Food Control 107 (2020) 106781

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Influence of carbohydrate- and protein-based foods on the formation of T

polar lipid fraction during deep-frying
Yih Phing Khora, Biow Ing Sima, Faridah Abasb, Oi Ming Laic, Yong Wangd,
Imededdine Arbi Nehdie, Hassen Mohamed Sbihie, Mohamed Mossad Gewike, Chin Ping Tana,f,∗
a
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia
b
Department of Food Sciences, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia
c
Department of Bioprocess Technology, Faculty of Biotechnology and Molecular Sciences, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia
d
JNU-UPM International Joint Laboratory on Plant Oil Processing and Safety (POPS), Department of Food Science and Engineering, Jinan University, Guangzhou,
510632, China
e
King Saud University, College of Science, Chemistry Department, Riyadh, Saudi Arabia
f
School of Biological Science and Food Engineering, Chuzhou University, Chuzhou, 239000, China

A R T I C LE I N FO A B S T R A C T

Keywords: The extents of the oxidation and polymerization processes were examined in refined, bleached, and deodorized
Polymerized triacylglycerol palm olein (RBDPO) to determine the impact of frying different foods on frying oil stability, particularly the
Oxidized fatty acid formation of polar lipid fraction and short chain fatty acid upon frying, and at the same time to evaluate its
Epoxy acids discarding point. Sliced potatoes (SP) and chicken breast meat (CBM) were fried for 200 min/day for seven
Frying
consecutive days using RBDPO at 180 °C without any oil replenishment. The amounts of total polar compound
Potato
Chicken breast meat
(TPC), polymerized triacylglycerols (PTG), and short-chain fatty acid (caprylic acid) that formed were sig-
nificantly (p < 0.05) higher in the RBDPO used to fry SP compared to CBM. The TPC in the RBDPO used to fry
SP exceeded the limit of rejection for human consumption (> 25% polar compounds) on the seventh day of
frying. In addition, the amounts of epoxy, keto, and hydroxy acids that formed were significantly (p < 0.05)
higher in the RBDPO used to fry CBM compared to SP. RBDPO also exceeded the safety limit when the con-
centration of epoxy acids respectively reached 7.4 g/kg and 8.8 g/kg after frying SP and CBM for seven days.

1. Introduction health if consumed in large quantities due to their detrimental effects

Frying is a commonly used and popular method to prepare food due
to the unique characteristics that are imparted to the fried foods such as
a golden color, savory flavor, and desirable texture, which are preferred
among consumers. However, since the frying process involves the
presence of oxygen and is done at high temperature, thermal and oxi-
dative reactions will occur simultaneously, which eventually degrade
the quality of the frying oil (Ben Hammouda et al., 2018). The forma-
tion of thermo-oxidative products must be closely monitored, as they
will affect the functional, sensory, and nutritional properties of the
frying oil. Moreover, in commercial frying, frying oil tends to be reused,
and a significant amount of the oxidized frying oil will be absorbed into
the fried foods and eventually ingested into the human body. These
thermo-oxidative products are generally recognized as threats to human
(Yu, Cho, & Hwang, 2018). The common chemical reactions that occur
in frying oil are oxidation, hydrolysis, and polymerization, which pro-
duce both volatile and nonvolatile compounds (Choe & Min, 2007;
Song, Kim, Kim, & Lee, 2017). Most volatile compounds will easily
evaporate together with steam, whereas the remaining nonvolatile
compounds will be trapped in oil to either undergo further chemical
reactions or become absorbed into the fried food (Choe & Min, 2007).
Monomeric oxidized triacylglycerols (oxTAGs), such as epoxy, keto,
and hydroxy fatty acids, are compounds that form from secondary lipid
oxidation. The analysis of oxTAGs has received relatively little atten-
tion, but they have been reported to be present in high amounts in
thermally oxidized foods (Mubiru, Shrestha, Papastergiadis, & De
Meulenaer, 2013). They are relatively stable compounds that result
from decomposition of primary oxidation products and have

∗
Corresponding author. Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor,
Malaysia.
E-mail addresses: sweet_appie@hotmail.com (Y.P. Khor), biowing.sim@gmail.com (B.I. Sim), faridah_abas@upm.edu.my (F. Abas),
omlai@upm.edu.my (O.M. Lai), twyong@jnu.edu.cn (Y. Wang), inahdi@ksu.edu.sa (I.A. Nehdi), hmsbihi@ksu.edu.sa (H.M. Sbihi),
mmossad@ksu.edu.sa (M.M. Gewik), tancp@upm.edu.my (C.P. Tan).

https://doi.org/10.1016/j.foodcont.2019.106781
Received 3 March 2019; Received in revised form 17 June 2019; Accepted 20 July 2019
Available online 20 July 2019
0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Y.P. Khor, et al.
demonstrated a high absorbability in both animals and humans
(Marmesat, Velasco, & Dobarganes, 2008). Among the oxTAGs, epoxy
fatty acids are of considerable concern because it is believed that all
lipid oxidation mechanisms may be the formation routes of epoxy fatty
acids, which may impose negative health effects (Liu, Cheng, et al.,
2018; Mubiru, Shrestha, Papastergiadis, & De Meulenaer, 2014).
The polar compounds (TPC) are a complex group of compounds that
represent the degree of deterioration of a frying oil. TPC can be further
classified into triacylglycerol (TAG) oligomers and TAG dimers, which
represent the polymerization reaction; monomeric oxidized triacylgly-
cerols (oxTAG) act as the indicator of the oxidative deterioration,
whereas diacylglycerols (DAG) and free fatty acids (FFA) illustrate the
hydrolytic alteration that occurs during the frying process (Farhoosh,
Einafshar, & Sharayei, 2009). Frying oils that share the same TPC might
possess a different polymerized TAG distribution (Zhu et al., 2018).
Therefore, it is vital to determine the exact distribution of the total
polar fraction to analyze the degree of polymerization in frying oils. In
addition, polymers impart a various negative effect toward the frying
oil, such as an increase in viscosity, a reduction of heat transfer, foam
production during deep-fat frying, and color deepening. The presence of
polymers will also increase the rate of oil absorption into fried foods
(Choe & Min, 2007).
Two main factors, namely, the high temperature and types of food
matrices, will lead to the formation of numerous thermo-oxidized by-
products during the frying process. Not only does the fatty acid com-
position or type of frying oil play an important role, but the constituents
of food substrates used for frying are also important. For instance,
proteins, carbohydrates, lipids, and water are worth investigating be-
cause they are directly involved in complex reactions that take place
during frying (Zhang, Qin, Li, Shen, & Saleh, 2015). To our knowledge,
studies normally evaluate the changes in PTG or oxTAG separately in
different types of frying oil during heat treatment, but studies that
evaluate the impact of different food substrates on the extents of both
oxidation and polymerization reactions together in frying oil are
lacking. Since palm olein is extensively used in frying applications, it is
important to have a fundamental understanding of the influence of food
types toward the oil stability during thermal processing. The main
objective of this study was to evaluate the effects of different foods
toward the changes in the formation of oxidation and polymerization
products in palm olein during frying.
2. Materials and methods
2.1. Materials
The refined, bleached, and deodorized palm olein (RBDPO) without
the addition of antioxidant was purchased from Moi Foods Malaysia
Sdn. Bhd. (Pulau Indah, Malaysia). Potatoes and chicken breast meat
were purchased from a local supermarket.
2.2. Chemicals
Analytical standards for the analysis of monomeric oxidized tria-
cylglycerols such as methyl heneicosanoate, methyl 12-hydro-
xystearate, and methyl 12-oxostearate were purchased from Sigma-
Aldrich (Darmstadt, Germany), while methyl trans-9,10-epoxystearate
was purchased from Santa Cruz Biotechnology, Inc. (Dallas, USA).
Platinum (IV) oxide hydrate and tetrahydrofuran (HPLC grade) were
acquired from Thermo Fisher Scientific (Waltham, USA). For the
column chromatography, silica gel 60 (0.063–0.200 mm) was pur-
chased from Merck (Darmstadt, Germany). For the analysis of fatty acid
composition, the Supelco's 37 component FAME mix and methyl oc-
tanoate were purchased from Sigma-Aldrich (Darmstadt, Germany). All
of the solvents and chemicals used for the analyses were of analytical
grade unless otherwise specified.
2
Food Control 107 (2020) 106781
2.3. Sample preparation
Fresh potatoes were peeled and sliced to a thickness of 2 mm while
chicken breast meat was cut to a dimension of 1.5 cm × 1.5 cm. The
samples were kept submerged in distilled water at room temperature.
Before frying, the samples were slightly blotted dry with tissue paper.
The samples were weighed into 100 g each batch. All samples were
freshly prepared on daily basis before the frying experiments.
2.4. Frying experiments
The frying experiments were conducted in replicates at 180 °C.
RBDPO (3.5 L) was poured into a Cornell batch fryer (Pulau Indah,
Malaysia). The temperature was raised to 180 °C during 15 min. A batch
of 100 g sliced potatoes (SP) or chicken breast meat (CBM) cubes were
fried for 2.5 min at 17.5 min intervals with a total of 3.5 h per day for
seven consecutive days. In other words, there were a total of 10 frying
cycles per day and 70 frying cycles after seven consecutive days. The
fryer was left uncovered during the frying process. For each day, at the
end of the frying experiment, the fryer was switched off and the oil was
allowed to cool down before covered with the lid. Oil samples (100 mL)
were collected each day after every 10 fryings and stored at – 20 °C for
further analyses. The frying experiments were conducted without any
oil replenishment.
2.5. Determination of epoxy-, keto-, and hydroxy fatty acids
2.5.1. Derivatization to fatty acid methyl esters (FAMEs)
The transesterification of frying oil samples was carried out at room
temperature by using sodium methoxide. Firstly, a 300-mg of frying oil
sample was weighed, followed by the addition of 3 mL of tert-butyl
methyl ether (TBME) and 1.5 mL of 0.2 M sodium methoxide in me-
thanol. The mixture was vortexed for 1 min and was allowed to stand at
room temperature for 2 min. Next, for the neutralization purpose,
0.1 mL of 0.5 M sulfuric acid was added and the mixture was vortexed
for 5 s. Then, another 3 mL of ultrapure water was added and vortexed
for 10 s. The mixture was brought for centrifugation at 4000 rpm at
5 min and only the supernatant was collected and evaporated to dryness
using nitrogen gas.
2.5.2. Solid-phase extraction
The solid-phase extraction (SPE) method was used to fractionate the
frying oil samples into two main fractions, namely, the polar and
nonpolar fractions. A silica-based SPE cartridge containing 1 g sorbent
was used in the analysis. After preconditioning the cartridge, 100 mg
FAMEs dissolved in 2 mL n-hexane-diethyl ether (98:2; v/v) were
transferred into the cartridge. The elution order started with the addi-
tion of 15 mL n-hexane-diethyl ether (98:2; v/v) to elute the nonpolar
fraction, followed by the addition of 25 mL diethyl ether to collect the
polar fraction. One milliliter of C21:0 (internal standard) with con-
centration of 500 μg/mL was pre-dissolved in TBME solution and was
added to the collected polar fraction. The mixture was vortexed and
subsequently evaporated using nitrogen gas.
2.5.3. Hydrogenation
The collected polar fraction was dissolved in 2 mL methanol. Next,
platinum (IV) oxide hydrate (metal catalyst) was added to facilitate the
hydrogenation process. Hydrogenation was performed by bubbling the
hydrogen gas into the sample at room temperature for 10 min. Then,
the remaining methanol was evaporated using nitrogen gas. The sample
was dissolved in 1.5 mL diethyl ether, filtered, and then analyzed using
a gas chromatograph equipped with a flame ionization detector (GC-
FID).
Y.P. Khor, et al.
2.5.4. Gas chromatography (GC)
An Agilent 6890 Series chromatograph (Agilent Technologies, Santa
Clara, USA) equipped with a split-splitless injector was used to analyze
the oxTAGs. The GC was operated in the split mode with a 20:1 split
ratio at 250 °C. A J&W DB-Wax fused-silica capillary column that was
60 m × 0.25 mm I.D. and film thickness of 0.25 μm (J&W Scientific,
Folsom, USA) was used for the analysis. The oven temperature was set
under an isothermal condition, which was 240 °C for 30 min. The flame
ionization detector was fixed at 250 °C, and the nitrogen as carrier gas
was set to 1 mL/min. The other gas flows were fixed as follows: hy-
drogen at 40 mL/min, air at 450 mL/min, and nitrogen (as auxiliary
gas) at 45 mL/min. The signal-to-noise ratios of each peaks were
monitored to ensure the reliability of the quantified data. The mono-
meric oxidized triacylglycerols contents were determined from the ca-
libration curve constructed by plotting the concentration of standards
versus the corresponding peak areas.
2.6. Determination of polymerized triacylglycerols
2.6.1. Silica column chromatography
Similar to the oxTAG analysis, for sample pretreatment, the frying
oil samples were fractionated into nonpolar and polar fractions first by
using the conventional silica gel-packed column. Ten grams of silica
adjusted to a water content of 5% (w/w) was filled into a 40 cm × 1 cm
I.D. glass column. The nonpolar fraction was eluted by using 75 mL of n-
hexane-diethyl ether (90:10, v/v) while the polar fraction was eluted by
using 75 mL of diethyl ether. For the collected polar fraction, the re-
sidual solvent was evaporated using a rotary evaporator under reduced
pressure. Next, the polar fraction was adjusted using tetrahydrofuran
(THF) to achieve a concentration of 50 mg/mL. The sample was filtered
and analyzed by using the high-performance size-exclusion chromato-
graph with a refractive index detector (HPLC-SEC/RI).
2.6.2. High-performance size-exclusion chromatography (HPSEC)
The polar fraction of oil was analyzed by HPSEC to determine the
polymerized triacylglycerols and their distribution. A Shimadzu high-
performance liquid chromatography (HPLC) (Kyoto, Japan) equipped
with a SIL-10AD injector, an LC-20AD pump, and a RID-10A refractive
index detector was used. To improve the peak resolution, two 100 Å
and 500 Å phenogel columns (30 cm × 0.78 cm I.D.) packed with
porous, highly cross-linked styrene-divinyl benzene copolymers (film
thickness 5 μm) (Phenomenex, Torrance, USA) were connected in
series. Tetrahydrofuran was selected as the mobile phase and the flow
rate was set to 1 mL/min. The oven temperature was set to 40 °C. Each
group of the polar fraction distributions was quantified using the fol-
lowing equations:
Poligomer = (Aoligomer / ∑A) PTPC
Pdimer = (Adimer / ∑A) PTPC
PoxTAG = (AoxTAG / ∑A) PTPC
PDAG = (ADAG / ∑A) PTPC
PFFA = (AFFA / ∑A) PTPC
where PTPC, Poligomer, Pdimer, PoxTAG, PDAG, and PFFA represent the per-
centages of total polar compound, TAG oligomers, TAG dimers,
monomeric oxidized triacylglycerols, diacylglycerols, and free fatty
acids found in the polar fraction of the oil sample respectively; Aoligomer,
Adimer, AoxTAG, ADAG, and AFFA represent the peak area of each specific
fractions; and ∑A represents the sum of all peak areas.
3
Food Control 107 (2020) 106781
2.7. Determination of oxidative stability
2.7.1. Peroxide value (PV), p-anisidine value (AV), and total oxidation
value (TOTOX)
The peroxide value (AOCS, 2013a) and the p-anisidine value (AOCS,
2013b) of the oil samples were evaluated by adopting the official
methods of the American Oil Chemists’ Society. Total oxidation value
(TOTOX) was calculated using the following formula:
TOTOX = 2PV + AV
where TOTOX represents the total oxidation value; PV represents the
peroxide value; and AV represents the p-anisidine value.
2.8. Determination of fatty acid composition
2.8.1. Derivatization
A base-catalyzed method was used for derivatization to FAMEs. The
derivatization was conducted at room temperature to avoid losses of
volatile FAMEs. Frying oil samples were each weighed to 100 mg and
were dissolved using 2 mL hexane that contained 1 mg/mL methyl tri-
decanoate (C13:0). Then, 0.1 mL 2 M potassium hydroxide in methanol
was added. The mixture was vortexed for 15 s, followed by cen-
trifugation at 4000 rpm for 5 min. Only the supernatant was collected;
it was then filtered and subsequently analyzed using a gas chromato-
graph with flame ionization detector (GC-FID).
2.8.2. Gas chromatography
The Agilent 6890 Series chromatograph (Agilent Technologies,
Santa Clara, USA) equipped with a split-splitless injector was used for
the FAME analysis. The injector was set to the split mode with a 20:1
split ratio at 250 °C, and an SGE BPX70 column with an internal dia-
meter of 25 m × 0.32 mm and film thickness 0.25 μm (Trajan Scientific,
Victoria, Australia) was chosen. The flame ionization detector was used
at 280 °C with a hydrogen flow rate of 40 mL/min, an air flow rate of
450 mL/min and a nitrogen flow rate, as an auxiliary gas, of 25 mL/
min. The analyses were run using nitrogen as a carrier gas at 10 psi. The
oven temperature ramp started with holding for 5 min at 100 °C, fol-
lowed by heating in an increment of 4 °C/min up to 240 °C and another
holding period of 20 min. A calibration curve for caprylic acid
(R2 = 0.99) was constructed separately to quantify its concentration.
2.9. Statistical analysis
Both the frying experiments and analyses were conducted in re-
plicates. The experimental data were analyzed using Minitab software
(Minitab Version 17.1, Minitab Pty Ltd, Sydney, Australia) and are
expressed as mean values ± standard deviations. A one-way analysis
of variance (ANOVA) and post hoc Tukey's test with a 5% significance
level was used to detect significant differences (p < 0.05) between the
mean values.
3. Results and discussion
To provide a clearer picture of the effect of frying different foods
toward the quality of frying oil, simple food ingredients such as fresh
potatoes (a carbohydrate-based food) and chicken breast meat with the
skin removed (protein-based food) were chosen as the food substrates
for the present study to avoid any other interference. Fresh potatoes
were chosen over French fries, as French fries are pre-fried and would
contain a certain amount of oil. In addition, chicken breast meat with
the skin removed was chosen rather than breaded chicken nuggets
because it is a lean protein source and contains the least fat compared to
other parts of the chicken. In addition, the breaded layer in chicken
nuggets is a source of carbohydrate, which might interfere with our
study. This was also done to prevent the complicated reactions that
Y.P. Khor, et al. Food Control 107 (2020) 106781

would occur when the oil from the pre-fried foods leaches out during
the frying process and interacts with the oil degradation compounds.
During the frying process, a new batch of samples was added to the
same oil without any oil replenishment. The impact of food substrates
on the formation of oxidized fatty acids, PTG and their distribution, as
well as the extent of oil deterioration, were studied.
3.1. Monomeric oxidized triacylglycerols
As monomeric oxidized triacylglycerols (oxTAG) constitute one of
the major fractions in the distribution of polymerized triacylglycerols, it
was important to focus and quantify the changes of all major oxidized
fatty acids that formed during the frying process. The oxTAGs were
formed from the homolytic β-scission cleavages of O–O, C–O, and C–C
bonds of hydroperoxides. Hydroperoxides are extremely unstable pri-
mary oxidation products that are formed by unsaturated TAGs as a
result of the hydrolysis process (Zhang et al., 2015).
Solid phase extraction is a pre-concentration technique that is used
to separate altered and nonaltered fatty acid methyl esters. Diethyl
ether was chosen as the eluent in SPE to elute the altered fatty acids
because it had a higher polarity index than hexane or the mixture of
both hexane and diethyl ether. The polarity was similar to the oxidized
fatty acids that possessed higher polarity than unaltered fatty acids.
This was important, as the solvent exhibited a stronger binding force
with the targeted analyte to ensure a complete elution from the sorbent
(Liu, Wang, et al., 2018). In addition, only the altered fatty acids that
composed the monomeric oxidized triacylglycerols were further ana-
lyzed by gas chromatography. Epoxy fatty acids are less polar and have
the shortest retention time, followed by keto fatty acids and ultimately
hydroxy fatty acids, which have the longest retention time.
Table 1 is an overview of the quantified data of epoxy, keto, and
hydroxy fatty acid concentrations in RBDPO that was fried using dif-
ferent foods at 180 °C with 10 frying cycles per day for 7 days. The
results demonstrated that epoxy, keto, and hydroxy fatty acids were not
detected in fresh palm olein. However, all oxidized fatty acids increased
significantly (p < 0.05) as the frying cycles increased in both RBDPO
fried with SP and CBM. It was notable that the respective appearances
of epoxy, keto, and hydroxy acids in RBDPO fried with CBM were
18.92%, 33.33%, and 51.85% higher than RBDPO fried with SP at the
end of the frying study. Final concentrations of 11.9 g/kg and 15.2 g/kg
for the sum of all epoxy, keto, and hydroxy fatty acids were detected in
RBDPO fried with SP and CBM, respectively, after 70 frying cycles. A
higher oxTAG formation was unfavorable in frying oil because it acted
as the pro-oxidant, causing a further oxidation process to occur
(Farhoosh et al., 2009).
Additionally, a study conducted on soft oil showed that oxTAG
ranged between 5.9% and 9.4% when the frying oil achieved 25% TPC
(Joaquín Velasco, Marmesat, Márquez-Ruiz, & Dobarganes, 2004).
However, in the current study, RBDPO appeared to be a more stable
frying oil, as it only yielded 1.2% of oxTAGs after 70 frying sessions.
Furthermore, it was observed that more epoxy acids were formed
compared to keto and hydroxy acids at the end of the frying study. In
European countries, the limit of rejection for epoxy acids was set as 7 g/
kg (Brühl, Weisshaar, & Matthäus, 2016). The epoxy acid concentra-
tions in RBDPO fried with both SP and CBM exceeded the limit of re-
jection when they achieved 7.4 g/kg and 8.8 g/kg, respectively, after 70
frying sessions.
Epoxy fatty acids were the predominant oxidized fatty acids that
formed during the thermo-oxidation process due to the abundance of
oleic acid in RBDPO. Epoxy fatty acids were generally more abundant
in thermo-oxidized monounsaturated oils than in polyunsaturated oils
(J. Velasco, Marmesat, Bordeaux, Marquez-Ruiz, & Dobarganes, 2014).
The unsaturated fatty acids were known to be precursors for the for-
mation of epoxy fatty acids, as they were very susceptible to oxidation
(Liu, Wang, et al., 2018; Mubiru et al., 2013). In short, oxTAG forma-
tion was significantly (p < 0.05) affected by the frying duration and
type of food substrate.
3.2. Total polar content
The occurrence of total polar content (TPC) in frying oil was due to
the degradation of triacylglycerols during heat treatment. TPC has been
extensively reported on as it is a widely used parameter to evaluate the
life span of frying oil (Zhang et al., 2015). Although various rapid
techniques had been developed to measure the TPC, in the current
study, conventional column chromatography was used to further ana-
lyze the collected polar fraction. The TPC increased linearly as the
number of frying cycles increased in both RBDPO fried with CBM
(R2 = 0.99) and SP (R2 = 0.98) (Table 2). The recommended range for
discarding the frying oils ranges from 25% to 27% depending on reg-
ulations set by the country. TPC values greater than 25% were obtained
after 70 frying sessions for palm olein used to fry SP. For palm olein
fried with CBM, the TPC value still fell within the safety limit after 70
frying sessions. This finding justified that the polymerization reaction
was slower in RBDPO when fried using protein-based food compared to
carbohydrate-based food. Similar findings have been reported whereby
rapeseed oil, as the frying medium, exhibited a lower TPC value when it
was fried with cod fillets compared to the sliced potatoes in fritters form
(Tynek, Hazuka, Pawlowicz, & Dudek, 2001). Since palm olein natu-
rally contains a high proportion of diacylglycerols that contribute to the

Table 1
Quantitative determination of epoxyFAMEs, ketoFAMEs, and hydroxyFAMEs (g/100g) in palm olein during deep-fat frying of sliced potatoes and chicken breast meat
at 180 °C.
Frying substrate Day EpoxyFAMEs KetoFAMEs HydroxyFAMEs Total

a a a
Sliced potatoes (Carbohydrate) 0 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00a
1 0.06 ± 0.00ab 0.04 ± 0.06ab 0.11 ± 0.08b 0.21 ± 0.12ab
2 0.16 ± 0.01bc 0.06 ± 0.00abc 0.15 ± 0.00bc 0.36 ± 0.02bc
3 0.26 ± 0.01cde 0.09 ± 0.00bcde 0.22 ± 0.03cde 0.57 ± 0.02cd
4 0.30 ± 0.02de 0.15 ± 0.02def 0.21 ± 0.03cde 0.65 ± 0.05de
5 0.67 ± 0.02g 0.16 ± 0.04ef 0.26 ± 0.02def 1.08 ± 0.05fg
6 0.68 ± 0.05g 0.16 ± 0.01ef 0.27 ± 0.03efg 1.11 ± 0.08g
7 0.74 ± 0.04g 0.18 ± 0.05fg 0.27 ± 0.03efg 1.19 ± 0.11g

Chicken breast meat (Protein) 1 0.07 ± 0.00ab 0.07 ± 0.01abcd 0.17 ± 0.00bcd 0.31 ± 0.01b
2 0.21 ± 0.01cd 0.13 ± 0.01cdef 0.24 ± 0.01def 0.58 ± 0.03cde
3 0.33 ± 0.02de 0.16 ± 0.01ef 0.27 ± 0.01efg 0.77 ± 0.04de
4 0.39 ± 0.06e 0.16 ± 0.03ef 0.29 ± 0.02efg 0.83 ± 0.11ef
5 0.52 ± 0.08f 0.18 ± 0.03fg 0.32 ± 0.02fgh 1.03 ± 0.13fg
6 0.63 ± 0.08fg 0.20 ± 0.02fg 0.35 ± 0.02gh 1.18 ± 0.12g
7 0.88 ± 0.01h 0.24 ± 0.05g 0.41 ± 0.08h 1.52 ± 0.26h

Data are expressed as the mean ± standard deviation (n = 4). Mean values with different superscripts in the same column are significantly different at p < 0.05.

4
Y.P. Khor, et al. Food Control 107 (2020) 106781

Table 2
Changes of polar fraction content and composition in palm olein during deep-fat frying of sliced potatoes and chicken breast meat at 180 °C.
Frying substrate Day Polar fraction content (%, g 100 g−1) Polar fraction composition (%, g 100 g−1)

TAG Oligomers TAG Dimers Oxidized TAG Monomers Diacylglycerols Free Fatty Acids

a a a a ab
Sliced potatoes (Carbohydrate) 0 8.38 ± 0.53 0.00 ± 0.00 0.27 ± 0.05 1.21 ± 0.05 6.54 ± 0.59 0.35 ± 0.11a
1 11.18 ± 0.29b 0.29 ± 0.06ab 1.46 ± 0.10b 2.40 ± 0.22b 6.72 ± 0.15ab 0.30 ± 0.01a
2 13.32 ± 0.43b 0.79 ± 0.07bc 2.29 ± 0.07c 3.39 ± 0.24c 6.55 ± 0.17ab 0.30 ± 0.03a
3 16.50 ± 0.24c 1.44 ± 0.15de 3.29 ± 0.03d 4.61 ± 0.17d 6.77 ± 0.24abc 0.38 ± 0.08a
4 19.07 ± 0.52cd 2.10 ± 0.17f 4.16 ± 0.12e 5.58 ± 0.15e 6.87 ± 0.34abc 0.36 ± 0.12a
5 21.24 ± 1.51d 2.93 ± 0.29h 5.08 ± 0.32fg 6.27 ± 0.38ef 6.77 ± 0.67ab 0.20 ± 0.09a
6 24.67 ± 0.64e 3.85 ± 0.18j 5.96 ± 0.11h 7.53 ± 0.35g 7.11 ± 0.25abc 0.21 ± 0.02a
7 27.45 ± 0.69f 4.70 ± 0.17k 6.87 ± 0.27i 8.40 ± 0.25h 7.31 ± 0.59abc 0.17 ± 0.20a

Chicken breast meat (Protein) 1 11.34 ± 0.34b 0.47 ± 0.10ab 1.64 ± 0.04b 2.13 ± 0.18b 6.79 ± 0.16abc 0.32 ± 0.06a
2 13.60 ± 0.63b 1.01 ± 0.14cd 2.54 ± 0.14c 3.44 ± 0.23c 6.29 ± 0.53a 0.31 ± 0.06a
3 16.58 ± 1.96c 1.55 ± 0.15e 3.47 ± 0.41d 4.26 ± 0.57d 7.01 ± 0.81abc 0.29 ± 0.06a
4 19.80 ± 1.16d 2.26 ± 0.16fg 4.32 ± 0.24e 5.49 ± 0.33e 7.47 ± 0.47abc 0.27 ± 0.03a
5 20.57 ± 1.99d 2.66 ± 0.33gh 4.70 ± 0.48ef 5.96 ± 0.36e 7.03 ± 0.88abc 0.22 ± 0.16a
6 23.86 ± 0.67e 3.16 ± 0.15hi 5.53 ± 0.16gh 6.95 ± 0.43fg 7.98 ± 0.19c 0.24 ± 0.27a
7 23.91 ± 1.03e 3.58 ± 0.44ij 5.53 ± 0.26gh 6.91 ± 0.34fg 7.67 ± 0.19bc 0.22 ± 0.15a

Data are expressed as the mean ± standard deviation (n = 4). Mean values with different superscripts in the same column are significantly different at p < 0.05.

Table 3 high initial TPC in fresh oil, it was inaccurate to solely judge oil quality
Changes of peroxide value, anisidine value, and TOTOX value in palm olein based on TPC (Ahmad Tarmizi, Hishamuddin, & Abd Razak, 2019).
during deep-fat frying of sliced potatoes and chicken breast meat at 180 °C. Therefore, determining the distribution of polar compounds in oil was
Frying substrate Day Peroxide value Anisidine value TOTOX value vital to provide further insights regarding the degradation level of the
(meq O2/kg) frying oil (Bansal et al., 2010b).

Sliced potatoes 0 1.40 ± 0.22a 2.20 ± 0.64a 5.00 ± 0.96a

(Carbohydrate) 1 2.50 ± 0.41ab 43.78 ± 1.57e 48.78 ± 1.69bc
3.3. Polymerized triacylglycerols and their distribution
2 4.37 ± 0.25cd 57.11 ± 2.34f 65.85 ± 2.58de
3 4.60 ± 0.84cde 62.93 ± 1.11f 72.13 ± 1.79e
4 4.75 ± 1.19cde 71.31 ± 3.05g 80.80 ± 4.59f The abundance of polymerized triacylglycerols (PTG) were sug-
5 5.75 ± 0.28def 79.47 ± 1.53h 90.96 ± 1.97g gested as a good indicator to gauge the history of the used frying oil
6 6.08 ± 0.63ef 82.79 ± 7.27hi 94.95 ± 6.95gh
(Matthäus, Haase, & Unbehend, 2009). These compounds are com-
7 6.66 ± 0.27f 86.23 ± 3.43i 99.56 ± 3.64h
Chicken breast 1 4.63 ± 0.25cde 36.89 ± 1.20bcd 46.14 ± 0.85bc
monly analyzed by high-performance size exclusion chromatography
meat (Protein) 2 6.63 ± 1.49f 45.55 ± 0.61e 58.80 ± 2.42d due to their higher molecular weight. With the presence of oxygen and
3 6.47 ± 0.57f 47.50 ± 0.81e 60.43 ± 1.43d heat treatment, many complex polymers with -C-C-, -C-O-C-, and -C-O-
4 3.92 ± 0.56bc 43.36 ± 0.58de 51.20 ± 1.56c O-C- linkages might form among TAG molecules (Zhang, Saleh, Chen, &
5 1.15 ± 0.19a 41.29 ± 2.24cde 43.59 ± 1.90b
Shen, 2012). Furthermore, the formation of positional and geometrical
6 – 36.07 ± 2.59bc –
7 – 32.67 ± 1.02b – isomers through autoxidation would yield thousands of individual new
compounds. Therefore, the definite molecular structure of the TAG
Data are expressed as the mean ± standard deviation (n = 4). Mean values polymers has scarcely been reported due to the complexity of the
with different superscripts in the same column are significantly different at polymers and the lack of effective techniques to separate the polymers.
p < 0.05. The fresh palm olein contained 0.27% TAG dimers before frying
(Table 2). This was regarded as normal because dimerization of tria-
cylglycerols took place during the deodorization step of the oil refining

Table 4
Determination of fatty acid composition in palm olein during deep-fat frying of sliced potatoes and chicken breast meat at 180 °C.
Frying substrate Day Fatty acid composition (relative percentages)

C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:2/C16:0 C8 (ppm)

Sliced potatoes 0 0.24 ± 0.00cd 1.53 ± 0.04bc 39.80 ± 0.01a 4.19 ± 0.00c 43.90 ± 0.05cd 10.33 ± 0.01j 0.26 ± 0.00i 0.00 ± 0.00a
(Carbohydrate) 1 0.24 ± 0.00de 1.49 ± 0.03b 40.26 ± 0.04b 4.23 ± 0.00cd 43.96 ± 0.02d 9.78 ± 0.02hi 0.24 ± 0.00h 26.54 ± 0.70b
2 0.24 ± 0.00cd 1.64 ± 0.07cd 40.66 ± 0.01c 4.27 ± 0.00cd 43.83 ± 0.05cd 9.29 ± 0.03g 0.23 ± 0.00g 33.25 ± 1.00bc
3 0.24 ± 0.00de 1.61 ± 0.02bc 41.07 ± 0.02d 4.32 ± 0.02cd 43.82 ± 0.02cd 8.86 ± 0.04f 0.22 ± 0.00f 42.72 ± 1.14de
4 0.24 ± 0.00de 1.77 ± 0.03e 41.54 ± 0.04e 4.37 ± 0.00cd 43.59 ± 0.03bcd 8.39 ± 0.03d 0.20 ± 0.00d 47.88 ± 1.11ef
5 0.24 ± 0.00ef 1.75 ± 0.07de 42.05 ± 0.02fg 4.42 ± 0.00cd 43.52 ± 0.08bc 7.90 ± 0.02c 0.19 ± 0.00c 59.83 ± 3.16gh
6 0.25 ± 0.00fg 1.80 ± 0.14e 42.58 ± 0.02h 4.48 ± 0.00cd 43.37 ± 0.11ab 7.39 ± 0.03b 0.17 ± 0.00b 69.26 ± 3.07ij
7 0.25 ± 0.00g 1.98 ± 0.06f 43.11 ± 0.01i 4.53 ± 0.00d 43.07 ± 0.06a 6.90 ± 0.01a 0.16 ± 0.00a 76.82 ± 0.99jk
Chicken breast meat 1 0.23 ± 0.00a 0.94 ± 0.00a 40.73 ± 0.07c 2.00 ± 0.31ab 45.73 ± 0.33ef 10.32 ± 0.11j 0.25 ± 0.00i 38.97 ± 1.89cd
(Protein) 2 0.23 ± 0.00ab 0.95 ± 0.01a 41.12 ± 0.10d 2.00 ± 0.18ab 45.74 ± 0.22ef 9.90 ± 0.04i 0.24 ± 0.00h 52.45 ± 1.30fg
3 0.24 ± 0.00bc 0.96 ± 0.01a 41.45 ± 0.11e 2.05 ± 0.16b 45.77 ± 0.23ef 9.46 ± 0.05gh 0.23 ± 0.00g 66.62 ± 3.39hi
4 0.24 ± 0.00de 0.97 ± 0.01a 41.88 ± 0.16f 1.70 ± 0.18a 46.20 ± 0.18g 8.91 ± 0.20f 0.21 ± 0.01ef 84.93 ± 1.00k
5 0.24 ± 0.00de 0.98 ± 0.01a 42.15 ± 0.10g 1.81 ± 0.18ab 46.06 ± 0.22fg 8.65 ± 0.10def 0.21 ± 0.00de 96.53 ± 7.42l
6 0.24 ± 0.00de 0.97 ± 0.02a 42.54 ± 0.08h 1.78 ± 0.01ab 45.62 ± 0.22e 8.73 ± 0.28ef 0.21 ± 0.01de 111.39 ± 4.74m
7 0.24 ± 0.00de 0.96 ± 0.01a 42.65 ± 0.03h 1.80 ± 0.14ab 45.71 ± 0.20ef 8.49 ± 0.32de 0.20 ± 0.01d 122.99 ± 6.56n

Data are expressed as the mean ± standard deviation (n = 4). Mean values with different superscripts in the same column are significantly different at p < 0.05.

5
Y.P. Khor, et al.
process due to high-temperature processing (Erickson, 1990). In addi-
tion, no TAG oligomers were detected in fresh palm olein, but they were
detected at concentrations of 4.70% and 3.58% in palm olein fried with
SP and CBM at the end of the frying study, respectively. According to
the European authorities, the limit of frying oil rejection for poly-
merized triacylglycerols (PTG) ranged from 12 to 13% with 10% PTG
recommended as the most appropriate level for oil replenishment
(Bansal et al., 2010a). RBDPO fried with SP exceeded the 10% limit
after 70 successive frying sessions, which occurred on the seventh day
of frying where the TPC values also exceeded the 25% limit of rejection.
PTG formation was also markedly affected by the types of food sub-
strates used during frying. RBDPO fried with SP resulted in a higher
PTG content than RBDPO fried with CBM. Similar studies have reported
that PTG formation was higher when frying French fries than chicken
nuggets (Bansal et al., 2010b). From day 6 to day 7, there were no
significant (p < 0.05) changes in PTG content or the distribution in
RBDPO samples fried using CBM, but in RBDPO fried with SP, the TPC
and its distribution consistently showed a significant increase, whereby
it achieved a concentration of 27.45% TPC and 11.57% PTG after the
frying studies.
A higher FFA concentration in RBDPO would greatly affect the
oxidative stability of the frying oil because FFA has been found to
possess a higher oxidative rate than the other esters (Farhoosh et al.,
2009). In current frying study, the effect of food substrate toward the
FFA content in both RBDPO samples was less apparent as there were no
significant (p > 0.05) changes in the FFA content throughout the 70
frying sessions. However, current studies rarely report only the FFA
content, especially for frying oils, because FFA is very volatile and could
easily be stripped from hot frying oil. In addition, it is easily degraded
or prone to react with other compounds (Zhang et al., 2015). Research
has suggested that there is no direct relationship between FFA and the
quality of used frying oil (Gertz, 2000). Therefore, FFA may not be
recommended as a reliable parameter to evaluate the extent of dete-
rioration of frying oil (Weisshaar, 2014).
Furthermore, a higher amount of DAG is undesirable in frying oil
because it acted as a pro-oxidant in the oil; in other words, it is a
precursor of volatile autoxidation products (Choe & Min, 2007). It was
noticeable that there were no significant (p > 0.05) changes in the
DAG content during the deep-fat frying process in palm olein fried using
both SP and CBM.
3.4. Oxidative stability of RBDPO
Peroxide value (PV) is commonly used to measure primary oxida-
tion products, such as hydroperoxides. The PV in RBDPO increased
significantly (p < 0.05) from day 1 to day 7 when frying SP (Table 3).
For RBDPO fried with CBM, the PV increased significantly (p < 0.05)
during the first two days of frying but decreased significantly
(p < 0.05) in subsequent days of frying. The PV could not be measured
on day 6 onwards due to the dark color of the oil that resulted from
polymerization. Since PV analysis involved the observation of color
changes during titration, the dark color of the oil sample would affect
the accuracy of the end-point determination during the titration. This
might be one of the limitations of the PV analysis. The decrease in PV
might be due to the fission of hydroperoxides that disintegrated and
were further converted into secondary oxidation products, such as al-
dehydes and ketones (Koh et al., 2011). In other words, the decrease in
PV occurred when the destruction rate of hydroperoxides exceeded the
formation rate. However, PV is considered to be an unstable parameter
to measure oil oxidation since new hydroperoxides might also form
during cooling and storage (Choe & Min, 2007; Matthäus et al., 2009).
For p-anisidine analysis, the value increased significantly
(p < 0.05) in RBDPO fried with SP, reaching a value of 86.23 after 70
frying sessions. However, for RBDPO fried with CBM, the p-anisidine
value increased from day 1 to day 3 and then decreased significantly
(p < 0.05) from day 4 onward. A similar trend was also reported in
6
Food Control 107 (2020) 106781
another study, which suggested that the decrease in the p-anisidine
value might be due to the elevated temperature that caused the car-
bonyl content, which was labile, to decrease (Enríquez-Fernández,
Álvarez de la Cadena y Yañez, & Sosa-Morales, 2011). The changes in
secondary oxidation products were also due to the onset of the ad-
vanced oxidation stage, during which the aldehydes were converted to
higher molecular weight compounds.
The TOTOX value provided adequate information regarding the
oxidative state of a frying oil because it described the development of
both primary and secondary oxidation products. The TOTOX value in-
creased significantly (p < 0.05) in RBDPO from day 1 to day 7 when
frying SP. When the RBDPO was fried with CBM, the TOTOX value
increased significantly (p < 0.05) from day 1 to day 3 but decreased
significantly (p < 0.05) afterward. Since the TOTOX value was cal-
culated as twice the peroxide value (PV) plus the anisidine value (AV),
the decrease was due to decreases in both PV and AV. Even though the
TOTOX value decreased after day 3, the value still surpassed the re-
jection limit (TOTOX value = 30) that was set by industries and official
authorities (Matthäus et al., 2009).
3.5. Fatty acid composition changes of RBDPO
Determining changes in fatty acid was equally important to gauge
the oxidation degree of the frying oil. However, this analysis should
normally be coupled with other analyses, such as TPC, PTG and oxTAG
to comprehensively evaluate the quality of the frying oil. According to
Table 4, fatty acid compositions in both RBDPO fried with SP and CBM
were significantly (p < 0.05) modified. The larger the number of
double bonds in fatty acids, the more unstable fatty acids are. Linoleic
acid showed a more significant decrease, which was a 33% reduction in
RBDPO fried with SP after 70 frying sessions compared to an 18% re-
duction in RBDPO fried with CBM. When there was a decrease in
polyunsaturated fatty acids (PUFA), there was an increase in saturated
fatty acids and oxidative degradation products. This could be justified
by the significant (p < 0.05) increases in lauric, myristic, palmitic, and
stearic acids in RBDPO when frying SP. In addition, this result is jus-
tified by the significant (p < 0.05) increase in caprylic acid, which was
quantified in both RBDPO fried with SP and CBM. Caprylic acid is a
type of short-chain fatty acid, which is a stable nonvolatile product that
results from the thermal decomposition of fatty acid hydroperoxides.
Research has shown that caprylic acid can form from the 9-hydroper-
oxides of all major unsaturated fatty acids, such as oleic, linoleic, and
linolenic acid (Brühl, 2014). The formation of caprylic acid in RBDPO
fried with CBM was 1.6 times higher than RBDPO fried with SP. The
formation of caprylic acid in RBDPO was of considerable concern be-
cause caprylic acid was not expected in fresh RBDPO. Studies have
shown that the formation of caprylic acid is well-correlated with the
formation of TPC. Therefore, the quantification of caprylic acid would
provide a better indication of the total alteration level of frying oils
after the frying process (Zhang et al., 2015).
4. Conclusion
In this study, if solely considering the distributions of oxTAG and
TPC, we would suggest using RBDPO for fewer than 60 frying sessions
without any oil replenishment, because the oxTAG and TPC contents
were close to the limit of rejection after 60 frying sessions. However,
the p-anisidine and TOTOX values were high in RBDPO fried with both
SP and CBM after 10 frying sessions. Therefore, from the sensory point
of view, RBDPO after being used for 10 frying sessions might not be
suitable for further human consumption due to the undesirable ran-
cidity of the oil. Based on the overall results, the oxTAG content ap-
proached the limit of rejection earlier during frying compared to TPC
and PTG. This justified the result that there was no single parameter
that was sufficient to illustrate the extent of deterioration of a frying oil.
It was also concluded that the degree of oxidation was more
Y.P. Khor, et al.
pronounced when the RBDPO was fried using CBM, whereas the poly-
merization rate was higher when RBDPO was fried using SP. The for-
mation of caprylic acid in RBDPO fried with CBM was higher than
RBDPO fried with SP. This study emphasized the importance of food
type in altering the stability of RBDPO during frying.
Conflicts of interest
The authors have declared no conflict of interest.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
The work was supported by the Putra Grant, Universiti Putra
Malaysia (Project number UPM/700-1/2/GIPP/2017/9532400). The
authors would like to extend their sincere appreciation to the Deanship
of Scientific Research, King Saud University for funding this research
through the Research Group Project number RGP-048.
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