6ef 59.3 ± 1.2i
28 93.1 ± 1g 84.1 ± 1.1i 90.5 ± 1.5h 89.9 ± 1.9h 121.4 ± 3.4c 110 ± 4d 79.9 ± 1.8j
32 99.6 ± 1.1h 110.5 ± 3.3g 140.4 ± 3.1d 130.3 ± 2.6e 159.1 ± 4.4b 148.1 ± 4.1c 94.5 ± 1.4i
Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; CV: carbonyl value; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: Seasame seed oil; TBHQ: tertiary
butyl hydroquinone.

that 0.4% Beneh kernel oil was better than 100 ppm TBHQ.[5] Furthermore, Sharayei et al. confirmed
that in CV assay, Beneh kernel oil at 1000 ppm concentration was not significantly different
(P > 0.05) from TBHQ (100 ppm) in canola oil blended with palm olein and virgin olive oils.[6]
AV is known as the triglycerides hydrolysis index. Free fatty acids concentration is elevated
during frying process which is due to hydrolysis of triglycerides or presence of carboxyl groups in
polymeric or oxidative products. By hydrolysis of triglycerides and increase of free fatty acids,
oxidation of edible oils is promoted, and thus, their shelf life is decreased.[29] Parameters resulting
from fitting of AV of various RSO samples during 32 h of frying process at 180°C are presented in
Table 4. As can be seen, the highest AV variation slope was observed in pure RSO (a = 0.103),
followed by samples containing 1%SSO (a = 0.099), 2%SSO (a = 0.098), 6%BHO (a = 0.086), 4%SSO
(a = 0.072), 4%BHO (a = 0.067), 1%BHO (a = 0.065), 1%, 2%, and 6% KHO (a = 0.06), 6%SSO
(a = 0.059), 2%BHO (a = 0.053), 100 ppm TBHQ (a = 0.05), and 4%KHO (a = 0.048). AV increase
in pure RSO and RSO samples containing 1%, 2%, 4%, and 6% of KHO, BHO, and SSO and
100 ppm TBHQ was 1047%, 582%, 553%, 459%, 586%, 601%, 572%, 662%, 842%, 932%, 903%,
692%, 598%, and 450% (Fig. 1). [No significant difference (P > 0.05) was observed among RSO
samples considering t0 AVs (0.32–0.34).] Thus, the most stable treatments against AV increase and
triglycerides hydrolysis in RSO were 4%KHO and 100 ppm TBHQ [with no significant difference
(P > 0.05)], followed by 2%KHO, 2%BHO, 6%KHO, 1%KHO, 1%BHO, 6%SSO, 4%BHO, 4%SSO,
6%SSO, 1% and 2% SSO, and pure RSO. Again, 4%KHO was determined as the best treatment.
Antioxidant activity of the first six treatments (4%KHO, 2%KHO, 2%BHO, 6%KHO, 1%KHO, and
1%BHO) was 98%, 81%, 79%, 77%, 77%, and 75% of that of TBHQ which agrees with the results of
CDV and CV. Regarding health-promoting effects of natural antioxidants, this is a remarkable
finding. In AV test, different results were found in RSO by increase in KHO, BHO, and SSO,
suggesting that by increase of these oils’ concentrations, their antioxidant activity was not necessarily
increased regularly. As mentioned before, this effect is attributed to impurities of these oils as
antioxidants. This result is in accordance with those reported by Sharayei et al. The authors found
out that there was not linear relation between increased Beneh kernel oil concentration in canola oil
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2997

Table 4. The parameters calculated from the linear relationship between the AV (mg/mL) and frying time
for the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process at 180°C.*
AV = a (time) + b
a ± SE b ± SE R2
RSO 0.103 ± 0.007 0.017 ± 0.01 0.968
RSO + 1%KHO 0.06 ± 0.004 0.135 ± 0.02 0.961
RSO + 2%KHO 0.06 ± 0.006 0.247 ± 0.01 0.989
RSO + 4%KHO 0.048 ± 0.003 0.283 ± .02 0.987
RSO + 6%KHO 0.06 ± 0.005 0.234 ± 0.01 0.989
RSO + 1%BHO 0.065 ± 0.004 0.184 ± 0.01 0.978
RSO + 2%BHO 0.053 ± 0.004 0.423 ± 0.02 0.985
RSO + 4%BHO 0.067 ± 0.005 0.227 ± 0.02 0.974
RSO + 6%BHO 0.086 ± 0.006 0.401 ± 0.03 0.987
RSO + 1%SSO 0.099 ± 0.004 0.348 ± 0.02 0.983
RSO + 2%SSO 0.098 ± 0.005 0.316 ± 0.02 0.981
RSO + 4%SSO 0.072 ± 0.004 0.193 ± 0.02 0.973
RSO + 6%SSO 0.059 ± 0.003 0.305 ± 0.03 0.993
RSO + 100 ppm TBHQ 0.05 ± 0.002 0.287 ± 0.02 0.982
*SE: standard error.
AV: acid value; BHO: beneh hull oil; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: seasame seed
oil; TBHQ: tertiary butyl hydroquinone.

Figure 1. Amount of increasing (%) in AV (mg/mL) of the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process
at 180°C. Means with the same lowercase letters are not significantly different at P < 0.05.
AV: acid value; BHO: beneh hull oil; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: Seasame seed oil; TBHQ: tertiary butyl
hydroquinone.

and AV variation.[5] In a similar work, it was revealed that during frying process, 100 ppm of Beneh
kernel oil and 100 ppm TBHQ have similar antioxidant effect in canola oil blended with palm olein
and virgin olive oils.[6]
Measurement of polar compounds is a reliable method to evaluate fried oils’ quality and their
reuse.[31] In many European countries, it is recommended that edible oil containing 24–27% TPC
should be abandoned.[32] Assuming that acceptable TPC of the oils in this study is 24%, the time
required for RSO samples to reach this point (t24) was calculated.[7] Primary TPC of oil samples was
determined between 4.7% and 4.9%, indicating no significant difference (P > 0.05). Figure 2 shows
t24 values of RSO samples at 180°C. As seen, t24 values for pure RSO and samples containing 1%, 2%,
4%, and 6% of KHO, BHO, SSO, and 100 ppm TBHQ were 14.9, 19.2, 20.3, 24.9, 18.8, 27.1, 27, 27.9,
28, and 25.5 h. Statistically, RSO containing 4% and 6% BHO was determined as the best sample in
polar compound assay, followed by those containing 1% and 2% BHO, 4%KHO, 100 ppm TBHQ,
1%, 2%, and 6%KHO, 2%, 4%, and 6% SSO, 2%SSO, and pure RSO. The results obtained in this
assay indicated that all levels of BHO and 4%KHO had equal or better antioxidant activity than
S2998 J. TAVAKOLI AND N. SORBI

Figure 2. The time required to TPC content of 24% (t24) for the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying
process at 180°C. Means with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: Seasame seed oil; TBHQ: tertiary butyl hydroquinone;
TPC: total polar compounds.

TBHQ in prevention of TPC increase during frying process. Sharayei et al. reported that by increase
in Beneh kernel oil in canola oil, t24 value was decreased which can be due to occurrence of
prooxidant state in canola oil. Moreover, the highest value of this factor was found in oil sample
containing 100 ppm TBHQ.[5] In another study, there was no significant difference (P > 0.05)
between 100 ppm Beneh kernel oil and 100 ppm TBHQ in preventing formation of polar com-
pounds in canola oil blended with palm olein and virgin olive oils.[6] The results of polar compound
assay indicated that hull oil of two wild Pistacia species can be ideal substitutes for TBHQ whose
health problems are well documented. Contrary to the results of CDV, CV, and AV assay, that the
best treatments were RSO samples containing KHO (4% and 2%), in this test, samples containing
BHO were the most resistant treatments against formation of polar compounds.
In the present study, there was no direct correlation between the increase in KHO and BHO
concentrations in RSO and improving its oxidative stability but in a study conducted by Ramadan
and Wahdan, it was found that with increasing concentrations of black cumin seed oil and coriander
seed oil (10% and 20%) in corn oil, oxidative stability of oil blends improved, which was due to
changes in the their fatty acid composition and increasing the their tocopherol content.[33] In
another study by Ramadan, it was found that increasing the amount of black cumin oil, cumin
oil, coriander oil, and clove oil in high linoleic sunflower oil had the same effect in improving
oxidative stability of oil blends, too.[34]

Antioxidant compounds (TT and TS) changes

The results of oxidation stability tests indicated that hull oil of the two wild pistachio species can be
regarded as good substitute for TBHQ. For better interpretation of oxidation stability assays (AV, CDV,
CV, and TPC), variation trend in TT and TS contents of RSO was investigated under treatment with
various concentrations of KHO, BHO, SSO, and 100 ppm TBHQ during 32 h frying process at 180°C.
Primary values of TT in different RSO samples were estimated in the range of 1008 and 1104 mg/kg,
showing that addition of various concentrations of KHO, BHO, and SSO into RSO had no significant
effect (P > 0.05) on TT values at t0 (Table 5). As is evident from Table 5, TT content variation of different
RSO samples showed a descending trend during frying process. The best sample in this assay was RSO
containing 100 ppm TBHQ in which, TT content was reduced to 52.4% of the primary level, followed by
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2999

Table 5. Changes in TT content (mg/kg) of the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process at 180°C.
RSO contain
Time (h) RSO 1%KHO 2%KHO 4%KHO 6%KHO 1%BHO 2%BHO
0 1040.2 ± 9d 1054 ± 15cd 1062 ± 13bc 1083 ± 14ab 1104.0 ± 18a 1036 ± 13d 1033 ± 10d
4 950 ± 10a 920 ± 11b 930 ± 11b 947 ± 7a 932 ± 19ab 766 ± 12c 764 ± 15c
8 820.4 ± 8b 711 ± 12d 723 ± 14d 797 ± 18b 760 ± 2c 644 ± 20f 622 ± 3f
12 635.4 ± 7b 540 ± 12d 539 ± 10d 629 ± 2b 615 ± 12c 496 ± 3e 507 ± 7e
16 580.2 ± 5b 490 ± 15d 496 ± 15d 539 ± 7c 505 ± 11d 444 ± 9e 406 ± 10f
20 521.9 ± 4b 435 ± 9d 434 ± 9d 522 ± 7b 440 ± 8d 350 ± 6g 336 ± 2h
24 482.2 ± 6b 402 ± 14d 408 ± 15d 445 ± 8c 391 ± 1e 320 ± 2g 329 ± 10g
28 439.2 ± 8c 361 ± 14 378 ± 13d 464 ± 2b 380 ± 14d 310 ± 10g 299 ± 2hi
32 402.5 ± 8c 359 ± 10d 362 ± 14cd 488 ± 15b 383 ± 12c 300 ± 4fg 286 ± 12fg
RSO contain
4%BHO 6%BHO 1%SSO 2%SSO 4%SSO 6%SSO 100 ppm TBHQ
0 1016 ± 13e 1008.0 ± 16e 1039 ± 15d 1040 ± 14d 1041 ± 8d 1038 ± 14d 1043 ± 7d
4 667 ± 25de 652 ± 5e 743 ± 8c 746 ± 9c 691 ± 9d 615 ± 15h 931 ± 5b
8 551 ± 3g 494 ± 4i 678 ± 9e 579 ± 10e 630 ± 11f 521 ± 17h 850 ± 6a
12 420 ± 12g 384 ± 5h 491 ± 9e 552 ± 13d 604 ± 8c 438 ± 12f 800 ± 5a
16 342 ± 2h 356 ± 15g 443 ± 6e 520 ± 11d 453 ± 9e 432 ± 13e 770 ± 6a
20 282 ± 4j 324 ± 3i 390 ± 10f 497 ± 14c 421 ± 11e 403 ± 15e 702 ± 3a
24 255 ± 3i 311 ± 3h 369 ± 9f 401 ± 10d 404 ± 7d 386 ± 9e 655 ± 7a
28 291 ± 8i 250 ± 6e 353 ± 7f 359 ± 18de 366 ± 9d 377 ± 15d 597 ± 8a
32 237 ± 11i 281 ± 14g 313 ± 17ef 335 ± 11e 292 ± 11fg 259 ± 7h 547 ± 7a
Mean ± SD (standard deviation) within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: seasame seed oil; TBHQ: tertiary butyl hydroquinone;
TT: total tocol.

RSO samples containing 4%KHO, pure RSO, 6%KHO, 1%KHO, 2%KHO, 2%SSO, 1%SSO, 1%BHO, 4%
SSO, 2%BHO, 6%BHO, 6%SSO, and 4%BHO (45.1%, 38.7%, 34.7%, 34.1%, 34%, 32.2%, 30.1%, 29%,
28.1%, 27.7%, 27.7%, 25%, and 23.4%). Moreover, primary TS values of the samples were 2401 ± 36,
2394 ± 40, 2380 ± 40, 2368 ± 40, 2351 ± 40, 2399 ± 43, 2387 ± 42, 2370 ± 44, 2355 ± 43, 2431 ± 30,
2454 ± 33, 2525 ± 33, 2587 ± 30, and 2403 ± 41 mg/kg. Remaining TS value in virgin RSO and RSOs
containing 1%, 2%, 4%, and 6% KHO, BHO, SSO, and 100 ppm TBHQ after 32 h of frying process was
reduced to 36.4%, 39.7%, 39.9%, 39.8%, 40%, 43.1%, 43.2%, 43.7%, 42.9%, 29.8%, 32.9%, 33%, 29.7%, and
42.9% of primary values (Fig. 3). Based on primary TT and TS values, it was concluded that increase in
TT and TS contents doesn’t account for suitable antioxidant effects of KHO and BHO, because RSO
contains large amount of TT and TS and addition of such compounds can’t explain superiority of some
samples. Therefore, tocopherol and esterol composition of KHO and BHO was qualitatively investigated
to reveal the reason for their high antioxidant activity.

Tocopherol and esterol composition

Tocopherol and esterol composition of KHO, BHO, and SSO is represented in Tables 6 and 7. The tables
show that tocol composition of the three oils is quite different from each other. In KHO, alpha tocotrienol
(1077 mg/kg) and delta tocotrienol (815 mg/kg) were dominant tocols, followed by delta tocopherol, beta
tocopherol, alpha tocopherol, and gamma tocopherol. The dominant tocol compound in BHO and SSO
was beta tocopherol (485 mg/kg) and gamma tocopherol (935 mg/kg). Sterol composition of KHO, BHO,
and SSO is represented in Table 7. The dominant esterol in the three oils was β-sitosterol. In KHO, the
major sterols were β-sitosterol (1140 mg/kg), Δ-stigmastenol, campesterol, Δ-7-avenasterol, sitostanol, Δ5-
avenasterol, stigmasterol, clerosterol, cholesterol, Δ5-24 stigmastadienol, and brassicasterol (202, 60, 33, 33,
29, 29, 28, 21, 17, and 10 mg/kg). In BHO, the major sterols were β-sitosterol (1014 mg/kg), Δ5-avenasterol,
Δ-7-avenasterol, Δ-stigmastenol, Δ5-24 stigmastadienol, sitostanol, clerosterol, campesterol, brassicasterol,
stigmasterol, and cholesterol (175, 172, 142, 67, 19, 18, 17, 9, 8, and 8 mg/kg). Similarly, β-sitosterol
(3387 mg/kg) was the major sterol in SSO, followed by campesterol, Δ-stigmastenol, Sitostanol,
S3000 J. TAVAKOLI AND N. SORBI

Figure 3. Amount of remaining (%) TS content of the RSO as affected by the KHO, BHO, SSO, and TBHQ during frying process at
180°C. The columns showing the similar components with the same lowercase letters are not significantly different at P < 0.05.
Error bars indicate standard deviations.
BHO: beneh hull oil; KHO: kolkhoung hull oil; RSO: refined soybean oil; SSO: seasame seed oil; TBHQ: tertiary butyl hydroquinone;
TS: total sterol.

Table 6. The tocol composition of the KHO, BHO, and SSO.

Tocol compounds KHO BHO SSO
α-Tocopherol (mg/kg oil) 36 ± 3b 106 ± 7a 4 ± 1c
β-Tocopherol (mg/kg oil) 55 ± 4b 485 ± 10a 15 ± 2c
γ-Tocopherol (mg/kg oil) 3 ± 1c 6 ± 1b 935 ± 12
δ-Tocopherol (mg/kg oil) 55 ± 1a 8 ± 1c 19 ± 1b
α-Tocotrienol (mg/kg oil) 1077 ± 20a 53 ± 2b 6 ± 1c
δ-Tocotrienol (mg/kg oil) 815 ± 13a 45 ± 4b 15 ± 2c
TT (mg/kg oil) 2041 ± 19a 703 ± 12c 994 ± 17b
Mean ± SD within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; KHO: kolkhoung hull oil; SSO: seasame seed oil.

stigmasterol, Δ5-avenasterol, Δ-7-avenasterol, Δ5-24 stigmastadienol, cholesterol, clerosterol, and brassi-

casterol (1134, 326, 230, 79, 103, 57, 28, 27, 18, and 12 mg/kg).
Tocols are classified into two groups, viz., tocopherols and tocotrienols. Tocopherols are good
antioxidants in room temperature because they block the radical chain of lipid peroxidation by
trapping lipid peroxyl radicals, thus extending the shelf life of fat-rich foods.[35] However, in frying
temperatures, tocopherols have lower efficiency and oil decomposition occurs in their presence. On
the other hand, stability of frying oils is provided by non-classical antioxidants such as Δ-7-avenasterol
and Δ-5-avenasterol because they possess ethylidene group in their structure. These compounds are
ineffective at room temperature and their antioxidant activity during frying process is attributed to
formation of stabilized tertiary alkyl radicals.[36–38] These radicals cut down radical chains formed
during frying process. The highest Δ-7-avenasterol and Δ-5-avenasterol content was observed in BHO
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S3001

Table 7. The sterol composition of the KHO, BHO, and SSO.

Sterol compounds KHO BHO SSO
Sitosterol (mg/kg oil) 1140 ± 9b 1014 ± 10c 3387 ± 15a
Δ-Stigmastenol (mg/kg oil) 202 ± 6c 142 ± 9a 326 ± 8b
Campesterol (mg/kg oil) 60 ± 4b 17 ± 4b 1134 ± 9a
Δ-7-Avenasterol (mg/kg oil) 33 ± 2b 172 ± 2c 57 ± 5a
Sitostanol (mg/kg oil) 33 ± 2b 19.2 ± 1c 230 ± 8a
Δ5-Avenasterol (mg/kg oil) 29 ± 2b 175 ± 1c 79 ± 7a
Stigmasterol (mg/kg oil) 29 ± 1b 8 ± 1c 103 ± 5a
Clerosterol (mg/kg oil) 28 ± 2a 18 ± 3b 18 ± 2b
Cholesterol (mg/kg oil) 21 ± 1b 8 ± 1c 27 ± 3a
Δ5-24 Stigmastadienol (mg/kg oil) 17 ± 2c 67 ± 1a 28 ± 2b
Brassicusterol (mg/kg oil) 10 ± 1ab 9 ± 1b 12 ± 2a
TS (mg/kg oil) 1602 ± 13.1c 1649 ± 12.2b 5401 ± 16a
Mean ± SD within a row with the same lowercase letters are not significantly different at P < 0.05.
BHO: beneh hull oil; KHO: kolkhoung hull oil; SSO: seasame seed oil.

(347 mg/kg), followed by SSO and KHO (136 and 57 mg/kg). Thus, antioxidant activity of BHO
compared to TBHQ is due to sufficient amount of Δ-7-avenasterol and Δ-5-avenasterol. However,
another factor seems to be involved in suitable antioxidant activity of KHO. Analysis of KHO tocol
structure indicated that this oil has a fairly different tocol composition compared to other edible oils.
Tocotrienols constitute 92.7% of TT content of KHO. Among ordinary oils, palm oil has high
tocotrienol content and, hence, possesses higher stability during frying process. It has been shown in
recent studies that tocotrienols have better antioxidant activity against spontaneous oxidation of frying
process than tocopherols. Tocotrienols have lipid tail three double bonds that act similar to Δ-7-
avenasterol and Δ-5-avenasterol.[39] In contrast to KHO, RSO lacks tocotrienols. Thus, by addition of
these compounds to RSO via KHO, it is expected that they cause RSO stability during frying process.
Presence of high tocotrienols in KHO can explain its suitable antioxidant effect during frying process.

Conclusion
This study was conducted to investigate the use of hull oil of two wild pistachio species found in Iran
(1%, 2%, 4%, and 6%) as natural antioxidant for improving oxidative stability of soybean oil during
frying process. Results of oxidation stability assays (TPC, AV, CV, and CDV) indicated that KHO in
level of 4% had the best antioxidant effect in RSO compared to TBHQ, followed by 4%BHO. The
importance of these results is more evident by noticing that KHO and BHO are not pure antioxidants.
The remarkable antioxidant effect of KHO and BHO during thermal process is due to high tocotrienol
content and presence of suitable amount of Δ-7-avenasterol and Δ-5-avenasterol, respectively. The
results obtained in this study revealed that hull oil of the two wild Pistacia species can be ideal
substitute for the synthetic antioxidant TBHQ. By this practice, not only oncogenic effect and
cardiovascular diseases caused by synthetic antioxidants are prevented, but also import of synthetic
antioxidants is avoided.

Funding
This research was funded by Jahrom University under Project Grant JU-1395/110.

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