Food Chemistry 306 (2020) 125560

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

Aromatic effects of immobilized enzymatic oxidation of chicken fat on T

flaxseed (Linum usitatissimum L.) derived Maillard reaction products
Chao-Kun Weia, Zhi-Jing Nia,b, Kiran Thakura, Ai-Mei Liaoc, Ji-Hong Huangc,d, Zhao-Jun Weia,e,
⁎

a
School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China
b
Biological Science and Engineering College, North Minzu University, Yinchuan 750021, People’s Republic of China
c
College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, People’s Republic of China
d
Henan Cooperation Science and Technology Institute, Zhengzhou 450001, People’s Republic of China
e
Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, People’s Republic of China

ARTICLE INFO ABSTRACT

Keywords: To control the oxidation in chicken fat by immobilized lipoxygenase (LOX), Maillard reaction products (MRPs)
Maillard reaction with chicken flavor were prepared and analyzed for flavor mechanism. > 50% activity of immobilized LOX was
Chicken fat retained after repeated use for five times or five weeks. The oxidized chicken fats were prepared by thermal, free
Lipoxygenase LOX, and immobilized LOX treatments. After addition of chicken fats, Maillard reaction produced more aliphatic
Immobilized enzyme
aldehydes and alcohols (126.0–839.5 ng/g and 493.5–2332.4 ng/g, respectively) which resulted in noticeable
Oxidation method
enhanced reaction, but the content of sulfur compounds such as thiols and thiophenes decreased significantly
Flavor
(870.8–1233.9 ng/g and 1125.0–2880.3 ng/g, respectively), and the structure of sulfur compounds could easily
form alkyl side chains. However, there was no significant difference in sensory and flavors between oxidized
chicken after treatments, which may be related to oxidized degree. The mechanism was proposed or aromatic
effects of oxidized chicken fat on flaxseed derived MRPs.

1. Introduction methanethiol, thiophene, and dimethyl disulfide in boiled beef; while,

The Maillard reaction is responsible for the preparation of meat
flavors, and research focus has been primarily directed towards im-
plications of this reaction on food browning and flavor formation
(Brehm, Jünger, Frank, & Hofmann, 2019; Hou et al., 2017). Further-
more, various researchers have showed the antioxidant and anti-in-
flammatory activity of Maillard reaction products (MRPs) from amino
acids, protein hydrolysates, and heterocyclic volatile compounds
formed during Maillard reaction in vitro and in food products (Qin et al.,
2018; Wakamatsu, Stark, & Hofmann, 2019). Flaxseed meal is a pro-
tein-rich by-product obtained during flaxseed oil extraction. Despite
insignificant applications in foods, flaxseeds proteins are recognized as
an important food ingredient (Bekhit et al., 2018). Several food pro-
ducts are fortified using flaxseed meal, especially dairy, cereal-based
foods as well as meat products (Teh, Bekhit, Carne, & Birch, 2014). The
formation of meat flavor depends on some characteristic flavor sub-
stances. For example, sulfur-containing volatile components such as
in another study, similar components such as 2-methylthiophene and 2-
furanmethanethiol in the Maillard reaction system of cysteine, ribose,
and polyunsaturated fatty acids (Elmore, Mottram, & Hierro, 2001;
Elmore, Campo, Enser, & Mottram, 2002). Some animal fats produce a
characteristic flavor of the meat when heated in the air; whereas,
heating in nitrogen does not lead to the corresponding flavor. This
shows that a certain degree of fat oxidation results in the generation of
meat flavor. The previous study showed that heating the beef after
removing triglycerides and phospholipids led to great alterations in
volatile components in terms of flavor characteristics. The changes of
volatile compounds were mainly manifested in the decrease of fatty
alcohols and aldehydes, and the increase of benzaldehyde and pyr-
azines (Aaslyng & Meinert, 2017). After removing triglycerides and
phospholipids from beef, heat treatment resulted into loss of volatile
components and barbecue aroma (Mottram & Edwards, 2010) which
was similar to the results obtained in pork and lamb (Guerrero et al.,
2014; Song et al., 2017).

Abbreviations: AV, acid value; DW, distilled water; DOC, deoxycholate; MDA, malondialdehyde; LOX, lipoxygenase; MRPs, Maillard reaction products; PV, peroxide
value
⁎
Corresponding author at: School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, People’s Republic of China.
E-mail addresses: kumarikiran@hfut.edu.cn (K. Thakur), aimeiliao@haut.edu.cn (A.-M. Liao), huangjihong@haut.edu.cn (J.-H. Huang),
zjwei@hfut.edu.cn (Z.-J. Wei).

https://doi.org/10.1016/j.foodchem.2019.125560
Received 20 May 2019; Received in revised form 13 September 2019; Accepted 16 September 2019
Available online 03 October 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
C.-K. Wei, et al.
Fatty acid oxidation and degradation occur during fat heating and
give rise to various degradation products. With the exception of alde-
hydes, other substances have a smaller contribution to meat odor due to
the higher odor threshold value. In fact, lipid degradation produces
aldehydes and ketones which interact with the Maillard reaction ma-
terials such as amino acids and reducing sugars during heat treatment
(Yang et al., 2015). However, the thermal oxidation of oils and fats
experiences some drawbacks such as high energy consumption, large
equipment volume, low production efficiency, and difficulty in product
quality control in the food industry.
Lipoxygenase (LOX) are responsible for the formation of hydroper-
oxides from polyunsaturated fatty acids such as linoleic and arachidonic
acids (Fofana et al., 2016). Since vegetable and animal oils contain a
certain proportion of unsaturated fatty acids which can be catalyzed by
LOX to prepare flavor precursors to participate in Maillard reaction.
Since the content of unsaturated fatty acids in vegetable oil is much
higher than found in animal oil. There are many reports on the pre-
paration of seasonings by catalytic oxidation of LOX with vegetable oil or
unsaturated fatty acid (Aziz, Akacha, Husson, & Kermasha, 2014). On the
other hand, there is limited knowledge on the use of animal oils as raw
materials for the preparation of seasonings by LOX catalytic oxidation.
LOX has limitations in practical applications, such as LOX needs to be
inactivated after the oxidation treatment is completed, and the conven-
tional heat treatment inactivation leads to the secondary oxidation of fat.
On the other hand, LOX is expensive and also leads to increased pro-
duction costs. Immobilized enzyme can effectively solve the aforemen-
tioned problems. In our previous studies, immobilized Alcalase and
Flavourzyme could facilitate the enzymatic hydrolysis of flaxseed pro-
tein, and prepare excellent substrates for Maillard reaction (Wei, Thakur,
Liu, Zhang, & Wei, 2018). Flaxseed derived MRPs were found acute non-
toxic and no adverse effect during 90-days period (Wei et al., 2019a).
Therefore, we simplified the oxidation process by using immobilized
LOX to generate the oxidized chicken fat with a suitable degree of
oxidation. The aim of this study was to utilize the resulting fat in
Maillard reaction to obtain the required chicken flavor. At the same
time, the effect of oxidized chicken fat on Maillard reaction process, and
sensory evaluation were determined. Furthermore, the volatile aro-
matic components based on molecular sensory science were also stu-
died. In order to reveal the changes in the species, molecular structure,
and content of volatile flavors of MRPs after the addition of oxidized
chicken fat, the mechanism of chicken flavor formation was elucidated.
2. Materials and methods
2.1. Materials and chemicals
Soy lipoxygenase (LOX) was purchased from Shanghai Zheng
Biotechnology Co. Ltd. (China). Chicken fat and flaxseeds were purchased
from a local market of Hefei city, China. Chicken fat obtained from sub-
cutaneous fat was separated from chicken carcass (AA Broiler). According
to our previous study, fresh chicken fat was boiled and filtered for its use
in the experiment (Wei, Fu, Liu, Zhang, & Liu, 2017). The chicken fat is a
uniform viscous liquid. After centrifugation, liquid oil (84.5%) and solid
fat (15.5%) were obtained. The chicken fat used in this study was whole
fat. Defatted flaxseed meal was obtained according to our previous study
(Wei et al., 2018, 2019b). The standard compounds (≥95%) which are
commonly used to identify the generated volatile flavor compounds were
purchased from J&K Chemical Ltd. (Beijing, China).
2.2. Immobilization of LOX
Immobilization of LOX enzyme was performed according to the
treatment conditions of Hsu, Foglia, and Piazza (1997) Briefly, the
mixture of sodium alginate (4%, w/v) in the form of droplets was added
into sodium borate buffer (0.2 mol/L, pH 9.0) and LOX (5 mg/mL in
sodium borate buffer). The obtained solution was dropped from 10 cm
2
Food Chemistry 306 (2020) 125560
height into 0.2 mol/L CaCl2 (400 mL) to form beaded particles at 20 °C.
The beads were covered with hexane and tetramethoxyortho-silicate
(1:1.5 v/v of the beads) was added. The obtained mixture was allowed
to stand for 12 h at 20 °C to complete the polymerization process. Fi-
nally, the immobilized LOX was filtered from the solution, air dried at
room temperature, and stored at 4 °C.
2.3. Determination of LOX enzyme activity
The assay for LOX activity was conducted by measuring the hy-
droperoxide formation from linoleic acid. LOX was accurately weighed
and added to distilled water adjusted to experimental pH for free LOX
(1:500, w/v) or immobilized LOX (1:100, w/v). Then after, the per-
oxide value (PV) was determined after addition of 1.0 g linoleic acid
and 0.6 g Tween 20 in order to react for 1.0 h at required temperature.
Enzyme activity (U/g) was calculated as substrate concentration. Our
study measured the enzyme activity in the temperature range of
15–35 °C at pH 9.0; pH range of 8–11 at 25 °C; a usage count and sto-
rage time at 25 °C and pH 9.0. In order to measure the peroxide value,
an inactivated enzyme of similar quality was taken as a blank group
after the above operation. Catalytic production of 1.0 mmol hydroper-
oxide per hour by 1.0 g enzyme can be defined as 1.0 U/g.
(PV PV0) m
X= ×
t m0 (1)
where X represented enzyme activity value; PV and PV0 represented
peroxide values of samples and blank group, respectively; m and m0
corresponded to the quality of samples and blank group; n represented
dilution multiples of enzymes.
2.4. Oxidative processing of chicken fat
For thermal treatment, 100 mL of chicken fat was heated in a
thermostatic bath at 110 °C for 2.5 h at 700 r/min stirring speed in a
round bottom flask and samples were collected at 30 min intervals for
150 min.
For free enzyme treatment, LOX solution (300 U/g substrates) was
mixed with 100 mL of chicken fat. The chicken fat was stirred at 20 °C
for 2.5 h; while, samples were collected at 30 min intervals for a total
time of 150 min at 700 r/min stirring speed. Each sample was in-
activated at 95 °C for 5 min.
For immobilized LOX treatment, 100 mL of chicken fat was mixed
with 100 mL of distilled water (DW) followed by addition of im-
mobilized LOX (300 U/g substrates). The chicken fat was stirred at
20 °C for 2.5 h; while, samples were collected at 30 min intervals for a
total time of 150 min at 700 r/min stirring speed. The immobilized
enzyme was recovered after the reaction was completed.
2.5. Determination of acid value (AV), PV, malondialdehyde (MDA), and
fatty acid composition
AV, PV, and fatty acid composition were analyzed according to
Moghtadaei, Soltanizadeh, and Goli (2018). MDA was analyzed as per
the method of Moudache, Nerín, Colon, and Zaidi (2017)
2.6. Preparation of MRPs
As reported in our recent study, flaxseed protein hydrolysates were
used as starting material, while chemical composition of flaxseed meal
and flaxseed protein hydrolysates have been analyzed. Flaxseed meal
revealed 38.57% protein content, 11.54% crude fiber content, 1.92%
fat content, and 3.12% moisture content, while flaxseed protein hy-
drolysates consisted of 34.81% protein content, 6.40% crude fiber
content, 0.32% fat content, 2.66% moisture content, 40.12% total sugar
content, and 6.10% ash content (Wei et al., 2018, 2019b). For the
Maillard reaction, four main ingredients such as 10.0 g of flaxseed
C.-K. Wei, et al.
protein hydrolysates, 3.0 g of D-xylose, 0.75 g of L-cysteine, and 0.3 g,
0.4 g, or 0.5 g of chicken fat were mixed with DW to obtain the con-
centration of 10% (w/v) followed by the initial pH of 7.5. The above
suspensions were allowed to stand at 120 °C with stirring for 120 min
followed by immediate cooling.
2.7. Sensory evaluation
For sensory evaluation, 14 trained panelists (six males and eight fe-
males, aged within 23–48 years) were selected from Laboratory of Sensory
Science, Hefei University of Technology (Hefei, China) by confirming the
criteria for descriptive analysis. Prior to the formal sensory evaluation of
the samples, all panelists discussed flavors (meaty, umami, peculiar smell,
and total acceptance) in detail until everyone agreed with the aroma and
taste attributes of the study. Sensory evaluation criteria were based on
previous research methods such as meaty; umami; and total acceptance
(Ogasawara, Katsumata, & Egi, 2006), as well as peculiar smell (Un-
acceptable taste in the sample). The taste criteria were as follow: The
meaty is lean chicken which is cooked at 121 °C for 60 min, which is tasted
at normal atmospheric temperature (20 °C). The umami is sodium gluta-
mate solution (1%, w/v). The MRPs sample solutions (0.5%, w/w) were
suspended into umami solution (1.0% (w/v) sodium glutamate, and 0.5%
(w/v) sodium chloride). The members evaluated 10.0 mL of each sample
in a triangle test with two umami soups as two blanks. The sensory eva-
luation was repeated three times per sample. The panelists were instructed
to score on a scale from 0 (not detectable) to 10 (strongly detectable) with
10-point category scales properties. Each panelist was asked to take a rest
(10 min) for sensory recovery between each set of four different samples.
The evaluation was performed at room temperature (25 °C) under a dim
light to mask the color difference between samples. All the samples were
evaluated in one-time evaluation within one day.
2.8. Measurement of reaction progress
For sample preparation, same procedure was followed as mentioned
in Section 2.6. The freshly prepared samples were diluted 50-fold by
using DW and the absorbance was measured at 420 nm and 294 nm
representing the browning index (Hou et al., 2017).
2.9. Volatile compounds analysis by gas chromatography-mass
spectrometry
For this, 2 μL of 1,2-dichlorobenzene (50 μg, in 1 mL of methanol) was
added to each MRPs sample (3 mL) as an internal control. The volatile
components of each MRPs solution were extracted with 75 μm carboxen/
poly-dimethylsiloxane SPME-fibre (50 °C, 5 min) and further identified
through comparison of data obtained from gas chromatography-mass
spectrometry (GC–MS) (Agilent GC–MS 7890 and DB-WAX 30 m ×
0.25 mm × 0.25 μm, or Agilent, DB-5MS 30 m × 0.25 mm × 0.25 μm,
Agilent) and NIST 08 (Gaithersburg, MD, USA). Kovats indices (KIs) were
obtained by applying C7-C30 standard under the same condition and all the
compounds were analyzed with the available standard compounds for
identification. Chromatographic conditions were used as per the methods
given by Hou et al. (2017) and Eric et al. (2014). The column flow rate was
set as 1.0 mL/min, using helium as a carrier gas. The column temperature
program was maintained at 40 °C for 2 min, 40–80 °C at 3 °C/min,
80–120 °C at 4 °C/min, and 120–230 °C at 10 °C/min for DB-WAX column;
whereas, at 40 °C for 2 min, 40–100 °C at 2 °C/min, 100–150 °C at 10 °C/
min, and 150–280 °C at 20 °C/min for DB-5MS column. The GC was
equipped with a mass spectrometric detector which was set at a scanning
range of 35 to 450 m/z.
Sv
Cv = × Ci
Si (2)
Where Cv and Ci represented the concentration of volatile compound and
internal standard, respectively; Sv and Si corresponded to the peak area of
3
Food Chemistry 306 (2020) 125560
volatile compound and internal standard.
2.10. Aromatic components analysis by gas chromatography-olfactometry
The volatile compounds were sampled with SPME-fibre (75 μm,
carboxen/poly-dimethylsiloxane). Three trained evaluators performed
the gas chromatography–olfactometry (GC-O) (DB-5MS capillary
column, 30 m × 0.25 mm × 0.25 μm, Agilent) analyses using a DATU
2000 high-resolution olfactometer system (DATU Inc., Durham, NC).
Chromatographic conditions were used as per the method given by Eric
et al. (2014). The column temperature program was maintained at 40 °C
for 2 min, 40–100 °C at 2 °C/min, 100–150 °C at 10 °C/min, and
150–280 °C at 20 °C/min for DB-5MS column. Nitrogen was used as
carrier gas at the rate of 1.0 mL/min. The used capillary column, pro-
grammed oven temperatures and desorption of the fiber were similar to
those in the aforementioned GC–MS analysis.
2.11. Statistical analysis
Results were analyzed by using one-way ANOVA through SPSS
Statistics 20.0 (SPSS, Inc., Chicago, IL, USA). Data were expressed as
mean ± SD (n = 3). Significance was considered at ± 5% (P < 0.05).
3. Results and discussion
3.1. Characteristics of immobilized LOX
LOX can specifically oxidize unsaturated fatty acids. Natural oils
from animal and vegetable contain large amounts of unsaturated fatty
acids. These oils can serve as a good substrate for LOX to produce
oxidized fats (Sande, Colen, Santos, Ferraz, & Takahashi, 2017). On the
other hand, LOX has few drawbacks such as high cost, and inactivation
after the reaction. It is possible to overcome the disadvantages of the
above free enzymes based on immobilized enzyme technology.
LOX activities were observed as 46.37 × 104 U/g at pH 9.0 and
25 °C for free enzyme. After immobilization, the oxidized effect of im-
mobilized enzyme was evaluated. As shown in Fig. 1, LOX demon-
strated similar stability for pH 9.0 and temperature 25 °C before and
after immobilization. Altogether, the immobilized LOX could retain half
of the potential after subsequent usage (5 times). Consequently, the
immobilized LOX showed 60% activities after storage at 4 °C.
3.2. Effects of oxidation methods on AV, PV, MDA, and fatty acid
composition
Lipid oxidation utilizes a free radical mechanism to generate vola-
tile compounds such as aldehydes (Tenyang et al., 2013). Based on the
pre-experiment, 300 U/g substrate LOX was selected for free enzyme
and immobilized enzyme. The effects of thermal, free LOX, and im-
mobilized LOX treatments on the oxidation of chicken fat were char-
acterized by AV, PV, MDA, and fatty acids. The purpose was to select a
treatment process with stable oxidation and relatively low oxidation
degree on the premise of ensuring that the oxidized chicken fat is sui-
table for the subsequent Maillard reaction. As shown in Fig. 2 A, the
release efficiency for free fatty acids increase linearly within 30 min of
thermal treatment. Free and immobilized LOX treatment of chicken fat
varied from heat treated chicken fat. Free fatty acids release was stable
after 30 min of enzyme treatment. It can be noticed that the thermal
treatment of chicken fat was more intense in initial reaction stage, re-
leasing a large amount of free fatty acids; and the enzyme treatment of
chicken fat was more stable in releasing free fatty acids. As shown in
Fig. 2B, for the initial 30 min of oxidation treatment, the PV of heat-
treated chicken fat was higher than that of enzyme-treated chicken fat.
For 30–60 min of oxidative treatment, the PV of enzyme-treated
chicken fat was increased rapidly than heat-treated chicken fat. How-
ever, the trend of change in PV had shown a stable rapid increase.
C.-K. Wei, et al.
Fig. 1. Relative enzyme activities of immobilized LOX at different pH (A), temperatures (B), usage count (C), and storage time (D). The values followed by different
letters were significantly different (P < 0.05).
Hydroperoxide was also formed in the early stage. As shown in Fig. 2C,
the formation trend of MDA was positively correlated with oxidation
time, and the MDA content of free enzyme-treated chicken fat was
significantly higher than that of heat treatment and immobilized en-
zyme treatment (P < 0.05). PV indicates the degree to which chicken
fat oxidizes to form peroxides and aldehydes, while MDA indicates the
degree to which chicken fat oxidizes to form final products. Therefore,
in order to obtain oxidized chicken fat with suitable PV and MDA, the
choice of oxidation time is based on the treatment time of PV rising to a
stable level and MDA as small as possible. At the same time, taking
untreated chicken fat as a control, we determined the content of un-
saturated and saturated fatty acids in chicken fat treated with heat
treatment for 30 min, chicken fat treated with free enzyme for 60 min,
and chicken fat treated with immobilized enzyme for 60 min. The re-
sults showed that the unsaturated fatty acids of the oxidized chicken fat
decreased significantly compared with the untreated chicken fat
(P < 0.05); while, the saturated fatty acids increased significantly
(P < 0.05), but there was no significant difference between the three
oxidized chicken fats (P > 0.05) (Fig. 2D). Therefore, the heat-treated
chicken fat at 30 min and the enzyme-treated chicken fat at 60 min
were selected for the Maillard reaction-related experiment.
3.3. Effects of oxidation methods on reaction progress
The dicarbonyl compounds formed in the intermediate stage were
measured at 294 nm, while the protein melanin developed in the ad-
vanced stage was measured at 420 nm (Cao et al., 2017). The effects of
thermal treatment, free LOX treatment, and immobilized LOX treatment
on the Maillard reaction process were studied with the MRPs without
chicken fat (NCF) and added untreated chicken fat (UCF) as control. As
shown in Fig. 3 A and B, the content of the intermediate reaction
4
Food Chemistry 306 (2020) 125560
products (294 nm) and the final reaction products (420 nm) were ac-
celerated significantly (P < 0.05) by giving different treatments to the
oxidized chicken fat compared with the control groups. This may be due
to accelerated pathway of the intermediate products of Maillard reaction,
such as aldehydes, after adding chicken fat or oxidizing chicken fat in
Maillard reaction, which speeds up the reaction system and produces
more dicarbonyl compounds (294 nm) and protein melanin (420 nm).
The stated inference was further confirmed in Section 3.5. Whereas, the
similarity observed between the intermediate reaction products (294 nm)
and the final reaction products (420 nm) absorbance (shown in Fig. 3A
and B) indicated that the colorless products were the precursors of the
brown final reaction products in the reactions. On the other hand,
Maillard reaction rate was reduced significantly (P < 0.05) due to the
excess addition of the oxidized chicken fat (such as 0.5 g). This may be
due to the excessive oxidation of chicken fat which may block the re-
action between reducing sugars and peptides. The Maillard reaction rates
of chicken fat obtained under different oxidation modes were as follow:
thermal treatment > free enzyme treatment > immobilized enzyme
treatment. The Maillard reaction involving the oxidized chicken fat after
the immobilized LOX treatment had better controllability.
3.4. Effects of oxidation methods on sensory characteristics
Sensory evaluation is a direct indicator of whether the MRPs can be
used as a chicken flavor in the presence of chicken fat. The sensory
properties of MRPs with NCF, UCF, chicken fat of thermal treatment,
chicken fat of free LOX treatment, and chicken fat of immobilized LOX
treatment were assessed by the well-trained team. To prepare chicken
flavor using sensory evaluation after heat-treatment, free enzyme and
immobilized enzyme treated oxidized chicken fat samples were used as
a raw material for Maillard reaction.
C.-K. Wei, et al.
Fig. 2. Effects of oxidation methods of chicken fat on AV (A), PV (B), MDA (C), and fatty acid composition (D). RCF indicated that raw chicken fat was not oxidized.
TT indicated that the chicken fat was oxidized by thermal treatment. FET indicated that the chicken fat was oxidized by free enzyme treatment. IET indicated that the
chicken fat was oxidized by immobilized enzyme treatment. Saturated fatty acids (ΣSFA) include myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0),
while unsaturated fatty acids (ΣUSFA) include palmitoleic acid (C16:1), oleic acid (C18:1) and linoleic acid (C18:2). The values on the same fatty acid followed by
different letters were significantly different (P < 0.05).
As shown in Fig. 3C–F, by comparing the MRPs of UCF and NCF, it
was found that peculiar smell increased significantly (P < 0.05), total
acceptance decreased significantly (P < 0.05), while meaty and
umami did not change significantly (P > 0.05). By comparing the
MRPs with oxidized chicken fat and UCF, the peculiar smell of the
chicken fat was significantly reduced (P < 0.05); while, the meat,
umami, and total acceptance were improved (P < 0.05). By comparing
the effects of different oxidized chicken fats on the meaty flavor of
MRPs, there were no significant difference (P > 0.05) in umami, pe-
culiar smell, and total acceptance between the MRPs supplemented
with free enzyme-treated chicken fat and with immobilized enzyme-
treated chicken fat. To illuminate the effect of the amount of added
oxidized chicken fat, as the amount in the reaction system increased
from 0.3 g to 0.5 g, the meaty, umami, and peculiar smell increased
significantly (P < 0.05); however, there was no significant change in
overall acceptability (P > 0.05). The results showed that oxidized
chicken fat was prepared by thermal and enzymatic treatment, and
Maillard reaction in the presence of oxidized chicken fat, did not cause
the difference in sensory evaluation of MRPs in general.
3.5. Aromatic components change derived from lipid oxidation and
degradation
Based on the flaxseed protein hydrolysates, the Maillard reactions
with NCF and UCF were used as controls to study the influence of
various oxidative methods on the generation of flavor components in
Maillard reaction. The volatile components produced by the reaction
system are mainly derived from three aspects: fat oxidative
5
Food Chemistry 306 (2020) 125560
degradation, Maillard reaction, and Maillard reaction with fat oxidation
degradation interaction (Yang et al., 2015).
The oxidation of fat in the air follows the free radical reaction pro-
cess. The fat is oxygenated to form a primary oxidation product (hy-
droperoxide), which is then degraded to produce volatile oxides such as
aldehydes, ketones, acids, esters, and hydrocarbons (Neff, Warner, &
Byrdwell, 2000). Generally, it is believed that aliphatic aldehydes, ke-
tones, alcohols, acids, and lactones are flavor substances produced by fat
oxidation. These volatile flavor ingredients have an outstanding con-
tribution to the characteristic flavor of different meats. However, many
hydrocarbons are produced during fat oxidation and degradation. These
volatile components have high odor activity values and little contribu-
tion to flavor which is not listed in Table 1. The present study could
detect many aliphatic compounds, including 26 volatile flavor com-
pounds released during lipid oxidation and degradation such as 13 al-
dehydes, 2 ketones, 7 alcohols, 1 acid, and 3 lactones from the MRPs
(Table 1). In terms of content and type, aldehydes and alcohols have the
highest content and the most diverse types, followed by ketones, acids,
and lactones. According to the previous studies on the volatile compo-
nents of oxidized chicken fat and oxidized chicken fat added to the
“cysteine-glucose” model reaction system, it can be concluded that these
compounds should be derived from the oxidative degradation of chicken
fat or the further reaction of oxidized chicken fat in the presence of
Maillard reaction (Wang, Yang, & Song, 2012).
As shown in Table 1, after the addition of UCF in the Maillard re-
action system, the content of the aldehydes, alcohols, ketones, acids,
and lactones was significantly (P < 0.05) higher as compared to the
one without chicken fat (126.0–839.5 ng/g, and 493.5–2332.4 ng/g of
C.-K. Wei, et al. Food Chemistry 306 (2020) 125560

Fig. 3. Effects of oxidation methods of chicken fat at 294 nm (A) and 420 nm (B) and on meaty (C), umami (D), peculiar smell (E), and total acceptance (F). NCF
indicated no chicken fat was added to the MRPs. UCF indicated addition of non-oxidized chicken fat 0.4 g to the MRPs. CF 0.3 g indicated adding 0.3 g oxidized
chicken fat to the MRPs. Others indicated the corresponding samples. The values followed by different letters were significantly different (P < 0.05).

aldehydes and alcohols); whereas, for TT 0.4 g, FET 0.4 g, and IET 0.4 g,
high significant (P < 0.05) difference was observed in these aromatic
components compared with UCF. This data was further confirmed in
the following Section 3.3 which demonstrates that the addition of
chicken fat could accelerate the reaction rate by producing more in-
termediate products such as aldehydes and alcohols in Maillard reac-
tion. On the other hand, the difference in content between TT 0.4 g, FET
0.4 g, and IET 0.4 g was more complicated.
Linoleic acid (C18:2) is one of the main fatty acids in chicken fat. As
shown in Fig. 4 A, linoleic acid oxidizes to form 9-OOOH and 13-OOOH
hydrogen peroxides, which further degrades to form aldehydes such as
(E,E)-2,4-decadienal, 2-octenal, and hexanal (Jayasena, Ahn, Nam, &
Jo, 2013). We identified 2-octenal (No. 8, Table 1) and hexanal (No. 3,
Table 1) in the reaction system. On the other hand, many unsaturated
aldehydes were found in the reaction system, which have high re-
activity and are prone to retro-aldol reaction to produce many kinds of
saturated aldehydes (Adams, Kitryte, Venskutonis, & De Kimpe, 2011).
Retro-aldol reaction of (E)-2-heptenal (No. 6, Table 1) was shown in
Fig. 4 B.
Aldehydes are very important flavor substances, which represent
one of the key factors causing different aroma to food, and serve as the
intermediates for continuation of the Maillard reaction. The smell of
short-chain aliphatic aldehydes is usually grass-like, while that of long-
chain aliphatic aldehydes is usually greasy. Among aldehydes, the
contents of saturated aldehydes such as hexanal, octanal, and nonanal
are higher, while the contents of unsaturated aldehydes such as (E)-2-
heptenal, (E)-2-octenal, and (E,E)-2,4-hexadienal are lower. Because the
unsaturated aldehyde has high reactivity and ease to participate in the
Maillard reaction. In the presence of Maillard reaction, unsaturated
aldehydes such as (E)-2-heptenal, (E)-2-octenal, and (E,E)-2,4-hex-
adienal can react with hydrogen sulfide or ammonia to form thio-
phenes, thiols or pyridines with alkyl side chains (Elmore et al., 2002).
In the reaction system, the content of alcohols derived from lipid oxi-
dation was also higher. In particular, Fan et al reported that 1-octene-3-

6
 Table 1
Flavor compounds identified from “chicken fat, xylose, cysteine, and flaxseed protein hydrolysates” thermal reaction products, which were generated from lipid oxidation and degradation or the Maillard reaction or the
interaction between the Maillard reaction and lipid oxidation and degradation.
1 3 4
C.-K. Wei, et al.

No. Flavor compounds KIs Amouts (ng/g) Odors Identification methods

2 2 2 2 2
WAX 5MS NCF UCF TT 0.4 g FET 0.4 g IET 0.4 g

Aldehydes 605.2 ± 44.2a 731.2 ± 69.5b 1319.1 ± 115.1c 1282.1 ± 105.8c 1444.7 ± 134.5c
1 3-methyl-butanal – – – – 52.7 ± 4.5 – 36.3 ± 2.7 fruity, green MS/O/S
2 Pentanal – 725 – – 33.5 ± 2.6a 35.8 ± 3.2a 58.7 ± 6.3b grass, pungent KI/O/S
3 Hexanal 1045 778 – 58.5 ± 4.3a 144.7 ± 15.1b 195.3 ± 20.6c 178.1 ± 19.3c grass, green MS/KI/O/S
4 Heptanal 1156 864 – – 36.8 ± 2.8 – 63.0 ± 5.2 fatty, green MS/KI/O/S
5 Octanal 1255 978 264.9 ± 21.2a 294.7 ± 26.0a 439.6 ± 36.6b 404.4 ± 32.3b 476.3 ± 38.7bc citrus, green MS/KI/O/S
6 (E)-2-heptenal 1325 946 – – – 33.6 ± 2.6 – fatty MS/KI/O/S
7 Nonanal 1367 1089 287.7 ± 18.6a 265.8 ± 28.7a 414.0 ± 35.4b 382.9 ± 25.5b 427.8 ± 44.0b green, fatty MS/KI/O/S
8 (E)-2-octenal 1406 1030 – – – 32.2 ± 3.0 – green, fatty MS/KI/O/S
9 Decanal 1471 1182 – – 36.4 ± 3.4 – 54.7 ± 4.9 green, fatty MS/KI/O/S
10 (E)-2-nonenal 1502 1127 – – 51.4 ± 4.6 35.8 ± 2.7 – green, fatty MS/KI/O/S
11 (E)-2-decenal 1598 1130 – 37.6 ± 2.8a 62.7 ± 5.3b 86.8 ± 9.7c 81.5 ± 7.6c fatty, tallowy MS/KI/O/S
12 2-dodecenal 1612 1215 52.6 ± 4.7a 42.5 ± 3.9b – 75.3 ± 6.5c – fatty, pungent MS/KI/O/S
13 (E,E)-2,4-hexadienal 1811 1352 – 32.1 ± 3.5a 47.3 ± 5.1b – 68.3 ± 5.8c green, fatty MS/KI/O/S

Alcohols 787.4 ± 80.7a 1280.9 ± 126.6b 2794.2 ± 266.1c 2924.4 ± 282.2 cd 3119.8 ± 293.7d
14 1-pentanol 1222 751 135.2 ± 15.2a 172.0 ± 16.1b 336.7 ± 28.7c 420.6 ± 38.3d 376.4 ± 43.0e green, fruity MS/KI/O/S
15 3-hexanol 1284 789 45.3 ± 3.7a 57.4 ± 6.3b – 56.8 ± 6.8b 71.3 ± 5.2c green, fruity MS/KI/O
16 4-methyl-2-pentanol – 804 37.8 ± 2.5a 45.3 ± 5.1b 73.1 ± 6.9c 33.8 ± 4.2a 57.2 ± 6.1b green, fruity KI/O/S
17 1-hexanol 1336 850 68.9 ± 7.4a 246.1 ± 27.0b 351.0 ± 33.2c 327.5 ± 36.6c 432.7 ± 42.8d green, fruity MS/KI/O/S
18 1-octen-3-ol 1427 955 150.5 ± 16.7a 224.4 ± 17.4b 417.3 ± 39.4c 486.6 ± 41.3d 457.1 ± 38.7 cd mushroom MS/KI/O/S
19 1-heptanol 1459 957 166.7 ± 14.2a 289.5 ± 32.4b 772.6 ± 81.8c 862.9 ± 74.8 cd 934.6 ± 102.1d green, fruity MS/KI/O

7
20 1-octanol 1566 1034 183.0 ± 21.3a 246.2 ± 22.3b 843.5 ± 76.3c 736.2 ± 80.2c 790.5 ± 55.8c fatty, citrus MS/KI/O

Ketones 0 96.9 ± 6.9a 259.3 ± 18.2b 319.6 ± 25.9c 290.6 ± 19.6d

21 2-heptanone 1148 877 – 45.2 ± 2.7a 137.4 ± 7.9b 168.3 ± 12.7c 157.2 ± 11.2c green, sweet MS/KI/O/S
22 2-nonanone 1353 1145 – 51.7 ± 4.2a 121.9 ± 10.3b 151.3 ± 13.2c 133.4 ± 8.4b fruity, fatty MS/KI/O/S

Acids 96.4 ± 7.2a 176.7 ± 20.1b 251.0 ± 17.4c 222.3 ± 21.6c 275.1 ± 26.3 cd
23 hexanoic acid 1816 974 96.4 ± 7.2a 176.7 ± 20.1b 251.0 ± 17.4c 222.3 ± 21.6c 275.1 ± 26.3 cd sweat, pungent MS/KI/O

Lactones 0 97.3 ± 9.1a 205.4 ± 18.2b 249.0 ± 25.6c 203.0 ± 15.3b

24 γ-hexalactone 1653 1150 – 37.9 ± 2.3a 112.3 ± 9.4b 135.4 ± 14.5c 96.2 ± 7.7b sweet MS/KI/O/S
25 γ-heptalactone 1760 1235 – 33.3 ± 3.8a 54.5 ± 6.2b 68.3 ± 7.2c 74.4 ± 5.2c sweet MS/KI/O
26 γ-nonalactone 1986 1367 – 26.1 ± 3.0a 38.6 ± 2.7c 45.3 ± 4.1d 32.4 ± 2.4ab sweet, coconut MS/KI/O/S

Thiols 1665.4 ± 141.2a 794.6 ± 66.8b 431.5 ± 37.8c 493.0 ± 36.7d 502.3 ± 46.1d
27 1-pentanethiol 1024 845 241.3 ± 15.5a 163.6 ± 11.4b 45.8 ± 3.5c 54.1 ± 3.3c 52.7 ± 6.2c garlic MS/KI/O
28 1-hexanethiol 1114 911 207.4 ± 16.3a 144.2 ± 9.2b 97.2 ± 10.8c 139.4 ± 9.5b 111.3 ± 8.7d garlic MS/KI/O
29 1-heptanethiol 1275 856 167.3 ± 9.7a 89.2 ± 6.7b 31.3 ± 2.4c 45.1 ± 3.1d 37.5 ± 2.3c garlic MS/KI/O
30 2-furfuraylthiol 1407 910 212.6 ± 23.6a 125.7 ± 13.8b 74.5 ± 5.4c 97.6 ± 6.5d 105.5 ± 8.6d meaty MS/KI/O/S
31 3-thiophenethiol 1548 947 836.8 ± 76.4a 271.9 ± 25.7b 182.7 ± 15.7c 156.8 ± 14.3 cd 195.3 ± 20.4c meaty MS/KI/O/S

Thiophenes 5680.1 ± 476.2a 4555.1 ± 393.1b 2954.7 ± 279.5c 2799.8 ± 246.7c 2976.9 ± 244.5c
32 2-methylthiophene 1076 766 345.5 ± 26.0a 277.1 ± 30.6b 161.8 ± 11.6c 207.4 ± 22.5d 187.9 ± 15.3 cd sulfury MS/KI/O/S
33 3-methylthiophene – 780 42.7 ± 4.6 – – – – sulfury KI/O/S
34 2,5-dimethylthiophene 1133 886 36.0 ± 5.7 – – – – garlic, sulfury MS/KI/O/S
35 2-ethylthiophene 1143 865 68.4 ± 4.3 38.8 ± 2.9 – – – garlic MS/KI/O/S
36 2-butylthiophene 1334 1067 – 26.7 ± 3.2a 21.1 ± 3.5b 38.4 ± 4.3c 20.7 ± 4.0b meaty MS/KI/O
37 2-pentylthiophene 1437 1168 – – – 23.4 ± 1.7 – meaty MS/KI/O/S
38 2-heptylthiophene 1644 1369 58.2 ± 5.5 41.3 ± 2.7 – – – meaty MS/KI/O
(continued on next page)
Food Chemistry 306 (2020) 125560
 Table 1 (continued)
1 3 4
No. Flavor compounds KIs Amouts (ng/g) Odors Identification methods

2 2 2 2 2
C.-K. Wei, et al.

WAX 5MS NCF UCF TT 0.4 g FET 0.4 g IET 0.4 g

39 2-thiophenecarboxaldehyde 1738 1084 544.3 ± 78.2a 416.5 ± 29.5b 382.2 ± 42.3bc 378.7 ± 32.8bc 428.3 ± 36.7b burnt MS/KI/O
40 5-methyl-2-thiophenecarboxaldehyde 1751 1131 1895.2 ± 127.4a 1527.4 ± 136.8b 873.1 ± 73.8c 1009.2 ± 84.9c 950.1 ± 73.2c burnt MS/KI/O/S
41 3-methyl-2-thiophenecarboxaldehyde 1779 1136 2689.8 ± 224.4a 2227.3 ± 187.4b 1516.5 ± 148.4c 1142.7 ± 100.5d 1389.9 ± 115.3c burnt MS/KI/O/S

Thiazoles 212.3 ± 16.9a 127.5 ± 9.7b 68.4 ± 7.3c 45.2 ± 2.8d 70.8 ± 6.6c
42 2-acetylthiazole 1621 1007 212.3 ± 16.9a 127.5 ± 9.7b 68.4 ± 7.3c 45.2 ± 2.8d 70.8 ± 6.6c rice MS/KI/O/S

Nitrogen-containing heterocyclics 749.8 ± 69.9a 1258.1 ± 106.2b 1910.3 ± 171.3c 1824.3 ± 160.5c 1859.8 ± 186.5c
43 2-methylpyrazine 1252 808 427.8 ± 37.1a 233.1 ± 25.6b 252.8 ± 13.2b 210.6 ± 23.5c 263.5 ± 19.7b roasted, nutty MS/KI/O/S
44 2-pentylpyridine 1267 904 67.5 ± 5.2a 312.4 ± 17.9b 426.9 ± 57.1c 467.0 ± 40.3c 441.1 ± 52.2c burnt, fatty MS/KI/O/S
45 2,5-dimethylpyrazine 1301 908 56.2 ± 6.8a 45.7 ± 3.2b 49.8 ± 6.7ab 42.4 ± 2.5b 46.3 ± 3.6b roasted, popcorn MS/KI/O/S
46 2-hexylpyridine 1564 962 92.7 ± 11.2a 177.4 ± 16.4b 556.5 ± 42.9c 527.8 ± 48.7c 497.4 ± 38.9c nutty MS/KI/O
47 2-acetylpyrrole 1945 1041 105.6 ± 9.6a 489.5 ± 43.1b 624.3 ± 51.4c 576.5 ± 45.5c 611.5 ± 72.1c licorice, nutty MS/KI/O/S

Oxygen-containing heterocyclics 4981.0 ± 457.3a 5425.3 ± 478.8ab 9488.3 ± 810.7c 9113.2 ± 865.5c 9265.1 ± 708.4c
48 2-pentylfuran 1208 967 396.4 ± 31.2a 487.1 ± 38.6b 675.7 ± 72.3c 522.5 ± 60.9b 638.4 ± 53.2c earthy, green MS/KI/O/S
49 Furfural 1440 842 1587.5 ± 166.1a 1897.8 ± 204.5b 3276.4 ± 269.5c 2933.1 ± 320.4c 3102.9 ± 186.8c caramel MS/KI/O/S
50 2-acetylfuran 1477 876 1656.8 ± 134.7a 1388.3 ± 95.7b 2673.5 ± 220.8c 2791.4 ± 239.7c 2534.7 ± 214.4c sweet MS/KI/O/S
51 5-methyl-2-furancarboxaldehyde 1545 959 552.3 ± 45.8a 634.2 ± 56.1b 1476.6 ± 113.3c 1236.5 ± 95.4d 1515.2 ± 130.3c caramel MS/KI/O
52 2-furanmethanol 1648 950 274.9 ± 17.3a 380.5 ± 28.0b 523.8 ± 41.6c 607.6 ± 53.1d 577.3 ± 47.2d sweet MS/KI/O
53 5-methyl-2-furanmethanol 1689 966 513.1 ± 62.2a 637.4 ± 55.9b 862.3 ± 93.3c 1022.4 ± 96.0d 896.7 ± 76.5c sweet MS/KI/O

1
KIs, Kovats indices determined using the n-alkanes C7-C30 on DB-Wax and DB-5MS column (30 m × 0.25 mm × 0.25 μm) in the GC–MS and GC-O analysis.
2
NCF indicated MRPs without chicken fat. UCF indicated adding untreated chicken fat 0.4 g to the MRPs. TT 0.4 g indicated adding oxidized chicken fat 0.4 g by thermal treatment to the MRPs. FET 0.4 g indicated
adding oxidized chicken fat 0.4 g by free enzyme treatment to the MRPs. IET 0.4 g indicated adding oxidized chicken fat 0.4 g by immobilized enzyme treatment to the MRPs. Means within different letters are significantly

8
(P < 0.05) different in the same line. “–”, not detected.
3
Odor detected by the panelists in GC-O analysis using the DB-5MS column.
4
KI, identified by Kovats indices (KI); MS, identified by search of mass spectra in the NIST 08 database and manual interpretation; O, identified by odor characteristics; and S, identified by comparison of the
abovementioned analytical parameters with the authentic chemicals injected.
Food Chemistry 306 (2020) 125560
C.-K. Wei, et al. Food Chemistry 306 (2020) 125560

Fig. 4. Possible formation pathways. (A) linoleic acid (C18:2) oxidation and degradation. (B) Formation of hexanal, (E)-2-octenal, and (E,E)-2,4-decadienal from (E)-2-
Heptenal via hydration and retro-aldol reaction. (C) Strecker degradation of amino acids and the production of H2S, NH3, and CH3CHO from cysteine. (D) Alkyl
thiols, (E) thiophenethiols, (F) alkyl thiophenes, and (G) thiophenecarboxaldehydes. -R represents an alkyl group (–CnH2n+1, n = 1–7). ARP represents Amadori
rearrangement reaction. RA represents retro-aldol reaction.

9
C.-K. Wei, et al.
alcohol was one of the sources of characteristic flavor of chicken soup
(Fan et al., 2019). Aldehydes in oxidized fats can be reduced to alcohols
due to the influence of reduced Maillard reaction products. Hydroper-
oxide of glycerides produced in oxidized fats can also change the de-
gradation pathway to produce reducing alcohols. The main source of
aldehydes is Strecker degradation and its reverse aldehyde reaction
produces unsaturated aldehydes for NCF as shown in Fig. 4C (Mottram,
1998). Therefore, it can be seen from Table 1 that aldehydes are pro-
duced in both types and quantities: oxidized chicken fat > UCF >
NCF. And no matter how the chicken fat is prepared, it has strong re-
action activity in Maillard reaction system in order to produce more
aldehydes and other flavor substances.
3.6. Aromatic components derived from the Maillard reaction or the
interaction between the Maillard reaction, lipid oxidation, and degradation
Aromatic components resulting from the interaction of Maillard
reaction or Maillard reaction with fat oxidative degradation comprised
in Table 1. The main compounds with high content in these flavor
mixes were 5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophe-
necarboxaldehyde, furfural, 2-acetylfuran, 5-methyl-2-furancarbox-
aldehyde, and 5-methyl-2-furanmethanol. There were more sulphur-
containing compounds (5 thiols, 10 thiophenes, and 1 thiazoles) than
nitrogen-containing and oxygen-containing heterocycles. However, it
can be seen from Table 1 in terms of content, the following trend was
observed: oxygen-containing heterocycles > sulphur-containing com-
pounds > nitrogen-containing heterocycles.
As shown in Table 1, after the addition of UCF in the Maillard re-
action system, the contents of the nitrogen-containing and oxygen-
containing heterocycles were significantly (P < 0.05) high as com-
pared to the ones without chicken fat; whereas, for TT 0.4 g, FET 0.4 g,
and IET 0.4 g, high significant (P < 0.05) difference was observed in
these aromatic components compared with UCF. On the other hand, the
content of the thiols, thiophenes, and thiazoles were significantly
(P < 0.05) lower as compared to the ones without chicken fat
(870.8–1233.9 ng/g and 1125.0–2880.3 ng/g of thiols and thiophenes);
while, for TT 0.4 g, FET 0.4 g, and IET 0.4 g, slight significant
(P < 0.05) difference was observed in these aromatic components
compared with UCF. Similarly, the difference in the content between TT
0.4 g, FET 0.4 g, and IET 0.4 g was more complicated. Therefore, fat
oxidative degradation products inhibited the formation of thiols, thio-
phenes, and thiazoles. On the other hand, fat oxidative degradation and
Maillard reaction interact to generate some new heterocyclic com-
pounds with alkyl chains or elevated levels of the original heterocyclic
compounds in the reaction system with oxidized fat.
Oxygen-containing heterocyclics are important intermediate pro-
ducts of Maillard reaction, mainly from the degradation of sugar, which
can be detected in many Maillard reaction systems (Yu, Tan, & Wang,
2012). However, many studies have reported that although the content
of oxygen-containing heterocycles is high, they generally have high
odor thresholds (Cao et al., 2017; Lee, Jo, & Kim, 2010). This results in
the fact that oxygen-containing heterocycles have little effect on the
flavor of Maillard reaction products.
Sulphur-containing compounds play an important role in meat
flavor production during Maillard reaction, and these compounds
generally have low odor thresholds. The main sulphur-containing
compounds identified in the reaction system were thiophenes, the
highest content of which was 2-methylthiophene, 2-thiophenecarbox-
aldehyde, 5-methyl-2-thiophenecarboxaldehyde and 3-methyl-2-thio-
phenecarboxaldehyde. Previously, thiophenes such as 2-methylthio-
phene, 5-methyl-2-thiophenecarboxaldehyde, and 3-methyl-2-
thiophenecarboxaldehyde have been detected in the “glutathione-glu-
cose” system (Lee et al., 2010; Wang et al., 2012). Therefore, it was
10
Food Chemistry 306 (2020) 125560
assumed that the main source of thiophenes in the system was the
Maillard reaction. Thiols are lower than thiophenes in content and
species, but many reports indicate that thiophenes have lower odor
thresholds. For instance, previous studies on chicken soup found that
some thiols such as methanethiol, 2-methyl-3-furylthiol, 2-furylthiol
are the key aroma components (Fan et al., 2019). It is worth noting that
the identified thiol and thiophene compounds with alkyl chains such as
1-heptanethiol and 2-heptylthiophene are rarely found in the literature
report on the ‘peptide/amino acid-reducing sugar’ model reaction. So,
these compounds may be formed after lipid oxidation degradation
products take part in Maillard reaction. As shown in Fig. 4C, Strecker
degradation in the presence of cysteine produces H2S and NH3
(Jayasena et al., 2013; Mottram, 1998). The oxidation and degradation
of fatty acids and the retro-aldol reaction of their products are prone to
produce large amounts of aliphatic aldehydes. These aliphatic alde-
hydes occur in the presence of H2S as shown in Fig. 4 D to form a
variety of alkyl thiols (No. 27–29, Table 1) (Zhao, Wang, Xie, Xiao,
Chenet al., 2019, Zhao, Wang, Xie, Xiao, Du et al., 2019). The formation
pathway of 3-thiophenethiol (No. 31, Table 1) was shown in Fig. 4E
(Mottram, 1998; Zhao, Wang, Xie, Xiao, Chenet al., 2019). On the other
hand, unsaturated aldehydes react with H2S, then cyclize and rearrange
to form alkyl thiophenes (No. 32–33, 35–38, Table 1) (Fig. 4F) (Lee
et al., 2010; Yu et al., 2012). In addition, 2-thiophenecarboxaldehyde
(No. 39, Table 1) with high content were identified in the products, and
the formation pathway was shown in Fig. 4G (Lee et al., 2010; Zhao,
Wang, Xie, Xiao, Du et al., 2019).
Five nitrogen-containing heterocycles (2 pyridines, 1 pyrrole and 2
pyrazines) were detected, of which 2-pentylpyridine and 2-hexylpyr-
idine belong to alkyl pyridines. Pyrazines is usually produced at high
temperatures and contributes to roast and nutty aroma (Zou, Liu, &
Song, 2018). It is also an important aroma component in roast meat.
Like the formation of alkylthiophenes, 2-pentylpyridine and 2-hex-
ylpyridine were mainly derived from the products of lipid oxidation and
degradation, which participated in Maillard reaction. After analyzing
flavor compounds, a proposed flow diagram involving aromatic effects
of oxidation of chicken fat on flaxseed derived Maillard reaction pro-
ducts mechanisms is presented in Fig. 5. The meaty components shown
in Fig. 5 were all sulfur-containing flavors, while these sulfur elements
were derived from H2S produced by Strecker degradation of cysteine.
On the other hand, dicarbonyl compounds that catalyze Strecker de-
gradation were produced during Maillard reaction. Oxidation and de-
gradation of chicken fat, degradation of cysteine, and formation of ty-
pical meaty components were discussed in previous detail.
4. Conclusion
To summarize, the oxidized chicken fats were prepared by thermal,
free LOX, and immobilized LOX treatments. > 50% activity of im-
mobilized LOX was maintained after subsequent use and the immobilized
enzymes displayed higher functionalities on immobilized LOX properties.
Chicken fat or oxidized chicken fat could react with both the amino
group-containing precursor in the flaxseed protein hydrolysates as well
as with cysteine and reducing sugar. After the addition of chicken fats to
the reaction system, the Maillard reaction was noticeably enhanced
producing more aliphatic aldehydes and alcohols, especially with the
addition of oxidized chicken fat. From the sensory and flavors evaluation,
the addition of immobilized LOX and free LOX oxidized chicken fat had
shown no obvious differences in the MRPs, which may be related to the
degree of oxidation of three kinds of chicken fat. Chicken fat or oxidized
chicken fat could react with both the amino group-containing precursor
in the flaxseed protein hydrolysates and with cysteine and reducing
sugar. This reaction has resulted in the increased amount of flavor in-
gredients in the system, but the content of sulfur compounds such as
C.-K. Wei, et al. Food Chemistry 306 (2020) 125560

Fig. 5. Proposed mechanism for aromatic effects of oxidation of chicken fat on flaxseed derived Maillard reaction products. FPH represents flaxseed protein hy-
drolysates. RA represents retro-aldol reaction. SD represents Strecker degradation. AR represents Amadori rearrangement.

thiols and thiophenes decreased significantly, and the structure of sulfur
compounds was likely to form alkyl side chains. This study proposed the
mechanism for flavor effects of oxidation of chicken fat on flaxseed de-
rived MRPs. Our results concluded that the MRPs imparted a meaty and
the characteristic flavor to the chicken.
Technology in Anhui Province (17030701058, 18030701158, 1703070-
1024, and 17030701028), and Zhongyuan Scholars in Henan Province
(192101510004).
Declaration of Competing Interest

Funding The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
This study was supported by the Major Projects of Science and ence the work reported in this paper.

11
C.-K. Wei, et al.
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