Food Chemistry 344 (2021) 128637

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Action of phytosterols on thermally induced trans fatty acids in peanut oil

Qin Guo a, 1, Tian Li a, b, 1, Yang Qu a, c, Xinping Wang a, d, Li Liu a, Hongzhi Liu a, Qiang Wang a, *
a
Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing
100193, PR China
b
Citrus Research Institute, Chinese Academy of Agricultural Sciences/Southwestern University, Chongqing 400712, PR China
c
College of Life Science and Technology, Xinjiang University, Urumqi 830046, PR China
d
College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: The effects of six phytosterols on thermally induced trans fatty acids (TFAs) in peanut oil were investigated.
Phytosterols Peanut oil, triolein, trilinolein and trilinolenin heated at 180 ◦ C for 12 and 24 h with or without phytosterols
Heat treatment were analyzed by GC-FID. The atomic net charge distribution, frontier molecular orbital energy (FMOE), and
Density functional theory
bond dissociation energy (BDE) of six phytosterols were calculated by density functional theory. Results showed
Trans fatty acids
Peanut oil
that six phytosterols inhibited the formation of trans oleic acid, trans linoleic acids, trans linolenic acids, and total
TFAs. The anti-isomerization effects of phytosterols were mainly associated with hydroxyl site activities, which
were affected by the double bond position in the main skeleton of cyclopentane tetrahydrophenanthrene and the
number of double bonds on the C17 branch chain. The FMOE difference and BDE of phytosterol molecules were
closely related to their anti-isomerization rates. The anti-isomerization mechanisms of phytosterols on TFAs in
peanut oil were proposed.

1. Introduction reduce TFAs in oils and fats.

Peanut oil is one of the most widely consumed vegetable oils and its
global consumption reached 6 million tons in 2018. Remarkably, peanut
oil consumption has increased at a rate of more than 1.6% per year.
Compositionally, peanut oil has a very high proportion of unsaturated
fatty acids (UFAs). In particular, oleic acid (C18:1-9c) and linoleic acid
(C18:2-9c,12c) collectively constitute more than 85% of the total fatty
acid content in peanut oil (Dun, Yao, Deng, Li, Li, Fan, et al., 2019;
Wang, Liu, Hu, Mzimbiri, Yang, & Chen, 2016). These UFAs are iso­
merized to trans fatty acids (TFAs) when peanut oil is subjected to the
thermal processing found in refining and cooking (Bhardwaj, Passi,
Misra, Pant, Anwar, Pandey, et al., 2016; Cui, Hao, Liu, & Meng, 2017;
Dorni, Sharma, Saikia, & Longvah, 2018). The intake of TFAs causes
more than 500,000 people worldwide to die from coronary heart disease
each year, increases the risk of heart disease by 21% and mortality by
28% (WHO, 2018), which evokes an international health alarm. To
combat this problem, the WHO issued a “replacement” action guidance
program on May 14th, 2018, planning to completely eliminate the trans
fats used in the global food supply chain by 2023. Thus, all countries in
the world are actively exploring new technologies and methods to
The TFAs in peanut oil are mainly composed of trans oleic acid and
trans linoleic acids, which are isomerized from C18:1-9c and C18:2-
9c,12c, respectively (Xie, Jiang, Li, Guo, Cheng, Qian, et al., 2019). The
amount of TFAs in peanut oil increases with both heating temperature
and time. Previous studies showed that the C– – C double bonds in UFAs
of edible oils isomerized to TFAs by both a free radical isomerization
mechanism (Chatgilialoglu, Ferreri, Guerra, Samadi, & Bowry, 2017;
Chatgilialoglu, Ferreri, Melchiorre, Sansone, & Torreggiani, 2014) and
proton transfer isomerization mechanism (Li, Yuan, Li, Wang, & Ha,
2013; Li, Zhang, Li, Wang, Xu, Deng, et al., 2013). Regardless of these
two proposed mechanism, the isomerization processes of C18:1-9c oc­
curs via one path (C18:1-9c → C18:1–9 t), while that of C18:2-9c,12c
occurs via two paths (C18:2-9c,12c → C18:2-9c,12 t → C18:2–9 t,12 t and
C18:2-9c,12c → C18:2–9 t,12c → C18:2–9 t,12 t). C18:1-9c and C18:2-
9c,12c require approximately 294.5 kJ/mol and 284.2–286.4 kJ/mol in
energy, respectively, to isomerize to TFAs (Li, Yuan, Li, Wang, & Ha,
2013; Li et al., 2013). Previous work from our group confirmed that the
UFAs found in edible oils were isomerized first to mono-trans isomers.
After this initial isomerization, they formed double- and multi-trans
isomers (Guo, Jiang, Deng, Li, Jin, Ha, et al., 2017; Guo, Jiang, Jin, Li,

* Corresponding author.
E-mail address: wangqiang06@caas.cn (Q. Wang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.foodchem.2020.128637
Received 31 January 2020; Received in revised form 13 October 2020; Accepted 11 November 2020
Available online 16 November 2020
0308-8146/© 2020 Elsevier Ltd. All rights reserved.
Q. Guo et al.
Wang, Wang, et al., 2017). Despite this breakthrough in understanding
TFAs formation, thus far, there has been no clear evidence to suggest
what approach to inhibit TFAs formation might be more effective.
At present, the most direct and effective method to reduce TFAs is to
decrease the thermal processing temperature and time. These ap­
proaches have been favored since they have lower costs when compared
to improving processing equipment, using precious metal catalysts, and/
or adding antioxidants. However, if the actual heating temperature is
too low or the heating time is not long enough, the texture, flavor, color,
and/or taste of the oil and fat will be altered. The addition of nutritional
antioxidants to vegetable oils may be another simple, alternative
method to reduce the amount of TFAs (Guo, Ha, Li, Jin, Deng, Li, et al.,
2015; Tsuzuki, Nagata, Yunoki, Nakajima, & Nagata, 2008) and has also
attracted the attention of the oil and fat industry. Studies have found
that natural and synthetic antioxidants such as tocopherols, sesamol,
TBHQ, and BHT not only have antioxidant effects when added to oils,
but also significantly reduce TFAs formation in thermally heated oils
(Tsuzuki, 2011). Due to the potential safety problems of synthetic an­
tioxidants, the development of natural antioxidants has become an
inevitable trend in the oil and fat industry. However, the mechanism
behind their ability to reduce TFAs formation remains unclear.
Phytosterols are 3-hydroxy steroid compounds with cyclopentane
hydrofluorene as the main carbon skeleton. The melting point of phytos­
terols are above 100 ◦ C, which is relative stable to heat. Stigmasterol,
β-sitosterol, campesterol and brassicasterol are the mainly phytosterols
naturally existed in most vegetable oils (Barrera-Arellano, Badan-Ribeiro,
& Serna-Saldivar, 2019; de Figueiredo, Bonafe, Martins, Martins, Mar­
uyama, de Oliveira Santos Junior, et al., 2018). Phytosterols are demon­
strated to have various bioactive properties including reducing intestinal
cholesterol absorption, cancer risk and cardiovascular problems (Shahzad,
Khan, Md, Ali, Saluja, Sharma, et al., 2017). Recently, phytosterols have
become highly valued by both academic and business circles of oils and
fats, and their function in vegetable oils, such as nutritional health and
antioxidant effects has been continuously explored (Moreau, Nyström,
Whitaker, Winkler-Moser, Baer, Gebauer, et al., 2018). Whether there are
other new functions of phytosterols with different structure remains to be
clarified. We hypothesized that phytosterols with different structure could
inhibit the formation of TFAs in vegetable oils, and clarified the action
relationships of plantsterols on inhibiting the formation of TFAs, then their
new anti-isomerization effect could be proposed, which could not only
expand their applications in the fields of food, medicine or biology, but also
provided a theoretical basis for developing new anti-isomers of oils and fats
to precisely reduce TFAs content.
Density functional theory (DFT) is a popular computational tool for
studying the properties of molecules and predicting their chemical re­
actions (Tzeng & Hu, 2014; Yao, Cao, Liu, Zhou, Li, & Wang, 2019).
Thus, the aim of this work was to study the effects of six phytosterols on
thermally induced TFAs in peanut oil and three simulated oils (triolein,
trilinolein and trilinolenin) combined by GC-FID and DFT. The TFAs in
peanut oil and three simulated oils were analyzed by GC-FID. DFT was
used to calculate the atomic net charge distribution, frontier molecular
orbital energy (FMOE), and bond dissociation energy (BDE) of phytos­
terols, and confirmed the reactive active sites of phytosterols reacted
with the UFAs. The possible inhibition pathways of phytosterols on TFAs
in peanut oil were delineated.
2. Materials and methods
2.1. Materials
Peanut oil was prepared in our lab using low-temperature pressing
with good quality (color: L*=90.6, a*=-6.76, b*=76.21; moisture and
volatiles: 0.05%; acid value: 0.68 mg/g; peroxide value: 0.23 mmol/kg).
Pure triolein (99%), trilinolein (99%), trilinolenin standards (99%) and
mixed standards GLC-674 as well as an internal standard (undecanoic
acid triglyceride) were all purchased from NU-CHEK Prep, Inc. (Elysian,
2
Food Chemistry 344 (2021) 128637
MN, USA). Stigmasterol (≥95%), β-sitosterol (≥98%), campesterol
(≥98%), brassicasterol (≥97%), and fucosterol (≥98%) were purchased
from Shanghai yuanye Bio-Technology Co.,Ltd (Beijing, China), delta7-
avenasterol (≥98%) was purchased from EMMX Biotechnology LLC
(Lake Forest, CA, USA). C18:1 isomer (C18:1–9 t), C18:2 isomers (C18:2-
9c,12c, C18:2–9 t,12c, C18:2-9c,12 t, C18:2–9 t,12 t), C18:3 isomers
(C18:3-9c,12c,15c, C18:3–9 t,12c,15c, C18:3-9c,12 t,15c, C18:3-
9c,12c,15 t, C18:3–9 t,12 t,15c, C18:3–9 t,12c,15 t, C18:3-9c,12 t,15 t,
and C18:3–9 t,12 t,15 t) and conjugated linoleic methyl ester mixture
(No. O5632) were all purchased from Sigma-Aldrich (St. Louis, MO,
USA). Isooctane was obtained from Fisher Scientific (Fair Lawn, NJ,
USA) and was used as a chromatographic organic solvent.
2.2. Thermal processing procedures
Natural peanut oil phytosterols were removed through molecular
distillation (Winkler & Warner, 2008). Briefly, 1.0 g of peanut oil
(without any natural phytosterols), triolein, trilinolein, and trilinolenin
with 1% of a selected phytosterol (stigmasterol, β-sitosterol, campes­
terol, brassicasterol, fucosterol, or delta7-avenasterol) were transferred
into a 3 mL micro-glass ampoule. The ampoules without air (N2, 5 mL/
min, 2 min) were melted and sealed by a propane-oxygen flame, and
then heated at 180 ◦ C for 12 h and 24 h in a silicone oil (viscosity 500
cSt) bath. Samples without any phytosterol were used as controls.
2.3. Trans fatty acid analysis using GC-FID
Oil samples were methylated using a methanolic KOH solution as
previously described by Zhang et al. (2015), and analyzed by established
methods in our lab using a GC-2010 chromatograph (Shimadzu, Kyoto,
Japan) equipped with an CP-SIL 88 column (100 m × 0.25 μm × 0.2 mm;
Supelco, Bellefonte, PA, USA) and a flame ionization detector (FID) (Guo
et al., 2015). The initial temperature of 60 ◦ C was maintained for 5 min
and then increased to 160 ◦ C at a rate of 25 ◦ C/min. After a time of 5 min
at 160 ◦ C, the temperature was again raised at a rate of 2 ◦ C/min to
achieve a final temperature of 225 ◦ C. The sample was maintained at this
final temperature for 15 min. The injection volume was 1 μL with a split
ratio of 1:10, and helium (99.999%) was used as the carrier gas with a
flow rate of 6.3 mL/min. The injector and interface temperatures were
both 230 ◦ C. The identities of TFAs contained in the samples were
determined by comparing retention times to those of known standards.
2.4. Quantum chemical calculations
Calculations were performed using Gaussian 09 software supplied by
High Performance Computing Cluster of Nanchang University Advanced
Research Institute (Inspur Tiansuo TS10000). Geometry optimization of
phytosterol molecules was conducted using the DFT method with the
hybrid exchange functional of Becke’s 3 parameters (B3) and the Lee-
Yang-Parr’s nonlocal correlation functional (LYP) for finding the stable
optimization geometries in association with the 6–311++G* basis
(Fig. 1). Atomic charge distribution, frontier molecular orbital energy,
and bond dissociation energy were all calculated at B3LYP/6–311++G*.
2.5. Statistical analyses
All experiments were performed in triplicate following a completely
randomized design. Sigmaplot 12.0 software (Systat Software Inc., San
Jose, CA, USA) was used for data plotting. One-way analysis of variance
(ANOVA) was applied to all samples to verify any significant differences
at a significance level of 0.05 using SPSS (Version 16.0, SPSS Inc., Chi­
cago, IL, USA).
Q. Guo et al.
Fig. 1. Molecular structures of the six selected phytosterols.
3. Results and discussion
3.1. Effects of phytosterols on the TFAs in heated peanut oil
To elucidate the effects of phytosterols on the formation of TFAs,
peanut oil with six varieties of phytosterols were heated at 180 ◦ C for 12
and 24 h. The stigmasterol, β-sitosterol, campesterol and brassicasterol are
the four representative phytosterols commonly found in vegetable oils,
fucosterol and delta7-avenasterol were selected from the consideration of
the molecular structure of phytosterols (Fig. 1). The linoleic acid in peanut
oil isomerized to form trans linoleic acids during heating, which is
consistent with previous study (Bhardwaj et al., 2016). Plantsterols with
different structures could inhibit the formation of trans linoleic acids in
peanut oil during heating (Supplemental Table 1 and Supplemental Fig. 1).
As shown in Fig. 2, equal quantities of C18:2-9c,12 t and C18:2–9 t,12c
were observed in unheated peanut oil, and their amounts increased with
increased heating time. However, neither trans oleic acid nor trans linolenic
acid were detected in the heated peanut oil. It maybe that peanut oil had
the lowest linolenic acid content, and oleic acid was relatively more stable
than either linoleic acid or linolenic acid (Tsuzuki, Nagata, Yunoki,
Nakajima, & Nagata, 2008). We noted that the amounts of C18:2–9 t,12c
and C18:2-9c,12 t were not influenced by any of the six phytosterols in the
unheated peanut oil (Fig. 2A and 2B). After heating at 180 ◦ C for 12 h,
phytosterols significantly reduced the amounts of both C18:2–9 t,12c and
C18:2-9c,12 t, with the exception of campesterol. Notably, delta7-
avenasterol had the best inhibitory effect on these two trans isomers
(22.33% and 25.19%, respectively), followed by stigmasterol (10.05% and
10.97%), brassicasterol (10.03% and 9.57%), and fucosterol (10.05% and
10.84%). β-sitosterol and campesterol had related lower inhibition rates
(4.07 and 1.29) at 180 ◦ C for 12 h. The results indicated that these phy­
tosterols had similar effects on the formation of C18:2–9 t,12c and C18:2-
9c,12 t. The inhibition rates of each phytosterol on C18:2–9 t,12c and
C18:2-9c,12 t increased significantly at 24 h than that of 12 h. Among the
six phytosterols, the inhibition rates of delta 7-avenasterol could reach
3
Food Chemistry 344 (2021) 128637
39.62% and 42.31% for these two isomers, respectively. The similar
inhibitory effects of the six tested phytosterols on the total TFAs were
shown in Fig. 2C. These findings indicated that the tested phytosterols
exhibit the better anti-isomerization effects. Inhibition rates increased with
increased heating time in the following order: delta7-avenasterol > sig­
masterol, brassicasterol, and fucosterol > β-sitosterol > campesterol.
3.2. Effects of phytosterols on the TFAs in heated triolein, trilinolein, and
trilinolenin
To further demonstrate the effects of the six tested phytosterols on
the formation of TFAs in peanut oil, we next used triolein, trilinolein,
and trilinolenin to imitate the peanut oil heating process (Fig. 3). Only
C18:1–9 t was observed in heated triolein, and the amount of C18:1–9 t
increased with heating time (Fig. 3A), which was similar to the results
observed in peanut and other edible oils (Li et al., 2013). Unexpectedly,
no C18:1–9 t was formed in triolein with phytosterols heated at 180 ◦ C
for 12 h and 24 h, except for campesterol. The anti-isomerization effect
of campesterol increased with increased heating time.
During heating, trilinolein was isomerized to equal parts C18:2-
9c,12 t and C18:2–9 t,12c, and the amount of both increased with
increased heating time. These results were similar to those observed in
heated peanut oil (Fig. 3B and 3C). However, the amount of both in
heated trilinolein was slightly higher than that observed in peanut oil.
This may have been due to trace elements or polyphenols that existed in
the peanut oil, which would have reduced the formation of trans isomers
(Guo et al., 2015). The six tested phytosterols were able to inhibit the
formation of both C18:2–9 t,12c and C18:2-9c,12 t. Moreover, the anti-
isomerization effects of all six of them in heated trilinolein was consis­
tent with that observed in peanut oil. The anti-isomerization rates of
stigmasterol (40.0% and 37.93%), β-sitosterol (33.33% and 27.59%),
campesterol (23.0% and 22.61%), brassicasterol (40.0% and 37.93%),
fucosterol (40.67% and 37.93%), and delta7-avenasterol (46.67% and
44.86%) for C18:2–9 t,12c and C18:2-9c,12 t at 24 h were significantly
Q. Guo et al.
Fig. 2. Effects of the six selected phytosterols on the formation of trans fatty
acid isomers (TFAs) in heated peanut oil.
higher than that observed at 12 h. Delta7-avenasterol exhibited the
highest anti-isomerization effect when compared to the other five phy­
tosterols. The anti-isomerization effects of the six phytosterols on total
trans linoleic acids (Fig. 3D) were similar to those observed for C18:2-
9c,12 t and C18:2–9 t,12c (Fig. 3B and Fig. 3C).
Although no trans linolenic acid was formed when peanut oil was
heated to 180 ◦ C for 12–24 h, trilinolenin was also heated at the same
temperature and for the same time to further clarify the anti-
isomerization effects of phytosterols. C18:3–9 t,12c,15c, C18:3-
4
Food Chemistry 344 (2021) 128637
9c,12c,15 t, and C18:3–9 t,12c,15 t were all formed in heated trilinole­
nin. The amount of C18:3-9c,12c,15 t was higher than that of C18:3–9
t,12c,15c (Fig. 3E and 3F), while the C18:3–9 t,12c,15 t content was the
lowest (Fig. 3G). These findings were similar to those observed in pre­
vious studies (Guo, Jiang, Jin, et al., 2017). When heated at 180 ◦ C for
12 h, the formation of C18:3–9 t,12c,15c and C18:3-9c,12c,15 t were
reduced by the six phytosterols. The anti-isomerization rate of stig­
masterol, β-sitosterol, campesterol, brassicasterol, fucosterol, and
delta7-avenasterol for C18:3–9 t,12c,15c at 12 h was 13.64%, 10.61%,
7.21%, 12.12%, 13.64% and 35.13%, respectively, which were reduced
to 13.58%, 10.43%, 4.35%, 12.17%, 13.91% and 20.68% at 24 h,
respectively. The anti-isomerization rate changes of six plantsterols for
C18:3-9c,12c,15 t at 12 h and 24 h were similar to that of C18:3–9
t,12c,15c. The reason for the decreased anti-isomerization rate of six
phytosterols at 24 h may be that the content of total trans linolenic acids
produced at 24 h was about twice than that of 12 h, and linolenic acid
contains three double bonds, which required to consume the more
phytosterols for anti-isomerization. Compared with oleic acid (one
double bond) and linoleic acid (two double bond), the amount of added
phytosterols to linolenic acid is too little to meet the effective dose of
anti-isomerization, so the inhibition rate is reduced compared to 12 h.
The anti-isomerization effects of the six phytosterols on total trans
linolenic acid were similar to those of C18:3–9 t,12c,15c, C18:3-
9c,12c,15 t, and C18:3–9 t,12c,15 t (Fig. 3H).
Collectively, the above results indicated that the anti-isomerization
effects of each phytosterol in triolein and trilinolein were similar to
those observed in peanut oil, further confirming that phytosterols have
anti-isomerization effects in edible oils, and these effects were not equal
and followed the order of: delta7-avenasterol > sigmasterol, brassicas­
terol and fucosterol > β-sitosterol > campesterol.
3.3. Atomic net charge distribution of phytosterols
According to molecular structure theory, the larger the difference in
charge between atoms, the more easily electrons transition, dissociate
their inter-atomic bonds, and allow a chemical reaction to occur. On the
contrary, the chemical reaction may not easily occur. In quantum
chemistry, the site of the larger difference in charge is the molecular
active site (Mitra, Saha, & Roy, 2009). In this study, the atomic net
charge distribution of six plantsterols was calculated by the accurate
quantum chemical calculation NBO layout method. The sites with larger
net charge differences of the six plantsterol molecules were concentrated
on the hydroxyl site according the data showed in Supplemental Fig. 2.
Given this, we speculated that the hydroxyl site of the plantsterols were
the active sites, and that these were the sites that exerted their anti-
isomerization effects. The activity order of the six plantsterol hydroxyl
sites was as follows: Delta7-avenasterol (1.18477 eV) > stigmasterol
(1.18432 eV) = brassicasterol (1.18432 eV) = fucoid (1.18432 eV) >
β-sitosterol (1.18424 eV) > campesterol (1.14733 eV).
3.4. Frontier molecular orbital energies of phytosterols
Quantum chemical molecular orbital theory holds that the highest
occupied orbital (HOMO) and the lowest empty orbital (LUMO) distri­
bution visually show the main active site of antioxidants (Agnihotri &
Mishra, 2009). As shown in supplemental Fig. 3, the electronic cloud
mainly distributed on the active hydroxyl site, indicating that the hy­
droxyl site was an active site for anti-isomerization. The HOMO and
LUMO characterize the ability of a group to supply electrons and accept
electrons, respectively. For the HOMO, the electronic cloud density of
the hydroxyl site in delta7-avenasterol was the highest, followed by
stigmasterol, fucosterol, and brassicasterol. The electronic cloud den­
sities of the hydroxyl sites in β-sitosterol and campesterol were lower
than the other plantsterols. Taken together, these results indicated that
the oxygen atom of delta7-avenasterol was the easiest to extract elec­
trons from the hydrogen atom, such that the hydrogen atom lost
Q. Guo et al.
Fig. 3. Effects of the six selected phytosterols on the formation of trans fatty acid isomers (TFAs) in heated triolein, trilinolein, and trilinolenin.
electrons and the dehydrogenation reaction occurred.
According to molecular orbital theory, the frontier molecular orbital
energy level difference △E (EHOMO-ELUMO) is an important metric for
characterizing molecular activity parameters. Among them, EHOMO is
the highest occupied orbital energy, which characterizes the ability of
molecules to push electrons. The larger the EHOMO value, the stronger
the ability to push electrons. ELUMO is the lowest empty orbital energy,
5
Food Chemistry 344 (2021) 128637
which characterizes the ability of molecules to absorb electrons. The
smaller the ΔE value, the easier the electron transition in the molecule,
resulting in greater chemical reactivity. As shown in Table 1, the △E of
delta7-avenasterol was the smallest, indicating that delta7-avenasterol
had the highest activity. The △E of stigmasterol and fucosterol were
equal, which were both slightly higher than that of brassicasterol. The
△E of β-sitosterol was lower than that of campesterol, but the △E of
Q. Guo et al. Food Chemistry 344 (2021) 128637

Table 1 bond position in the main skeleton of cyclopentane tetrahydrophenan­

The partial properties, △E and hydroxyl BDE of phytosterols. threne. Moreover, that its effect on both △E and hydroxyl BDE was
Species molecular Double C17 △E Hydroxyl greater than the side-chain double bond. The effects of the C7 = C8
formula bond branch (kcal/ BDE double bond of plantsterols on △E and hydroxyl BDE was greater than
number carbon mol) (kcal/ that of either C5 = C6 or C2 = C3.
(double number mol)
Taken together, these results indicate that the position of the double
bond
position) bond in the main skeleton of cyclopentane tetrahydrophenanthrene as
well as the double bond number in C17 branch chain play key roles in
Stigmasterol C29H48O 2(C5 = 10 151.1670 66.2669
C6, C22 =
the plantsterol molecular anti-isomeric activity, especially for the
C23) former.
β-sitosterol C29H50O 1(C5 = 10 153.9846 65.9588
C6) 3.6. Relationship between the calculated parameters and anti-
Campesterol C28H48O 1(C2 = 9 159.9685 67.8476
isomerization rates of phytosterols
C3)
Brassicasterol C28H46O 2(C5 = 9 151.2612 66.3177
C6, C22 = Oleic acid is relatively stable in peanut oil, no C18:1-9c was detected
C23) under the experiment conditions. Although triolein was isomerized to
Fucosterol C29H48O 2(C5 = 10 151.1670 66.2669
form C18:1-9c under the same heating conditions. However, C18:1-9c
C6, C24 =
C25)
had not been detected in the presence of phytosterols (except campes­
delta7- C29H48O 2(C7 = 10 108.0948 63.1287 terol). Only data on the anti-isomerization rate of campesterol was not
avenasterol C8, C24 = representative for the correlation analysis. Thus, the correlations be­
C25) tween the calculated parameters and the anti-isomerization rates of
Note: △E, frontier molecular orbital energy difference; BDE, bond dissociation plantsterols in peanut oil and trilinolein were analyzed to further show
energy. their relationship (Fig. 4). Hydroxyl site charge difference in plantsterol
molecules and their anti-isomerization rates in peanut oil were 0.33,
these two plantsterols were higher than those of the other plantsterols. 0.30, and 0.31 for C18:3–9 t,12c, C18:3-9c,12 t, and total trans linoleic
The activity order of the six plantsterols was consistent with the results acid at 12 h, respectively. These increased to 0.47–0.66 at 24 h. The
of the atomic net charge distribution and frontier molecular orbital correlation coefficients for trilinolein were 0.33, 0.32, and 0.33 for
distribution analyses. C18:3–9 t,12c, C18:3-9c,12 t, and total trans linoleic acid at 12 h,
respectively, and slightly increased at 24 h. Lower correlation co­
efficients indicated that the hydroxyl site charge difference might not be
3.5. Bond dissociation energies of phytosterols a critical calculated parameter in anti-isomerization reactions. The
correlation coefficients of △E in plantsterol molecules and its anti-
According to thermodynamic theory, bond dissociation energy (BDE) isomerization rate in peanut oil ranged from 0.56 to 0.89 for C18:3–9
refers either to the energy that needs to be absorbed by chemical bond t,12c, C18:3-9c,12 t, and total trans linoleic acid at both 12 h and 24 h of
cleavage or the energy that needs to be released by chemical bond syn­ heating, while the correlation coefficients of hydroxyl BDE ranged from
thesis. Therefore, the smaller the BDE, the more easily the chemical bond is 0.63 to 0.85 under the same conditions. The correlation coefficients of
either broken or formed, and the more likely a chemical reaction will △E and hydroxyl BDE in trilinolein had certain differences when
occur. The hydroxyl BDE of delta7-avenasterol is 63.1287 kcal/mol compared with those of peanut oil. The relative higher correlation co­
(Table 1), which is significantly lower than that of the other five plant­ efficients of △E and hydroxyl BDE and their anti-isomerization rates
sterols. This result indicated that delta7-avenasterol was more prone to suggest that △E and hydroxyl BDE may be the key calculated param­
chemical reaction. The hydroxyl BDEs of stigmasterol and fucosterol were eters regarding phytosterol molecules in anti-isomerization reactions.
equal, which were both slightly lower than brassicasterol. The hydroxyl At present, the isomerization of UFAs via free radical isomerization
BDE of campesterol was higher than that of β-sitosterol, which were both or proton transfer isomerization were reported (Chatgilialoglu, Ferreri,
lower than the other four plantsterols. The hydroxyl BDE changes among Melchiorre, Sansone, & Torreggiani, 2014; Li, Yuan, Li, Wang, & Ha,
the six plantsterols were consistent with the results of the atomic net charge 2013). Free radical isomerization includes reversible radical addition or
distribution and △E analyses. Thus, the reaction activity order of the six hydrogen abstraction. Trans linoleic acids are the main trans isomers
plantsterols was further confirmed to be as follows: delta7-avenasterol > produced in peanut oil during heating. It was confirmed that the linoleic
stigmasterol = fucoido ≈ brassicasterol > β-sitosterol > campesterol. acid in peanut oil first isomerized to C18:3–9 t,12c and C18:3-9c,12 t,
The structures of the six plantsterols have certain effects on both the and then stepwise to C18:3–9 t,12 t. Radical catalysts (NO2•, RS•, and
△E and hydroxyl BDE (Table 1). When compared with both stigmasterol ROO•) accelerate the isomerization reaction via reversible radical
and fucosterol, the molecular weight and double bond numbers were the addition. To this end, Tzeng and Hu (2014) showed that the cis–trans
same. The difference of double bond position in the C17 branch chain isomerization of linoleic acid was more prone to reverse addition re­
had no influence on either the △E or hydroxyl BDE of these two mol­ actions than that of hydrogen abstraction, and that β-carotene and
ecules. In the stigmasterol and brassicasterol molecules, the double bond lycopene protected linoleic acid isomerization by intercepting the
numbers and positions were the same. The carbon number in the C17 isomerization-causing radicals. Chatgilialoglu et al. (2017) reported that
branch chain had no effect on either their △E or hydroxyl BDE. When the thiyl radical induced isomerization rate of linoleic acid for the C9
the main skeleton of cyclopentane tetrahydrophenanthrene was the and C13 positions was twice that at the C10 and C12 positions in
same, the double bonds on the C17 branch chain increased both the △E addition reactions.
and hydroxyl BDE of the molecule. The double bond number of the In this study, the calculated BDE values in linoleic acid molecular for
molecule and the difference of double bond position in the main skel­ C8-H8, C11-H11 and C14-H14 were 83.2, 68.1 and 83.2 kcal/mol,
eton of cyclopentane tetrahydrophenanthrene had significant effects on suggesting hydrogen dissociation was more easily at C11-H11 than that
the △E and hydroxyl BDE when compared with β-sitosterol, campes­ of C8-H8 and C14-H14. The hydroxyl BDE of six phytosterols ranged
terol, and brassicasterol. The greater the number of double bonds in the from 63.13 to 67.85 kcal/mol (Table 1), which were lower than C8-H8,
molecule, the smaller the △E and hydroxyl BDE. The structure of C11-H11 and C14-H14 of linoleic acid. It was indicated that the hy­
fucosterol and delta7-avenasterol further confirmed that the △E and droxyl hydrogen of phytosterols dissociated first to form hydroxyl rad­
hydroxyl BDE were significantly affected by the difference of double icals in the reaction system, and then can quickly combine with C11 free

6
Q. Guo et al. Food Chemistry 344 (2021) 128637

Fig. 4. Relationship between the calculated parameters and anti-isomerization rate of phytosterols (PO: peanut oil, L: trilinolein; HNC: hydroxyl site net charge
difference of phytosterols, △E: the frontier molecular orbital energy level difference of phytosterols; BDE: bond dissociation energy of phytosterols; the asterisk
represents significance at the 0.05 level).

Fig. 5. Proposed anti-isomerization mechanisms of phytosterol on trans fatty acid isomers (TFAs) in heated peanut oil.

7
Q. Guo et al.
radicals compared with C8 and C14. The BDE values for C9-H9, C10-
H10, C12-H12 and C13-H13 were calculated as 104.2, 104.8, 104.8 and
104.2 kcal/mol, indicating hydrogen transfer occur more easily at C9 →
C10, C13 → C12. If the heat was enough, the hydroxyl hydrogen of
phytosterols dissociated first to form hydroxyl radicals in the reaction
system, and then can add at C9 and C13, which had relative lower steric
hindrance compared with C10 and C12.
Given this, we proposed different anti-isomerization mechanisms for
the effects of phytosterols on linoleic acid (Fig. 5). For reversible radical
addition, the hydrogen of hydroxyl site in phytosterols dissociated, and
phytosterol free radical added at either the C9 or C13 of the C– – C bond to
generate a relatively stable free radical adduct. This adduct had difficulty
undergoing C–C bond rotation owing to the steric hindrance of phytos­
terols. This, in turn, resulted in lower C18:3–9 t,12c, C18:3-9c,12 t, and
C18:3–9 t,12 t. In the hydrogen abstraction mechanism, an bis-allylic
hydrogen was abstracted by phytosterol free radicals; other phytosterol
free radicals added at the C11 position to form a stable adduct, preventing
or slowing the formation of trans linoleic acid isomers. Our previous studies
found that the main, rate-limiting step in the proton transfer isomerization
of linoleic acid was the formation of single-trans linoleic acid transition
state 1 (Li, Yuan, Li, Wang, & Ha, 2013). The mechanism by which phy­
tosterols inhibited proton transfer isomerism may be the combination of
phytosterol free radicals with C11, which increased the difficulty of proton
transfer owing to steric hindrance. A distinguishing feature of these three
mechanisms was that reversible radical addition may result only in
geometrical isomers; comparatively, hydrogen abstraction and proton
transfer reactions can lead to both geometrical and conjugated isomers.
4. Conclusions
The six phytosterols exhibited anti-isomerization effects on oleic
acid, linoleic acids, and linolenic acids. The anti-isomerization rates of
six phytosterols increased with heating time in peanut oil. The anti-
isomerization effects of phytosterols was mainly associated with the
activities of hydroxyl sites. The hydroxyl site reaction activities of
phytosterols were significantly affected by the double bond position in
the main skeleton of cyclopentane tetrahydrophenanthrene as well as by
the number of double bonds on the C17 branch chain. Increasing double
bonds in phytosterol molecules resulted in higher anti-isomerization
activity. Finally, we proposed inhibition mechanisms of phytosterols
on TFAs in heated peanut oil.
CRediT authorship contribution statement
Qin Guo: Writing - original draft. Tian Li: Methodology, Supervi­
sion. Yang Qu: Writing - review & editing. Xinping Wang: Software,
Validation. Li Liu: Writing - review & editing. Hongzhi Liu: Writing -
review & editing. Qiang Wang: Supervision, Resources.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This study was supported by a research grant from the Natural Sci­
ence Foundation of China (31772097), and the Special National Key
Research and Development Plan (2017YFC1600600).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
Food Chemistry 344 (2021) 128637
org/10.1016/j.foodchem.2020.128637.
References
Agnihotri, N., & Mishra, P. C. (2009). Mechanism of scavenging Action of N-
acetylcysteine for the OH radical: A quantum computational study. The Journal of
Physical Chemistry B, 113(35), 12096–12104.
Barrera-Arellano, D., Badan-Ribeiro, A. P., & Serna-Saldivar, S. O. (2019). Chapter 21 -
Corn Oil: Composition, Processing, and Utilization. In S. O. Serna-Saldivar (Ed.),
Corn (Third Edition, pp. 593–613). Oxford: AACC International Press.
Bhardwaj, S., Passi, S. J., Misra, A., Pant, K. K., Anwar, K., Pandey, R. M., & Kardam, V.
(2016). Effect of heating/reheating of fats/oils, as used by Asian Indians, on trans
fatty acid formation. Food Chemistry, 212, 663–670.
Chatgilialoglu, C., Ferreri, C., Guerra, M., Samadi, A., & Bowry, V. W. (2017). The
reaction of thiyl radical with methyl linoleate: Completing the picture. Journal of the
American Chemical Society, 139(13), 4704–4714.
Chatgilialoglu, C., Ferreri, C., Melchiorre, M., Sansone, A., & Torreggiani, A. (2014).
Lipid geometrical isomerism: From chemistry to biology and diagnostics. Chemical
Reviews, 114(1), 255–284.
Cui, Y., Hao, P., Liu, B., & Meng, X. (2017). Effect of traditional Chinese cooking methods
on fatty acid profiles of vegetable oils. Food Chemistry, 233, 77–84.
de Figueiredo, L. C., Bonafe, E. G., Martins, J. G., Martins, A. F., Maruyama, S. A., de
Oliveira Santos Junior, O., Biondo, P. B. F., Matsushita, M., & Visentainer, J. V.
(2018). Development of an ultrasound assisted method for determination of
phytosterols in vegetable oil. Food Chemistry, 240, 441–447.
Dorni, C., Sharma, P., Saikia, G., & Longvah, T. (2018). Fatty acid profile of edible oils
and fats consumed in India. Food Chemistry, 238, 9–15.
Dun, Q., Yao, L., Deng, Z., Li, H., Li, J., Fan, Y., & Zhang, B. (2019). Effects of hot and
cold-pressed processes on volatile compounds of peanut oil and corresponding
analysis of characteristic flavor components. Lwt-Food Science and Technology, 112,
Article 107648. https://doi.org/10.1016/j.lwt.2018.11.084.
Guo, Q., Ha, Y., Li, Q., Jin, J., Deng, Z., Li, Y., & Zhang, S. (2015). Impact of additives on
thermally-induced trans isomers in 9c,12c linoleic acid triacylglycerol. Food
Chemistry, 174, 299–305.
Guo, Q., Jiang, F., Deng, Z., Li, Q., Jin, J., Ha, Y., & Wang, F. (2017). Reaction pathway
mechanism of thermally induced isomerization of 9,12-linoleic acid triacylglycerol.
Journal of the Science of Food and Agriculture, 97(6), 1861–1867.
Guo, Q., Jiang, F., Jin, J., Li, Q., Wang, F., Wang, Q., & Ha, Y. (2017). Highly sensitive
method for the quantification of trans-linolenic acid isomers in trilinolenin of edible
oils using an ionic liquid capillary column. Journal of the Science of Food and
Agriculture, 97(14), 4697–4703.
Li, A., Yuan, B., Li, W., Wang, F., & Ha, Y. (2013). Thermally induced isomerization of
linoleic acid in soybean oil. Chemistry and Physics of Lipids, 166, 55–60.
Li, C., Zhang, Y., Li, S., Wang, G., Xu, C., Deng, Y., & Wang, S. (2013). Mechanism of
formation of trans fatty acids under heating conditions in triolein. Journal of
Agricultural and Food Chemistry, 61(43), 10392–10397.
Mitra, I., Saha, A., & Roy, K. (2009). Quantitative structure-activity relationship
modeling of antioxidant activities of hydroxybenzalacetones using quantum
chemical, physicochemical and spatial descriptors. Chemical Biology & Drug Design,
73(5), 526–536.
Moreau, R. A., Nyström, L., Whitaker, B. D., Winkler-Moser, J. K., Baer, D. J.,
Gebauer, S. K., & Hicks, K. B. (2018). Phytosterols and their derivatives: Structural
diversity, distribution, metabolism, analysis, and health-promoting uses. Progress in
Lipid Research, 70, 35–61.
Shahzad, N., Khan, W., Md, S., Ali, A., Saluja, S. S., Sharma, S., … Al-Ghamdi, S. S.
(2017). Phytosterols as a natural anticancer agent: Current status and future
perspective. Biomedicine & Pharmacotherapy, 88, 786–794.
Tsuzuki, W. (2011). Effects of antioxidants on heat-induced trans fatty acid formation in
triolein and trilinolein. Food Chemistry, 129(1), 104–109.
Tsuzuki, W., Nagata, R., Yunoki, R., Nakajima, M., & Nagata, T. (2008). cis/trans-
isomerisation of triolein, trilinolein and trilinolenin induced by heat treatment. Food
Chemistry, 108(1), 75–80.
Tzeng, Y. Z., & Hu, C. H. (2014). Radical-induced cis-trans isomerization of fatty acids: A
theoretical study. Journal of Physical Chemistry A, 118(25), 4554–4564.
Wang, Q., Liu, H., Hu, H., Mzimbiri, R., Yang, Y., & Chen, Y. (2016). Chapter 3 - Peanut
Oil Processing Technology. In Q. Wang (Ed.), Peanuts: Processing Technology and
Product Development (pp. 63–81). Academic Press.
WHO. (2018). An action package to eliminate industrially-produced trans-fatty acids.
Geneva: WHO.
Winkler, J. K., & Warner, K. (2008). The effect of phytosterol concentration on oxidative
stability and thermal polymerization of heated oils. European Journal of Lipid Science
and Technology, 110(5), 455–464.
Xie, Y., Jiang, S., Li, M., Guo, Y., Cheng, Y., Qian, H., & Yao, W. (2019). Evaluation on the
formation of lipid free radicals in the oxidation process of peanut oil. Lwt-Food
Science and Technology, 104, 24–29.
Yao, Y., Cao, R., Liu, W., Zhou, H., Li, C., & Wang, S. (2019). Molecular reaction
mechanism for the formation of 3-chloropropanediol esters in oils and fats. Journal of
Agricultural and Food Chemistry, 67(9), 2700–2708.
Zhang, M., Yang, X., Zhao, H. T., Dong, A. J., Wang, J., Liu, G. Y., … Zhang, H. (2015).
A quick method for routine analysis of C18 trans fatty acids in non-hydrogenated
edible vegetable oils by gas chromatography-mass spectrometry. Food Control, 57,
293–301.