Journal of Cereal Science 111 (2023) 103689

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

Effects of transglutaminase and glucose oxidase on the properties of frozen

dough: Water distribution, rheological properties, and microstructure
Wen-Tao Guo a, Xue-Fei Yang a, Yi-Shun Ji b, Bin Hu b, Wan-Guang Li b, Xi-Yang Zhong a,
Shao-Tong Jiang a, Zhi Zheng a, *
a
School of Food and Biological Engineering, Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei University of Technology, Hefei, 230601,
China
b
China National Supervision and Examination Center for Foodstuff Quality, Hefei, 230031, China

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

Keywords: The effects of transglutaminase (TG) and glucose oxidase (GOX) on the water distribution, rheological properties,
Frozen dough and microstructure of dough after freeze-thaw cycles were studied. The results showed that, when TG (1%, w/w)
Freeze-thaw cycle and GOX (0.006%, w/w) were added to the dough simultaneously, the syneresis rate of the dough after 8 freeze-
Transglutaminase
thaw cycles reduced by 12.9%, and the extensibility and maximum resistance of the frozen dough increased by
Glucose oxidase
9.6 mm and 14.7 g, respectively. Moreover, the G′ and G′′ values of the frozen dough were significantly
increased, and the hardness and freezable water content were reduced by 60.1% and 2.9%, respectively. The LF-
NMR analysis showed that the tightly bound water content increased by 4.9%, and the semi-bound water content
decreased by 4.0%. The number of free sulfhydryl groups in the dough treated with the two enzymes decreased
by 16.3 μmol/g. Fourier IR revealed that the content of α-helix increased by 3.1%, β-sheet increased by 5.8%, and
β-turn decreased by 4.8%. Scanning electron microscope images indicated that the gluten network of the two
enzyme-treated dough remained uniform and dense after multiple freeze-thaw disruptions.

1. Introduction
Although dietary patterns vary worldwide, flour-based products are
still a staple food for most people. Over the past decades, the technique
of freezing dough has been widely used as a cost-saving, quality-regu­
lated technology for processing and shipping flour products (Luo et al.,
2018). However, some studies have shown that during long-term frozen
storage, uneven water redistribution and ice crystal formation, resulting
in damage to the dough structure. At the same time, starch is dislodged
from the gluten network, its ability to retain air and hold water is
reduced, thus leading to product deterioration (Li et al., 2019).
To solve these problems, some researchers have found that the
quality of frozen dough can be increased by using frozen dough im­
provers. Common frozen dough improvers include thickeners and
emulsifiers (Fu et al., 2021). Thickeners can limit the movement of free
water, and maintain the structural stability of the dough with low
temperature condition (He et al., 2020). Yu et al. (2020) revealed that
pigskin gelatin can limit the water migration of frozen dough, and
control the growth of ice crystals, which can result in larger and softer
bread. Emulsifiers can form a film between gluten proteins, starch, and
water to maintain the low temperature stability of the dough (Kang
et al., 2021). Niu et al. (2017) found that soy lecithin and sodium
stearoyl lactate can interact with gluten proteins to increase the ductility
of frozen dough, and make bread softer.
However, both thickeners and emulsifiers have drawbacks, such as
expensive and difficult preparation. It has been suggested that enzymes
can be used to delay the deterioration of frozen dough on the grounds
that enzymes are eco-friendly, healthier, and cheaper than other addi­
tives (Asghar et al., 2011; Liang et al., 2021). Struyf et al. (2017) sug­
gested that α-amylase increased the volume of frozen dough by
increasing the content of maltose in the dough, and promoting yeast
fermentation capacity. Xylanase can reduce the freezable water content
of the dough during frozen storage, and preserve the quality of the
frozen dough by making the ice crystals formed inside the frozen dough
fine and uniform (Kim and Yoo, 2020). Transaminase (TG) can promote
the intermolecular or intramolecular crosslinking of glutamine and
lysine residues to form high-molecular-weight gluten (Niu et al., 2018).
One study concluded that TG can strengthen the quality of whole wheat

* Corresponding author.
E-mail address: zhengzhi@hfut.edu.cn (Z. Zheng).

https://doi.org/10.1016/j.jcs.2023.103689
Received 10 February 2023; Received in revised form 25 April 2023; Accepted 27 April 2023
Available online 28 April 2023
0733-5210/© 2023 Elsevier Ltd. All rights reserved.
W.-T. Guo et al.
dough by improving its protein network during frozen storage, making
the frozen dough products less firm (Kang et al., 2021). The ratio of
high-molecular-weight gluten subunits was dramatically increased by
TG treatment in another investigation, and the decrease in viscoelas­
ticity that causes frozen dough was slowed (Tang et al., 2016). Glucose
oxidase (GOX) maintains frozen dough structure by oxidizing free sulf­
hydryl groups to disulfide bonds in dough, and results in a similar effect
to TG on the structure of frozen dough. Xiao et al. (2021) showed that
the oxidation of gluten protein by GOX could counteract the depoly­
merization effect of gluten due to the formation of ice crystals during
frozen storage, and result in larger volume of bread. However, the
combined addition of TG and GOX may increase the overall quality of
the frozen dough during multiple freeze-thaw cycles has not been
reported.
Therefore, we investigated the changes in the water distribution,
rheological properties, and microstructure of the frozen dough with the
combined addition of TG and GOX, after multiple freeze-thaw cycles.
The purpose of this research was to offer new perspectives for the cre­
ation of frozen dough improvers that are healthier, more efficient, and
cheaper.
2. Materials and methods
2.1. Materials
Huaimai 22 wheat (11.8% protein, 13.5% moisture, and 0.4% ash)
was obtained from Linquan County Jinhe Flour Co., Ltd. (Fuyang,
China). Yeast was purchased from Anqi Yeast Co. Ltd. (Yichang, China).
Transglutaminase (EC 2.3.2.13, 100 U/g, lyophilized powder) was
purchased from Beijing Solarbio Technology Co., Ltd. (Beijing, China),
and glucose oxidase (EC 1.1.3.4, 10,000 U/g, lyophilized powder) was
bought from Shanghai Yuanye Biological Technology Co., Ltd.
(Shanghai, China); both of these products were of analytical grade. The
amounts of TG and GOX that were added during the experiments were
obtained from experimental results. All the other chemicals were also of
analytical grade.
2.2. Preparation of the frozen dough
The basic dough formulation consisted of 100 g flour, 2 g yeast, and
50 g water. Enzyme-containing dough that contained 1% TG, 0.006%
GOX, or both 1% TG and 0.006% GOX (w/w, based on flour) were
named TG, GOX, and GT, respectively. The dough used as a control,
known as CON, contained no enzymes.
A dough mixer (Bear Electric Appliance, Foshan, China) was used to
combine the weighed components for 15 min. The resulting dough was
relaxed at 37 ◦ C for 20 min, after which it was placed in a polyethylene
bag. The freeze-thaw cycle treatment involved freezing the dough for 2 h
in a − 38 ◦ C refrigerator (BC-102SFA, Aucma, Qingdao, China), stabi­
lizing it for 24 h in a − 18 ◦ C refrigerator, thawing it for 2 h at 30 ◦ C, and
then returning to a − 38 ◦ C refrigerator again for 2 h, and the process was
carried out eight times.
2.3. Freeze-thaw stability
The freeze-thaw stability of dough is characterized by the syneresis
rate. For this study, we referred to the method presented by Xu with
appropriate modifications (Xu et al., 2009). A mixture of 3 g of dough
and 47 mL of water was placed in test tube, boiled in a water bath kettle
for 20 min, quickly cooled down to 25 ◦ C, poured into an empty test
tube, weighed, and kept at − 18 ◦ C for 24 h. Through 24 h, the samples
were thawed at 30 ◦ C for 5 h. The test tube was retained in a centrifuge
(KDC-160HR, ZONKIA, Hefei, China) at 7104 g for 20 min; data were
recorded from 0, 4, and 8 freeze-thaw cycles and calculated according to
the following equation:
2
Journal of Cereal Science 111 (2023) 103689
Gn − Gn+1
syneresis rate(%) = ∗ 100% (1)
Gn − G0
Weights of G0 and Gn represent the weights of the test tube empty,
the sample and test tube empty, respectively, and Gn+1 is the weight of
the test tube and the sample after centrifugation.
2.4. Tensile properties
The tensile property of the frozen dough was determined using an XT
Plus texture analyzer (Stable Micro Systems, Godalming Surrey, UK),
according to method of (Zhou et al., 2019). The dough (5.0 g) was
pressed into strips (3.0 cm long, 0.5 cm wide, and 0.5 cm high), and an
A/KIE adapter was connected to the texture analyzer.
2.5. Rheological properties
Using an AR1000 rheometer (TA Instruments, New Castle, USA), the
frozen dough’s rheological properties were tested, in accordance with
the procedure outlined by (Niu et al., 2018), which was modified
appropriately. A parallel plate (40 mm in diameter, with a 2 mm gap)
was selected as the test probe. The center of the parallel plate was filled
with dough (1–2 g), and a strain scan test was run to identify the dough
sample’s linear viscoelastic zone. Following the strain sweep readings,
the target strain was adjusted at 0.5%. The elastic modulus (G′ ), viscous
modulus (G′′ ), and tan δ (G"/G′ ) value were then measured as a function
of frequency during a frequency scan test that was conducted between
0.1 and 10.0 Hz.
2.6. Texture profile analysis
The textural characteristics of the frozen dough during 0–8 freeze-
thaw cycles were investigated using a texture analyzer with a P/36R
probe attached. Test conditions: trigger force 10.0 g, strain 50%, pre-test
speed 1.0 mm/s, test speed 5.0 mm/s, post-test speed 5.0 mm/s, and
interval time 5.0 s.
2.7. Determination of the freezable water content
The freezable water content of the dough was measured using dif­
ferential scanning calorimetry (DSC, TA, New Castle, USA). Appropriate
modifications were made according to (Liu et al., 2018). The central
piece of the frozen dough (10–20 mg) was enclosed in an aluminum
crucible and allowed to defrost at room temperature prior to testing.
Test conditions: sealed aluminum crucible, 25 ◦ C start temperature,
10 ◦ C/min temperature decline to − 40 ◦ C, 5 min of steady temperature,
then 10 ◦ C/min temperature increase to 20 ◦ C.
2.8. Water mobility
The water distribution was determined using low-field nuclear
magnetic resonance (LF-NMR, Niumag Corp., Shanghai, China), and the
method of (Xin et al., 2018) was referenced and modified as appropriate.
The frozen dough was taken out of the freezer and allowed to thaw at
room temperature before its center (2 g) was put in a sample vial and
connected to the NMR probe. Using the CPMG method, the transverse
relaxation time (T2) was measured. Test conditions: SW 200 kHz, SF 18
MHz, RFD 0.080 ms, RG1 20.0 db, DRG 13, PRG 1, TW 5000 ms, NS 8,
TE 0.20 ms, and NECH 15000.
2.9. Determination of free sulfhydryl(-SH) groups and secondary
structure
The –SH content of the frozen dough was measured using Ellman’s
reagent, as described by (Lu et al., 2021). To determine the solution’s
–SH concentration, the absorbance was measured at 412 nm, using the
W.-T. Guo et al.
following equation:
− SH(μmol / g) = (75.53 × A412 ) / C)
The protein content is C, and the absorbance at 412 nm is A412.
The secondary structure of the proteins in the dough was examined
using Fourier transform infrared spectroscopy (Nicolet 6700, Thermo
Nicolet, Waltham, USA) at a resolution of 4 cm− 1. The freeze-dried
dough was milled, combined with KBr, crushed, and formed into
flakes before being scanned between 500 and 4000 cm− 1.
2.10. SEM
The freeze-dried dough was cut into pieces, put on a test table, and
exposed to gold spray for 1 min. Using a scanning electron microscope
(EM-30, COXEM Co., Ltd., Daejeon, Korea) with an electron gun accel­
eration voltage of 7 kV, the cross-sections of the dough were examined.
Images were captured at a 1000 × magnification.
2.11. Statistical analysis
At least three runs of each experiment were performed. One-way
ANOVA was used for the statistical analysis with SPSS 26.0. Using
Duncan’s test, and P ＜0.05 was regarded as significant.
3. Results and discussion
3.1. Freeze-thaw stability
The syneresis rate of the enzymatically treated dough, at different
freeze-thaw cycles, is displayed in Fig. 1A. As the freeze-thaw cycles
proceeds, the syneresis rate of the dough also increased, continuously.
After eight freeze-thaw cycles, the syneresis rate of the TG dough
increased from 13.0% to 23.1%, that of the GOX dough increased from
12.8% to 20.1%, and that of the GT dough increased from 9.8% to
15.9%, all these were less than the syneresis rate of the CON dough. This
reason might be due to the freeze-thaw cycles that destroyed the
structure of the dough and reduced the water retention capacity of the
gluten protein network (Wang et al., 2022). Therefore, the simultaneous
addition of TG and GOX helped maintain the moisture content of the
frozen dough.
3.2. Tensile properties
The tensile properties of the enzyme-treated dough at different
numbers of freeze-thaw cycles are presented in Fig. 1B and C. The
extensibility decreased throughout during the freeze-thaw cycles
(Fig. 1B). Compared to the CON group, when eight freeze-thaw cycles
Fig. 1. Effects of the enzyme treatments on the syneresis rate and tensile properties of dough, during the freeze-thaw cycles. A: syneresis rate; B: extensibility; C:
resistance.
CON represents the control group, and TG, GOX, and GT represent the addition of the different enzymes. To denote a statistically significant difference (P < 0.05),
samples with the same freezing procedure are labeled with various lowercase letters.
Journal of Cereal Science 111 (2023) 103689
were reached, the TG dough’s extensibility decreased from 30.3 mm to
23.6 mm, that of the GOX dough decreased from 31.3 mm to 25.7 mm,
(2)
and that of the GT dough decreased from 32.9 mm to 28.3 mm, with the
least change in extensibility observed in the GT dough. Meanwhile, the
maximum resistance also tended to decrease (Fig. 1C). The maximum
resistance of the TG, GOX, and GT groups decreased by 20.7 g, 22.5 g,
and 17.8 g, respectively. The results indicated that the freeze-thaw cy­
cles reduced the dough’s strength, which was caused by the deteriora­
tion of the dough internal structure gluten network, however, TG and
GOX can strengthen the gluten protein network and reduce the amount
of damage attributed to the freeze-thaw cycles, and adding TG and GOX
at the same time has a higher effect. In a few more investigations, similar
results were attained (Adams et al., 2017).
3.3. Rheological property
The rheological properties of the dough containing different enzymes
at 0, 4, and 8 freeze-thaw cycles are shown in Fig. 2. The rheological
results of the all samples indicated that G′ was higher than G′′ in the
range class of 0–10.0 Hz. Meantime, the values of G′ and G′′ showed an
increasing trend in this frequency range. The dough G′ values were
higher than their G′′ values, demonstrating that their solid properties
were superior than liquid properties (Jia et al., 2019). The G′ and G′′ of
the GT dough were higher than those of the other groups at the same
frequency and number of freeze-thaw cycles. The ice crystals produced
as a result of freezing affect the polymerization of the starch and pro­
teins, and disrupted the internal network structure of the dough (Huang
et al., 2022), whereas the addition of the enzymes strengthens the gluten
protein formation network, which is not as easily disrupted by the ice
crystals produced as a result of freezing. Therefore, the addition of en­
zymes can mitigate the deterioration of frozen dough. In addition, when
TG and GOX were added in combination to the dough, gluten protein
cross-linking was promoted to resist the break attributed to the dough
recrystallization. Therefore, the simultaneous addition of the two en­
zymes leads to a higher protective influence on the frozen dough
viscoelasticity.
Tan δ represents the processing quality of the dough; a lower tan δ
indicates that the dough has higher plasticity. The tan δ of the GT group
was lower than that of the other three groups following the same number
of freeze-thaw cycles, and the tan δ of the CON group was the highest.
This indicated that the dough treated with enzymes had stronger plas­
ticity, and the dough after freezing had a higher quality; this also reflects
the higher viscoelastic protection of dough when TG and GOX were
added together. A decrease in the tan δ value as a result of TG addition to
the refined dough was also reported. Kim et al. (2020) found that both
the G′′ and G′ of dough decreased with increasing refrigeration time,
while the tan δ value increased with increasing refrigeration time, and
3
W.-T. Guo et al.
Fig. 2. Effects of enzyme treatment during freeze-thaw cycles on the rheological properties of dough.
Elastic modulus G′ (A-C), viscous modulus G′ (D-F), and tan δ (G-I). CON represents the control group and TG, GOX, and GT represent the addition of
different enzymes.
the TG and GOX significantly strengthened the rheological properties of
the dough, similar to our results. The addition of the enzymes can
significantly reduce dough’s tan δ, and the dough can still maintain a
relatively low tan δ after the freeze-thaw cycles, suggesting that the
enzymes could reduce the freezing damage dough it suffered.
3.4. Texture profile analysis
Table 1 shows the textural changes in the dough after enzyme
treatment and a different number of freeze-thaw cycles. Compared to the
CON group, the hardness of TG, GOX, and GT decreased by 11.6%, 9.6%,
and 26.1%, at 0 freeze-thaw cycles, respectively. After 8 freeze-thaw
cycles, the hardness of CON, TG, GOX, and GT increased by 162.3%,
129.3%, 120.1%, and 102.7%, respectively. Hardness is an important
parameter for determining dough quality; the smaller the hardness, the
softer the dough. The change in hardness was the same as that observed
in another study, Bai et al. (2022) found that the hardness of the dough
increases with multiple freeze-thaws. The freeze-thaw cycle reduced the
extensibility and flexibility of the dough, as seen by the decline in
springiness, cohesiveness, and resilience, although the impact of two
enzymes slowed down the degradation of the dough, which was similar
to the rheological and stretching results. Similar reports claim that the
addition of enzymes to whole wheat frozen cooked pasta was effective in
increasing the hardness of the noodles and reducing their springiness,
cohesiveness, and resilience (Kang et al., 2021).
4
Journal of Cereal Science 111 (2023) 103689
3.5. Freezable water content
The ice crystals inside frozen dough are formed by freezable water;
the less water that freezes, the less disrupt will be done to the frozen
dough (Zhu et al., 2022). As is shown in Table 1, during the freeze-thaw
cycle, the ΔH of the frozen dough containing enzymes was less than the
CON group, and the ΔH of the fresh GT group was 43.3 J g− 1, which was
13.2% less than CON group. The dough’s ΔH increased with the number
of freeze-thaw cycles. It could be observed that the freezable water (FW)
and non-freezable water (NFW) results of frozen dough containing
enzyme were higher than those of the CON group. This is mainly due to
the addition of enzymes, which reduces the mobility of water. On one
hand, the addition of enzymes that promote gluten crosslinking causes
water to be locked inside the dough; on the other hand, the addition of
enzymes causes part of the water to be absorbed, changing the dough’s
rheological properties. By densifying the gluten network, the enzymes
can inhibit the migration of water inside the dough and can reduce the
physical damage caused by freezing, thus improving the stability of the
dough structure during the freeze-thaw cycles. The amount of freezable
water in frozen dough can be greatly reduced by amylase, xylanase, and
glucose oxidase, according to some studies (Wang et al., 2018).
3.6. Water distribution
LF-NMR resonance can analyze the water distribution inside the
object. The three major groups T21 (0.01–3.05 ms), T22 (3.05–75 ms),
and T23 (75–500 ms) indicate tightly bound water, semi-bound water,
and free water, respectively (He et al., 2020), and the typical T2
W.-T. Guo et al. Journal of Cereal Science 111 (2023) 103689

Table 1
Texture analysis, enthalpy of melting, freezable and non-freezable water content of dough with multiple freeze-thaw cycles.
Sample Texture Hardness(g) Springiness Cohesiveness Resilience DSC ΔH (J⋅g− 1) FW (%) NFW (%)

0F/T CON 180.26± 0.80± 0.70± 0.067± 48.0± 39.7± 60.3±

6.15a 0.03b 0.01b 0.002b 1.54a 1.28a 1.28b
TG 159.40± 0.90± 0.78± 0.071± 44.3± 36.6± 63.4±
5.59b 0.02a 0.05a 0.002b 2.44ab 2.02ab 2.02ab
GOX 162.90± 0.90± 0.71± 0.077± 46.7± 38.6± 61.4±
8.07b 0.05a 0.01b 0.009ab 0.25ab 0.20ab 0.20ab
GT 133.23± 0.91± 0.79± 0.093± 43.3± 35.8± 64.2±
6.30c 0.04a 0.02a 0.014a 2.92b 2.42b 2.42a

4F/T CON 277.99± 0.67± 0.62± 0.053± 57.6± 44.6± 55.4±

1.20a 0.01b 0.01c 0.003c 1.37a 1.13a 1.13c
TG 250.64± 0.74± 0.66± 0.065± 55.0± 42.6± 57.4±
4.82b 0.01a 0.02bc 0.003ab 0.05b 0.04b 0.04b
GOX 253.79± 0.74± 0.64± 0.063± 56.5± 43.7± 56.3±
13.82b 0.05a 0.01b 0.003b 0.41b 0.34ab 0.34bc
GT 220.54± 0.76± 0.71± 0.071± 51.7± 40.0± 60.0±
11.38c 0.03a 0.02a 0.002a 1.28c 1.06c 1.06a

8F/T CON 472.91± 0.54± 0.51± 0.043± 59.2± 46.1± 53.9±

27.83a 0.01b 0.03a 0.002c 1.32a 4.00a 4.00b
TG 365.59± 0.62± 0.56± 0.055± 56.7± 44.1± 55.9±
1.30b 0.02ab 0.04a 0.004b 1.57ab 1.30ab 1.30ab
GOX 358.55± 0.66± 0.56± 0.059± 56.9± 44.2± 55.8±
23.55b 0.07a 0.01a 0.004b 1.73bc 1.43ab 1.43ab
GT 301.46± 0.68± 0.59± 0.067± 55.5± 43.2± 56.8±
9.04c 0.01a 0.03a 0.002a 1.80c 1.49b 1.49a

CON represents the control group and TG, GOX, and GT represent the addition of different enzymes. The mean minus the standard deviation (n ≥ 3) is used to express
data. This indicates that several superscripts are significantly different from one another within a column (P < 0.05).

relaxation distribution curves of the dough illustrated in Fig. 3A.
The changes in water distribution inside the dough with different
enzymes added under 0, 4, and 8 freeze-thaw cycles are displayed in
Fig. 3B–D. The freeze-thaw cycles lead to the tightly bound water
changes to semi-bound water, and the semi-bound water changes to free
water. Throughout the process, the T21 of all the dough reduced with the
number of freeze-thaw cycles, confirming that the content of bound
water decreased continuously. As for the T22, the CON group increased
from 80.2% to 87.1% after eight freeze-thaw cycles, while the GT group
increased from 76.4% to 83.1%, indicating that the two enzymes
decreased the bound water content in the dough, and the simultaneous
processing of TG and GOX is higher than that of a single enzyme. The

Fig. 3. Changes in the NMR spin-spin relaxation (T2) during the freeze-thaw cycles with different enzymes. (A) Typical T2 distribution curve, (B) T21 peak area
proportions, (C) T22 peak area proportions, and (D) T23 peak area proportions of; CON represents the control group, TG, GOX, and GT represent the addition of
different enzymes.

5
W.-T. Guo et al.
overall changes in dough T23 were not significant during the freeze-thaw
cycle, but the GT group still had the lowest free water content. This
demonstrates/that the addition of enzymes can effectively inhibit the
conversion of bound water to free water and that when TG and GOX are
used together, the results are higher.
It is speculated that the two enzymes may strengthen the cross-
linking of the gluten proteins to protect the starch higher, resulting in
a decrease in the amount of starch deterioration, and a delayed reduc­
tion in the content of freezable water, and a slower disruption of the
gluten protein network of the dough, which is corresponded to the DSC
results. The stabilization of dough water by the effect of enzymes had
also been reported by (Niu et al., 2018). Other studies found that
polysaccharides can stabilize water in frozen dough to reduce dough
deterioration, which is consistent with our conclusion (Hong et al.,
2021).
3.7. -SH contents and protein secondary structures
Changes in the –SH of the frozen dough after different enzyme
treatments at 0, 4, and 8 freeze-thaw cycles are displayed in Fig. 4A. At
0 freeze-thaw cycles, the –SH contents of TG, GOX, and GT decreased by
0.47, 0.12, and 1.36 μmol/g, respectively. Compared with the CON
group, these results demonstrated a decrease in the –SH content, which
may be related to the oxidation of –SH or associated with an increased
disulfide bond content. As the freeze-thaw cycles proceeded, the –SH
content increased. When freeze-thaw to 8 times, compared with the CON
group, the –SH content of TG, GOX, and GT decreased by 13.7, 9.16, and
16.28 μmol/g, respectively. This demonstrated that the freeze-thaw
cycles caused significant changes in the –SH content and that the
changes in the –SH content of the fresh dough after the addition of the
enzymes were significantly lower than that after the freeze-thaw cycles.
This suggests that the enzymes can curb the increase in –SH content (Ke
et al., 2020) and that the two enzymes are more effective when used
simultaneously.
To investigate more deeply the structural changes on the internal
proteins of the dough, the effects of added enzymes on the secondary
structures of the frozen dough proteins are shown in Fig. 4B–D. Specif­
ically, the amide I band (1600–1700 cm− 1) of proteins is the most
6
Journal of Cereal Science 111 (2023) 103689
valuable for assessing any changes in their secondary structure. The
absorption peaks in the regions 1650–1660, 1610–1640, 1660–1700,
and 1640–1650 cm− 1 were considered representative of α-helix, β-sheet,
β-turn, and irregular curl, respectively (Hong et al., 2021).
As the freeze-thaw cycle proceeds to 8 times, the α-helix and β-sheet
contents in all the dough decreased, while the β-turn content increased;
the irregular curl content did not change significantly. In the CON group,
the α-helix content decreased from 18.13% to 14.70%, the β-sheet
content decreased from 37.63% to 31.24%, and the β-turn content
increased from 30.03% to 39.93% after 8 freeze-thaw cycles. The results
indicate that the α-helix and β-sheet structures are sensitive to the
freeze-thaw cycle treatment and can be partially converted to β-turns
during this process (Niu et al., 2018). The α-helix and β-sheet contents of
the wheat proteins decreased less after eight freeze-thaw cycles,
following the addition of the enzymes; the α-helix and β-sheet contents
of the GT group decreased from 21.40% to 17.20%, and 41.39%–
35.67%, respectively. After 8 freeze-thaw cycles, the decrease was
obviously less than the CON group. Meanwhile, the β-turn content
increased from 23.96% to 32.64%, which is significantly lower than the
β-turn content increase in the CON group. Therefore, enzyme treatment
inhibited the transformation of regular to irregular structures in the
protein secondary structures, which led to the stabilization of the dough
structure; the dough was higher protected by the interaction of the two
enzymes.
3.8. Microstructures
The effects of the different enzymes on the microstructure of the
frozen dough after 0, 4, and 8 freeze-thaw cycles were photographed, as
shown in Fig. 5. All fresh dough had a complete gluten network structure
with starch granules dispersed on the gluten, which was sparser in the
CON group and more densely packed in the enzyme-supplemented
group, especially in the GT group. After eight freeze-thaw cycles, the
gluten network of the CON group displayed irregular large holes, and the
gluten network-wrapped starch granules were dislodged from the pro­
tein network. However, after 8 freeze-thaw cycles, the gluten network of
the GT group did not exhibit significant holes. The surface was uniform,
and the starch was still buried inside by the gluten network. The gluten
Fig. 4. Changes in –SH contents and protein sec­
ondary structures in the frozen dough with different
enzymes added at different times of the freeze-thaw
cycles. (A) –SH in the frozen dough with different
enzymes after the freeze-thaw cycles, (B) Secondary
structure content of the dough after 0 freeze-thaw
cycles, (C) Secondary structure content of the dough
after 4 freeze-thaw cycles, (D) Secondary structure
content of the dough after 8 freeze-thaw cycles; CON
represents the control group, TG, GOX, and GT
represent the addition of different enzymes. To
denote a statistically significant difference (P <
0.05), samples with the same freezing procedure are
labeled with various lowercase letters.
W.-T. Guo et al. Journal of Cereal Science 111 (2023) 103689

Fig. 5. Scanning electron micrographs of the dough after 0, 4, and 8 cycles of freezing and thawing, with various enzymes applied Scanning electron micrographs
after 0, 4, and 8 freeze-thaw cycles are A–D, E–H, and I–L, respectively. CON represents the control group and TG, GOX, and GT represent the addition of
different enzymes.

network in the GT group was more complete during the freeze-thaw administration, funding acquisition.
cycles, and the starch were deeply entrenched in its structure.
Following the addition of antifreeze proteins to the dough, reports of a
Declaration of competing interest
similar phenomenon have also been reported (Zhang et al., 2015). This
indicates that TG and GOX added simultaneously have a protective ef­
All authors disclosed no relevant relationships.
fect on the microstructure of frozen dough, which is the same as the
changes in the –SH and secondary structures.
Data availability

4. Conclusions
The data that has been used is confidential.

In this study, we found that based on the amount of flour added, 1%

TG and 0.006% GOX added simultaneously, effectively inhibited an
increase in the syneresis rate of frozen dough, decreased the content of
freezable water, and inhibited the water redistribution and ice crystal
reformation. The tensile and rheological results showed that enzyme
treatment slowed down the deterioration of the viscoelastic properties of
the frozen dough. Meanwhile, the addition of enzymes reduced the
broken caused to the gluten protein network and secondary structure
following the freeze-thaw cycles and effectively inhibited the deterio­
ration of the internal structure of the dough. SEM results also demon­
strated that the damage to the gluten network of the dough, after
enzyme treatment, was reduced during the freeze-thaw cycle. In
conclusion, TG and GOX can effectively mitigate the deterioration of
dough during the freeze-thaw cycles, while producing higher results.
Therefore, this study provides new ideas on how to more effectively
protect the structure of frozen dough.
Authorship contributions
Wen-Tao Guo: conceptualization; methodology; data curation;
formal analysis; writing-original draft. Xue-Fei Yang: methodology;
investigation. Yi-Shun Ji: methodology; investigation. Bin Hu: meth­
odology; investigation. Wan-Guang Li: methodology; formal analysis.
Xi-Yang Zhong: methodology; investigation. Shao-Tong Jiang: meth­
odology; investigation. Zhi Zheng: conceptualization, methodology,
investigation, formal analysis, review & editing, supervision, project
Acknowledges
This study was supported by the National Key Research and Devel­
opment Program of China (2018YFD0400600), Key Scientific and
Technological Project of Anhui Province of China (No.
202003b06020017; 202003b06020020; 202103b06020009) and
Fundamental Research Funds for the Central Universities of China
(Grant No. PA2020GDSK0058).
References
Adams, V., Ragaee, S.M., Abdel-Aal, E.M., 2017. Rheological properties and bread
quality of frozen yeast-dough with added wheat fiber. J. Sci. Food Agric. 97 (1),
191–198.
Asghar, A., Anjum, F.M., Allen, J.C., 2011. Utilization of dairy byproduct proteins,
surfactants, and enzymes in frozen dough. Crit. Rev. Food Sci. Nutr. 51 (4), 374–382.
Bai, N., Guo, X.-N., Xing, J.-J., Zhu, K.-X., 2022. Effect of freeze-thaw cycles on the
physicochemical properties and frying performance of frozen Youtiao dough. Food
Chem. 386, 132854.
Fu, Y., Liu, X., Xie, Q., Chen, L., Chang, C., Wu, W., Xiao, S., Wang, X., 2021. Effects of
Laminaria japonica polysaccharides on the texture, retrogradation, and structure
performances in frozen dough bread. LWT–Food Sci. Technol. 151, 112239.
He, Y., Guo, J., Ren, G., Cui, G., Han, S., Liu, J., 2020. Effects of konjac glucomannan on
the water distribution of frozen dough and corresponding steamed bread quality.
Food Chem. 330, 127243.
Hong, T., Ma, Y., Yuan, Y., Guo, L., Xu, D., Wu, F., Xu, X., 2021. Understanding the
influence of pullulan on the quality changes, water mobility, structural properties
and thermal properties of frozen cooked noodles. Food Chem. 365, 130512.
Huang, R., Huang, K., Guan, X., Zhang, J., Zhang, P., 2022. Incorporation of defatted
quinoa flour affects in vitro starch digestion, cooking and rheological properties of
wheat noodles. J. Cereal. Sci. 108, 103542.

7
W.-T. Guo et al.
Jia, F., Ma, Z., Wang, X., Li, X., Liu, L., Hu, X., 2019. Effect of kansui addition on dough
rheology and quality characteristics of chickpea-wheat composite flour-based
noodles and the underlying mechanism. Food Chem. 298, 125081.
Kang, M.J., Chung, S.-J., Kim, S.S., 2021. The effects of transglutaminase and
refrigerated storage on the physicochemical properties of whole wheat dough and
noodles. Foods 10 (7), 1675.
Ke, Y., Wang, Y., Ding, W., Leng, Y., Lv, Q., Yang, H., Wang, X., Ding, B., 2020. Effects of
inulin on protein in frozen dough during frozen storage. Food Funct. 11 (9),
7775–7783.
Kim, H.-J., Yoo, S.-H., 2020. Effects of combined alpha-amylase and endo-xylanase
treatments on the properties of fresh and frozen foughs and final breads. Polym. Bull.
(Heidelberg, Ger.) 12 (6), 1349.
Li, J., Zhu, Y., Yadav, M.P., Li, J., 2019. Effect of various hydrocolloids on the physical
and fermentation properties of dough. Food Chem. 271, 165–173.
Liang, Y., Qu, Z., Liu, M., Zhu, M., Zhang, X., Wang, L., Zhan, X., Wang, J., 2021. Further
interpretation of the strengthening effect of curdlan on frozen cooked noodles
quality during frozen storage: studies on water state and properties. Food Chem.
346, 128908.
Liu, M., Liang, Y., Zhang, H., Wu, G., Wang, L., Qian, H., Qi, X., 2018. Comparative study
on the cryoprotective effects of three recombinant antifreeze proteins from Pichia
pastoris GS115 on hydrated gluten proteins during freezing. J. Agric. Food Chem. 66
(24), 6151–6161.
Lu, L., Yang, Z., Guo, X.N., Xing, J.J., Zhu, K.X., 2021. Effect of NaHCO3 and freeze-thaw
cycles on frozen dough: from water state, gluten polymerization and microstructure.
Food Chem. 358, 129869.
Luo, W., Sun, D.W., Zhu, Z., Wang, Q.J., 2018. Improving freeze tolerance of yeast and
dough properties for enhancing frozen dough quality - a review of effective methods.
Trends Food Sci. Technol. 72, 25–33.
Niu, M., Hou, G.G., Kindelspire, J., Krishnan, P., Zhao, S., 2017. Microstructural,
textural, and sensory properties of whole-wheat noodle modified by enzymes and
emulsifiers. Food Chem. 223, 16–24.
Niu, M., Xiong, L., Zhang, B., Jia, C., Zhao, S., 2018. Comparative study on protein
polymerization in whole-wheat dough modified by transglutaminase and glucose
oxidase. LWT–Food Sci. Technol. 90, 323–330.
Journal of Cereal Science 111 (2023) 103689
Struyf, N., Verspreet, J., Verstrepen, K.J., Courtin, C.M., 2017. Investigating the impact
of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough
fermentation and bread sugar levels. J. Cereal. Sci. 75, 35–44.
Tang, X., Wang, F., Huang, W., Zou, Q., Jia, C., Jin, D., Omedi, J.O., Li, Z., 2016. The
combination of rhizopus chinensis lipase and transglutaminase affects the rheology
and glutenin macropolymer properties of frozen dough. Cereal Chem. 93 (4),
377–385.
Wang, M., Zeng, J., Huang, K., Tian, X., Gao, H., Zhang, K., 2022. Effects of freeze-thaw
treatments at different temperatures on the properties of gluten protein from
fermented dough. J. Food Process. Preserv. 46 (8), e16768.
Wang, X., Pei, D., Teng, Y., Liang, J., 2018. Effects of enzymes to improve sensory quality
of frozen dough bread and analysis on its mechanism. J. Food Sci. Technol. 55 (1),
389–398.
Xiao, F., Zhang, X., Niu, M., Xiang, X., Chang, Y., Zhao, Z., Xiong, L., Zhao, S., Rong, J.,
Tang, C., Wu, Y., 2021. Gluten development and water distribution in bread dough
influenced by bran components and glucose oxidase. LWT–Food Sci. Technol. 137,
110427.
Xin, C., Nie, L., Chen, H., Li, J., Li, B., 2018. Effect of degree of substitution of
carboxymethyl cellulose sodium on the state of water, rheological and baking
performance of frozen bread dough. Food Hydrocolloids 80, 8–14.
Xu, H.-N., Huang, W., Jia, C., Kim, Y., Liu, H., 2009. Evaluation of water holding capacity
and breadmaking properties for frozen dough containing ice structuring proteins
from winter wheat. J. Cereal. Sci. 49 (2), 250–253.
Yu, W., Xu, D., Zhang, H., Guo, L., Hong, T., Zhang, W., Jin, Y., Xu, X., 2020. Effect of
pigskin gelatin on baking, structural and thermal properties of frozen dough:
comprehensive studies on alteration of gluten network. Food Hydrocolloids 102,
105591.
Zhang, Y., Zhang, H., Wang, L., Qian, H., Qi, X., 2015. Extraction of oat (Avena sativa L.)
antifreeze proteins and evaluation of their effects on frozen dough and steamed
bread. Food Bioprocess Technol. 8 (10), 2066–2075.
Zhou, J., Yang, H., Qin, X., Hu, X., Liu, G., Wang, X., 2019. Effect of β-cyclodextrin on the
quality of wheat flour dough and prebaked bread. Food Biophys. 14 (2), 173–181.
Zhu, X., Yuan, P., Zhang, T., Wang, Z., Cai, D., Chen, X., Shen, Y., Xu, J., Song, C.,
Goff, D., 2022. Effect of carboxymethyl chitosan on the storage stability of frozen
dough: state of water, protein structures and quality attributes. Food Res. Int. 151,
110863.