Food Chemistry 358 (2021) 129869

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

Effect of NaHCO3 and freeze–thaw cycles on frozen dough: From water

state, gluten polymerization and microstructure
Lu Lu a, b, Zhen Yang a, b, Xiao-Na Guo a, b, Jun-Jie Xing a, b, Ke-Xue Zhu a, b, *
a
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, PR China
b
School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, PR China

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

Keywords: This study investigated the influence of NaHCO3 on the water state, gluten polymerization, microstructure and
Frozen dough quality of frozen steamed bread dough during freeze–thaw cycles. Results showed that the steamed bread made
Freeze–thaw cycles from alkaline (0.4% NaHCO3) frozen dough possessed a larger specific volume and smaller hardness after 4
Gluten polymerization
freeze–thaw cycles, than the non-alkaline dough group. The addition of NaHCO3 slowed the increase of freezable
Protein network analysis
AngioTool software
water content and water mobility of dough during freeze–thaw cycles, and the high amount of NaHCO3 (0.4%–
1%) showed the great effect. Compared with non-alkaline dough, the sodium dodecyl sulfate extractable protein
proportion and free sulfhydryl level of alkaline dough increased less after freeze–thaw cycles, indicating a
strengthened freeze–thaw tolerance of alkaline dough. Based on microstructure image and corresponding protein
network analysis (PNA) results, the protein area and total protein length in alkaline dough remained at a higher
level than non-alkaline group after 4 freeze–thaw cycles.

1. Introduction
Frozen dough can be manufactured on a large scale, off-site, and then
shipped to local restaurants or retail operations for on-site baking, thus
saving both of the equipment and labor costs (Ma et al., 2016). However,
some problems may occur during the processing of frozen dough, such as
low specific volume (Zhang, Zhang, & Wang, 2007), high hardness
(Wang, Pei, Teng, & Liang, 2017) and uneven porosity of bread
(Frauenlob et al., 2017). These problems can probably be attributed to
yeast inactivation and gluten deterioration, which are related to water
redistribution and ice recrystallization of frozen dough (Xin, Nie, Chen,
Li, & Li, 2018). Yeast inactivation induced a remarkable decrease of CO2
production and led to a low specific volume of the bread (Lu, Xing, Guo,
Sun, & Zhu, 2020). Gluten deterioration is one of the most challenging
problems in frozen dough (Wang, Lee, Xu, & Jin, 2016). During frozen
storage or freeze–thaw cycles, the gluten becomes dehydrated and
depolymerized through breakage of the inter-chain disulfide bonds (SS)
(Tang et al., 2019). Depolymerization of gluten results in the increased
of sodium dodecyl sulfate (SDS) soluble protein, the decreased of gas
retention, and the deterioration of the frozen dough (Wang, Lee, Xu, &
Jin, 2016).
Studies have focused on approaches to improving the freeze
tolerance of frozen dough, including enzymes (Wang, Pei, Teng, &
Liang, 2017), antifreeze proteins (Zhang, Zhang, & Wang, 2007; Chen
et al., 2020), hydrocolloids (Shyu, Hwang, & Hsu, 2008; Xin, Nie, Chen,
Li, & Li, 2018), and gluten network enhancement methods (María et al.,
2012; Fu, 2008; Peng et al., 2021). For instance, transglutaminase was
used to strengthen the gluten protein in frozen dough (María et al.,
2012), but it was difficult to maintain the enzyme activity in the frozen
dough system. Therefore, developing a simple, reliable, and low-cost
method is necessary to maintain the freeze–thaw tolerance of frozen
dough by strengthening the gluten network.
Sodium bicarbonate (NaHCO3), a kind of alkali, has been widely
used as a leavening agent during fermentation of dough. Traditionally,
homemade sourdough steamed bread involves the use of alkali to
neutralize the excessive sourness by lactic acid bacteria (Huang & Mis­
kelly, 1991). In addition, numerous studies have shown that alkali can
improve the quality of dough-based products, such as noodles and
bread. Leuschner, O’Callaghan, and Arendt (1997) showed that the high
specific volume of bread is dependent on the concentration of sodium
bicarbonate used in the recipe. Fu (2008) showed that NaHCO3 mark­
edly enhance the gluten strength, water absorption, and texture prop­
erties of noodles. Another study found that the addition of alkali in
noodles forms a more closed gluten network structure, mainly due to the

* Corresponding author.
E-mail address: kxzhu@jiangnan.edu.cn (K.-X. Zhu).

https://doi.org/10.1016/j.foodchem.2021.129869
Received 19 November 2020; Received in revised form 10 April 2021; Accepted 13 April 2021
Available online 20 April 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
L. Lu et al.
disulfide/sulfhydryl exchange of gluten (Li, Sun, Han, Chen, & Tang,
2018). Guo, Yang, and Zhu (2019) showed that the addition of NaHCO3
increase the specific volume of buckwheat steamed bread and promote
protein aggregation to form dehydroalanine-derived and disulfide cross-
linking. Xi, Xu, Wu, Jin, and Xu (2020) showed the hydrolysis of glu­
tenin macropolymers in steamed bread under alkaline conditions.
However, the freeze–thaw tolerance of alkaline frozen dough is still
unknown.
The overall aim of this paper was to study the effects of NaHCO3 (0,
0.2%, 0.4%, 0.6%, and 1% on flour weight basis, w/w) on the state of
water, gluten polymerization, and the quality of frozen steamed bread
dough during different freeze–thaw cycles. In particular, the specific
volume, and hardness of steamed bread made from frozen dough,
freezable water, and the water distribution of frozen dough were
analyzed. Furthermore, to further study the gluten polymerization of
frozen dough, molecular weight distribution, free sulfhydryl (SH), zeta
potential, and microstructure were analyzed.
2. Materials and methods
2.1. Materials
Edible NaHCO3 (Wei Meizi Co., Kunshan, China) and wheat flour
(Wudeli Flour Co., Henan, China) were obtained from the local super­
market. The wheat flour possessed a moisture content of 12.9 ± 0.05%
and a protein content of 11.5 ± 0.39% (dry basis, w/w). The compressed
yeast (Saccharomyces cerevisiae) was obtained from the Angel Yeast Co.
(Shandong, China). All chemicals and reagents were of at least analytical
grade.
2.2. Preparation of frozen dough and steamed bread
The frozen dough recipe consisted of 1000 g wheat flour, 450 g
deionized water, 30 g yeast, and edible NaHCO3. The different levels of
NaHCO3 (0, 0.2%, 0.4%, 0.6%, and 1.0% on flour weight basis, w/w)
was first dissolved with 100 g water and then mixed with other in­
gredients. All ingredients were mixed in a dough mixing machine (ARM-
2, Thunderbird Co., Surrey, British Columbia, Canada) for 6 min to make
a smooth dough. The mixed dough was manually divided and molded
into pieces of 60 g each, and then subjected to pre-fermentation in a
proofer (RE64D-32, Kolb Huizhou Co., Huizhou, China) at a tempera­
ture of 38 ◦ C and relative humidity of 75% for 20 min. Finally, the dough
was frozen in a refrigerator (DW-40L328, Shjingmi Co., Shanghai,
China) at − 40 ◦ C for 2 h. The freeze–thaw cycles treatment was con­
ducted at − 18 ◦ C for 6 d, thawing at 30 ◦ C for 1 h, and this part was
considered as one freeze–thaw cycle. The freeze–thaw cycles treatment
was repeated 1–4 times, i.e. 1–4 freeze–thaw cycles. The 0 freeze–thaw
samples are frozen dough without freeze–thaw cycles treatment.
In each of the freeze–thaw cycles, samples were fermented in a
proofer at a temperature of 38 ◦ C and relative humidity of 75% for 60
min. The fermented dough was steamed in a steam oven (SCC102,
Rational Co., Bavaria, Germany) for 15 min to make the steamed bread.
2.3. Characteristics of steamed bread
2.3.1. Specific volume of steamed bread
The specific volume of the steamed bread was determined according
to the AACC international method 10-16.01 (AACC, 2016). After the
dough was steamed, the steamed bread was cooled at room temperature
for 1 h, and then placed in a polyethylene box for specific volume
analysis using a volume tester (VolScan Profiler, SMS Co., London, UK)
within 2 h. The parameters were: vertical step was slow; rotation speed 1
rps; reference shape ellipse.
2.3.2. Hardness of the steamed bread
The hardness of the steamed bread was determined according to a
2
Food Chemistry 358 (2021) 129869
method reported by Lu, Xing, Yang, Guo, and Zhu (2020). The experi­
ments were conducted at 25 ◦ C using a texture analyzer (Model TA-plus,
SMS Co., London, UK) coupled with a cylindrical probe P36. The results
were calculated under the pattern of “return to start”. The selected test
conditions were as follows: distance strain of 25%, pretest speed of 5
mm/s, test speed of 2 mm/s, and post-test speed of 10 mm/s.
2.3.3. Color measurement of steamed bread
The color of the steamed bread made from frozen dough was
measured based on the method of Li et al. (2012). The analysis was
conducted using a chromameter (CR-400, Konica Minolta Holdings Inc.,
Japan) portable spectrophotometer equipped with D65 illuminant for
values of L* (lightness, white-black), a* (color-opponent dimension, red-
green), and b* (color-opponent dimension, yellow-blue). The ΔE was
calculated as follows:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = (ΔL)2 +(Δa)2 + (Δb)2 )Where ΔL, Δa, and Δb were the dif­
ferences of L*, a*, and b* values between the treatment sample and a
control sample (L value of 87.2, a value of − 2.19, and b value of 14.4).
The control sample was the steamed bread without NaHCO3 and
freeze–thaw cycles.
2.4. The pH measurement of frozen dough extract
The pH of the frozen dough extract was measured using a pH meter
(ST 3100, Ohaus Instruments, New Jersey, USA). A 10 g sample of frozen
dough was crushed with 90 mL of water. Subsequently, the pH of the
dough extract was determined using a pH probe at 20 ◦ C.
2.5. Measurement of freezable water content of frozen dough
A differential scanning calorimeter (DSC 8500, PerkinElmer, USA) in
a N2 flow was used to measure the freezable water content in the frozen
dough according to the method reported by Ding et al. (2015) with a
slight modification. About 10 mg of the frozen dough sample was placed
into a DSC pan and hermetically sealed. An empty pan was used as
reference. After cooling to − 90 ◦ C, the system routine was setup as
follows: cooling from 30 ◦ C to − 30 ◦ C at a rate of 10 ◦ C/min, holding at
− 30 ◦ C for 5 min, and then heating from − 30 ◦ C to 30 ◦ C at a rate of
10 ◦ C/min. Enthalpy (ΔH) of the melting peak was determined using
Pyris software (PerkinElmer, USA). The freezable water content (FW)
was calculated according to the formula:
FW (%) = ΔHw×Wt
ΔH
× 100%Where ΔH was the enthalpy of the melting
peak of the endothermic curve, J/g; ΔHw was the enthalpy of the
melting peak of water, 333 J/g; Wt was the total water content of the
frozen dough sample.
2.6. Measurement of water distribution of frozen dough
Water distribution was determined by low field nuclear magnetic
resonance (LF-NMR) according to the method reported by Xin, Nie,
Chen, Li, and Li (2018). An LF-NMR analyzer (MesoMR23-060 V-I,
Niumag Co., Shanghai, China) was used to analyze the T2 relaxation
time. After reaching 15 ◦ C, frozen dough samples of approximately 4 g
were wrapped in polytetrafluoroethylene bags and placed in a 15 mm
diameter glass tube. The transverse relaxation curves were obtained
using a CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence. The pa­
rameters were set as follows: echo time (TE) = 0.6 ms, the number of
sampling points (TD) = 90024, the interval time (TW) = 1000 ms, the
number of echoes (NECH) = 1500, number of slices (NS) = 2. The peak
area of the frozen dough was calculated by cumulative integration using
SciDAVis software 1.26 for Mac.
2.7. Zeta potential of gluten
The zeta potential of gluten of the frozen dough was tested by a zeta
L. Lu et al.
potential analyzer (Brookhaven Instruments, Nano Brook Omni) at 25 ◦ C
according to the method described by Chen et al. (2018). The gluten was
isolated from the frozen dough, then freeze-dried, ground, and sieved
through 80-mesh. Finally, the gluten powder samples were prepared
with a solid content of 0.1% in deionized water and subjected to zeta
potential measurement. Five runs were carried out for each
measurement.
2.8. Extractability of the gluten protein from steamed bread by SDS
The gluten protein extractability in SDS was analyzed by size-
exclusion high performance liquid chromatography (SE-HPLC) accord­
ing to the method of Huang, Zhao, Zhu, and Zhou (2017). For the
analysis under non-reducing conditions, freeze-dried steamed bread
powder containing 2.0 mg protein was accurately weighed and
dispersed in 1 mL of 0.05 M sodium phosphate buffer (PBS) containing
2.0% SDS at pH 6.8. For the analysis under reduction condition,
dithiothreitol (DTT) of 1.0% was added to the extract. After vortexing
for 60 min and centrifugation at 7, 690 × g for 10 min at 20 ◦ C, the
supernatant was collected and filtrated through a polyethersulfone
(Millex-HP, 0.45 μm) film. The SE-HPLC analysis was performed using
an LC-2010 system (Shimadzu, Kyoto, Japan). Briefly, 20 μL of the
sample was loaded on a TSK G4000-SWXL analytical column (Tosoh
Biosep, Tokyo, Japan). The elution solvent was PBS of 0.05 M (con­
taining SDS of 2.0% at pH 6.8) at a flow rate of 0.7 mL/min. The elution
curve was monitored in a column temperature at 30 ◦ C and at an
absorbance of 214 nm. SDS extractable protein (SDSEP) was calculated
according to the method reported by Wang, Lee, Xu, and Jin (2016) as
follows:
SDSEP(%) =
A1
× 100%
Ar
Where, A1 was the peak area of the protein extraction under the non-
reduction condition. Ar was the peak area of protein extraction under
the reduction condition.
2.9. Determination of free SH groups of gluten
The level of free SH groups was determined using a method reported
by Wang, Guo, and Zhu (2016). The steamed bread was freeze-dried,
ground, and sieved (80-mesh). The 0.4 g of steamed bread powder
was suspended in 10.0 mL 0.2 M of Tris-glycine buffer (containing 1.0%
SDS, 8 M urea and 3 mM EDTA at pH 8.0). After shaking for 60 min, the
slurry was centrifuged at 11,000 × g for 10 min and the supernatant was
collected. After that, the Tris-glycine buffer containing 0.1% (w/v)
DTNB was added to the supernatant in the dark and incubated for 20
min. The absorbance was measured immediately using an ultramicro
microplate spectrophotometer (Epoch, Biotek, Virginia, USA) at 412
nm. The absorbance values were converted to levels of the free SH
(µmol/g protein) using a calibration curve made from cysteine.
2.10. Microstructure of gluten network in steamed bread
A confocal laser scanning microscope (CLSM) (LSM 710, Leica Co.,
Wetzlar, Germany) was used for microstructure analysis. The micro­
structure of steamed bread samples was quantified by protein network
analysis (PNA), according to the method reported by Lucas, Petermeier,
Becker, and Jekle (2019). First, the steamed bread samples were
wrapped in gel and sliced into 20 μm pieces using a freezing microtome
(CM1850 UV, Leica Co.), and then stained using 0.1 mg/mL rhodamine
B (Innochem, Beijing, China) solution for 1 min. After that, a small
amount of deionized water was used to rinse the mixture, and then it was
dried with a filter paper. Then, the CLSM images were acquired in 1024
× 1024 pixel resolution. The protein network was quantified based on
the CLSM microscopic images. Finally, the protein area, junction
3
Food Chemistry 358 (2021) 129869
number, total protein length, average protein length, and end points
number were measured using AngioTool software (version 64, National
Cancer Institute, Bethesda, Maryland, USA).
2.11. Statistical analysis
Data was presented as the mean value and standard deviation from at
least three replicate measurements for each experiment. Specifically, the
repetition number of hardness test was 8 times. For CLSM, the repetition
number was 6 times. The number of repetitions in other experiments
were 3 or 4 times. Statistical analysis was performed by one-way
ANOVA and Duncan’s tests for statistical analysis of color, pH, and
CLSM data. A two-ways ANOVA followed by simple effects test and
multiple comparisons was performed for statistical analysis of the spe­
cific volume, hardness, freezable water, SDSEP, free SH, and zeta po­
tential. A value of P < 0.05 was considered as statistically significant. All
statistical evaluation was performed using the software SPSS statistics
25 (IBM, USA).
3. Results and discussion
3.1. Characteristics of steamed bread made from frozen dough
The specific volume and hardness of steamed bread made from
frozen dough are shown in Fig. 1A and B. There were differences in
specific volume and hardness among the different NaHCO3 addition.
Specifically, the average specific volume of steamed bread decreased
significantly from 2.37 to 2.17 mL/g, and the average hardness values
increased from 1022 to 1072 g with 0.6% addition of NaHCO3. The
negative effect of high NaHCO3 addition (0.6%) on the specific volume
of steamed bread may be related to the excessively strong network
structure in the dough. The CO2 formed from yeast was not able to
diffuse and to form a relatively big gas cell. Moreover, the addition of
NaHCO3 induced a color change in steamed bread (Table S1). After the
addition of 0.4% NaHCO3, the L* values decreased from 86.9 to 78.6, ΔE
values decreased from 0.37 to 15.8, whereas the b* values increased
from 14.8 to 28.1. The color change caused by the pigment turning
yellow under alkaline conditions and considered negative regarding to
the quality of the steamed bread (Xi, Xu, Wu, Jin, & Xu, 2020).
The specific volume and hardness of steamed bread after 0–4
freeze–thaw treatments were also different. After 2 freeze–thaw treat­
ments, the specific volume of steamed bread decreased significantly
from 2.48 to 2.33 mL/g, and the hardness values increased greatly from
483 to 1121 g (Fig. 1A and B). In line with our study, Xin, Nie, Chen, Li,
and Li (2018) reported that the freezing process led to a low specific
volume and high hardness of bread. However, there was a statistically
significant interaction (P < 0.05) between the freeze–thaw and NaHCO3.
For instance, the specific volume and hardness values of 0.4% addition
sample show no significantly difference (P > 0.05) between 0 freeze­
–thaw and 2 freeze–thaw cycles. These results indicated that the frozen
dough with 0.4% addition of NaHCO3 showed a high freeze–thaw
tolerance. In addition, a low negative effect was on the color of the
steamed bread in the groups with 0.4% NaHCO3 addition after 4
freeze–thaw treatments (Table S1).
3.2. Freezable water in frozen dough
The freezable water content is closely related to the size, amount,
and distribution of ice in the frozen dough (Kontogiorgos, Goff, &
Kasapis, 2008). Thus, it is necessary to know the status of freezable
water in frozen dough. Fig. 2A and B showed the influence of NaHCO3
on the freezing and melting curves of frozen dough after 4 freeze–thaw
cycles. As shown in Fig. 2A, the addition of NaHCO3 increased the
freezing temperature of the frozen dough and led to a slight decrease of
freezable water in 0 the freeze–thaw treatment (Fig. 2B). As shown in the
two-ways ANOVA analysis, there were differences in freezable water
L. Lu et al. Food Chemistry 358 (2021) 129869

Fig. 1. (A) Specific volume of steamed bread made from frozen dough. (B) Hardness of steamed bread made from frozen dough. Error bars represent the standard
deviations of determinations, Different superscript capital letter represent significant differences (P < 0.05) between 0 and 4freeze–thaw treatments. Differences (P <
0.05) between NaHCO3 concentration showed by different superscript lowercase letter. The 0, 1, 2, 3 and 4F/T mean 0, 1, 2, 3, 4 times of freeze–thaw treatment.

among the different NaHCO3 addition and the different NaHCO3 addi­
tion were divided into three homogenous groups (Tukey, P < 0.05).
Specifically, the average freezable water increased significantly from
64.0 to 54.6 mL/g with 0.6% addition of NaHCO3. These results may be
related to the interaction of NaHCO3 and water. Guo, Wei, and Zhu
(2017) also showed that there is a noncovalent interaction between an
ionic compound (such as NaHCO3) and water, which increase the water
absorption of the dough.
As shown in the two-ways ANOVA analysis, the freezable water of
frozen dough after 0–4 freeze–thaw treatments were also different (P <
0.05). Freezable water increased after 0–4 freeze–thaw treatments
(Fig. 2B). This could be related to the ice crystal recrystallization during
the freeze–thaw cycles. Ding et al. (2015) showed that freeze–thaw
reduce the total number of crystals in the dough, but increase the crystal
size. This led to damage to the gluten network and thus increased the
release of bound water of the dough. As shown in Fig. 2B, after 1
freeze–thaw cycle, the average freezable water increased from 46.5 to
52.2%. However, the freezable water of NaHCO3 addition (0.4%, 0.6%,
and 1%) samples showed no significantly difference (P > 0.05) between
0 freeze–thaw and 1 freeze–thaw, indicated that there was a signifi­
cantly interaction between the freeze–thaw and NaHCO3 (P < 0.05).
These results may suggested that dough with the addition of NaHCO3
had a high freeze–thaw tolerance, which levelled up further when the
amount of NaHCO3 increased. This may have resulted from the

4
L. Lu et al.
Fig. 2. (A) Freezing and melting curves of frozen dough after 4 freeze–thaw cycles. (B) Freezable water of frozen dough.
interchange of SH − SS in the gluten network or the noncovalent
interaction (hydrogen bonds or hydrophobic) between NaHCO3, water,
and gluten (Han, Ma, Li, & Sun, 2020).
3.3. Water mobility in frozen dough
A typical T2 distribution curve of frozen dough was shown in Fig. 3A.
There are three proton populations (T21, T22, and T23) in the curve. T21,
T22, and T23 are bound water, immobilized water and free water,
respectively. The corresponding peak area proportion of T21, T22, and
T23 was shown in Fig. 3B, C, and D, respectively.
As shown in Fig. 3A, the relaxation time of T2 shifted to the left in the
frozen dough with the added NaHCO3 of 0.4%, which indicated that the
addition of NaHCO3 decreased the value of T2. Han, Ma, Li, and Sun
(2020) showed that a short T2 represented a close bond between water
and solids, forming the strong water–solid interaction that can affect the
stability of foods. Similarly, in the present study, a short T2 was observed
after the addition of NaHCO3 in the frozen dough, indicating strong
5
Food Chemistry 358 (2021) 129869
interactions between solids and water in alkaline frozen dough. Some
other studies also showed that the addition of alkali could enhance the
interaction between water molecules and gluten, resulting in lower
mobility of water (Kontogiorgos, Goff, & Kasapis, 2008; Han, Ma, Li, &
Sun, 2020).
Water mobility is related to ice redistribution and determined the
properties of frozen dough (Peng, Li, Ding, & Yang, 2017). The freeze­
–thaw treatment induced the water redistribution in all samples
(Fig. 3B), while the T21 proportion of frozen dough without NaHCO3
decreased from 3.24 to 2.81% after 2 freeze–thaw cycles. Tang et al.
(2019) reported that T21 represents the bound water that is strongly
associated with the gluten matrix. Our results showed that the propor­
tion of bound water decreased in all samples after freeze–thaw treat­
ment. These results may be related to the redistribution of ice crystals
and the dehydration of gluten during freeze–thaw cycles. Some other
studies also showed that freeze–thaw treatment damaged the gluten
network and thus led to the release of bound water from the dough (Ding
et al., 2015). Fig. 3C and D showed the change of peak area proportion of
L. Lu et al.
Fig. 3. (A) The typical T2 relaxation time distribution curve of frozen dough after 4 freeze–thaw cycles. (B) Peak area proportions of T21. (C) Peak area proportions of
T22. (D) Peak area proportions of T23.
T22 and T23 in different freeze–thaw cycles. Kontogiorgos (2011) showed
that T22 is immobilized water with intermediate mobility, and fill with
the parallel sheets space in gluten, and T23 is free water with high
mobility that distribute in the void of the gluten network. As shown in
Fig. 3C and D, the peak area proportion of T22 decreased 1.0%, whereas
the T23 increased 0.7% after 4 freeze–thaw treatment in 0.2% NaHCO3
addition groups, when compared with 0 freeze–thaw treatment. How­
ever, the peak area proportion of T22 and T23 was relatively stable during
freeze–thaw cycles when NaHCO3 addition was high (0.4%–1%) (Fig. 3C
and D). This could be related to the alkali-induced high crosslinking
gluten network, which possessed a stronger interaction with water in
alkaline dough than in non-alkaline dough. Previous studies reported by
Li et al. (2018) showed that the addition of K2CO3 enhanced the inter­
action of water and the gluten network structure due to the presence of
disulfide/sulfhydryl exchange in the noodle system. Han, Ma, Li, and
Sun (2020) also showed that both glutenin and gliadin could contribute
to the K2CO3 enhanced hydrophobic interactions, water-solids in­
teractions, and molecular chain aggregation in gluten. Thus, our results
may indicate that high addition (0.4%–1%) of NaHCO3 could positively
inhibit the migration of water in frozen dough during freeze–thaw
treatment.
3.4. Gluten extractability by SDS in steamed bread made from frozen
dough
The protein extractability of the SDSEP is a good indicator of the
crosslinking polymerization degree of gluten protein (Hayta &
6
Food Chemistry 358 (2021) 129869
Schofield, 2004). Fig. 4A and B presents the SE-HPLC profiles obtained
under the non-reducing condition of steamed bread and the corre­
sponding SDSEP results. As shown in the two-ways ANOVA analysis,
there were differences (P < 0.05) in SDSEP among the different NaHCO3
addition. It was observed that the SDSEP value of steamed bread
decreased from 24.0 to 17.3%, as the amount of NaHCO3 levels from 0 to
1%. These results suggested that steamed bread with NaHCO3 showed
inferior solubility of gluten protein and a higher degree of gluten protein
crosslinking polymerization than steamed bread without NaHCO3.
These results were in line with Li et al. (2018). This may be due to the SH
oxidation and SH-SS interchange interaction were fortified under alka­
line conditions, and the dehydroalanine-derived crosslinking reactions
occurred in steamed bread with NaHCO3.
The SDSEP of steamed bread after 0–4 freeze–thaw treatments were
also different, as shown in Fig. 4B. After 2 freeze–thaw cycles, the
average SDSEP increased significantly from 18.4 to 19.1%. However, the
SDSEP in NaHCO3 addition groups (0.2%, 0.4%, 0.6%, and 1%) showed
no significantly difference (P > 0.05) between 0 freeze–thaw and 2
freeze–thaw as shown in Fig. 4B. Moreover, after 4 freeze–thaw cycles,
the SDSEP of steamed bread with 0.4% addition of NaHCO3 was 18.6%,
whereas the steamed bread without addition of NaHCO3 was 25.0%.
These results indicated that a high degree of crosslinking after freeze­
–thaw treatment in steamed bread with addition of NaHCO3. These re­
sults may be related to the stronger interaction between water and solids
in the alkaline frozen dough during freeze–thaw cycles than in frozen
dough without NaHCO3 (Figs. 2A and 3A), resulting in a lower mobility
of water as well as less recrystallization of ice crystals. Even after 4
L. Lu et al.
Fig. 4. (A) The SE-HPLC profiles of steamed bread obtained under the non-reducing condition after 4 freeze–thaw cycles. (B) Free SH of steamed bread. (C) SDSEP
(non-reduced condition) of steamed bread. (D) Zeta potential of gluten separated from frozen dough samples.
freeze–thaw cycles, the steamed bread with NaHCO3 still possessed a
higher freeze–thaw tolerance and degree of protein cross-linking. Thus,
the high-degree crosslinking network could be maintained by reducing
the damage to gluten proteins caused by ice crystals.
3.5. Free SH levels in steamed bread made from frozen dough
In order to further study the effect of NaHCO3 on the protein poly­
merization of streamed bread made from frozen dough, the level of free
SH was determined. The results of free SH levels of steamed bread shown
in Fig. 4C. The free SH of steamed bread made from frozen dough
decreased significantly from 2.41 to 1.63 μmol/g protein, as the rising
NaHCO3 addition levels from 0 to 0.6%, but increased to 1.85 μmol/g
protein when NaHCO3 addition was up to 1%. Free SH of protein plays a
highly important role in protein polymerization during hydrothermal
processes (Lagrain, Thewissen, Brijs, & Delcour, 2008; Wagner, Morel,
Bonicel, & Cuq, 2011). Under alkaline conditions, free SH is correlative
with SH oxidation, SH-SS interchange, β-elimination, and
dehydroalanine-derived crosslinking reactions of protein. In our results,
a decreased level of free SH in steamed bread with NaHCO3 could be
related to the SH oxidation and SH-SS interchange interaction, and an
increased level of free SH induced by a high amount of NaHCO3 could be
related to the β-elimination reaction of intramolecular SS. This obser­
vation is in accordance with the previous results, which found that the
β-elimination reaction can occur at alkaline pH in gluten model systems
and release the free SH under alkaline conditions (Guo, Yang, & Zhu,
7
Food Chemistry 358 (2021) 129869
2019; Deleu, Lambrecht, Vondel, & Delcour, 2019; Rombouts, Jansens,
Lagrain, Delcour, & Zhu, 2014).
Compare to the 0.4% NaHCO3 sample with 0 freeze–thaw, the
sample with 0.4% NaHCO3 after 4 freeze–thaw cycles showed a slight
increase in free SH. Moreover, after the 4 freeze–thaw, the sample with
0.4% NaHCO3 was obvious lower in free SH than the sample without
NaHCO3 (Fig. 4C). This may be indicated that the freeze–thaw cycles
induced the depolymerization of the alkaline gluten protein network,
but the tight crosslinking network of steamed bread was still main­
tained. This may have resulted from the decreased freezable water and
water distribution, which resulted in an enhanced freeze–thaw tolerance
of the frozen dough with the addition of NaHCO3. In addition, after 4
freeze–thaw cycles, the free SH in the 1% NaHCO3 addition group
decreased and stayed at a lower level (1.6 μmol/g protein). These results
might be suggested that β-elimination reactions (which release free SH)
were quantitatively less important than free SH oxidation reactions
(which consume free SH) in frozen dough after 4 freeze–thaw cycles
(Huang & Miskelly, 1991; Rombouts, Lagrain, Brijs, & Delcour, 2010;
Guo, Wei, & Zhu, 2017).
3.6. Zeta potential in gluten isolated from frozen dough
As shown in Fig. 4D, the value of zeta potential was changed from
positive to negative for gluten isolated from frozen dough containing
NaHCO3. The change of absolute value of zeta potential increased from
6.8 to 27.4 mV, as the addition of NaHCO3 from 0 to 0.6%, but decreased
L. Lu et al. Food Chemistry 358 (2021) 129869

to 23.8 mV when NaHCO3 addition levels was up to 1%. This could be addition of 0, 0.4%, and 1% decreased from 5.73, 7.62 and 8.62 to 5.43,
related to the increased change of pH in the environment (Table S2). 6.98 and 8.44 after 4 freeze–thaw cycles. Although NaHCO3 induced
Zeta potential was used to express the potential of charged particles and more negative charges and electrostatic repulsion of gluten, the gluten
verify the electrostatic interaction in the solution. With a higher absolute network still strengthened with the addition of NaHCO3. This may show
zeta potential value, surface charges, and electrostatic interactions be­ that the electrostatic interaction was a minor factor influencing the
tween protein molecules increased (Chen et al., 2018). In this study, the gluten network in alkaline frozen dough.
result indicated that NaHCO3 enhance the electrostatic repulsion be­
tween gluten-gluten, as well as decrease the interaction of glutens.
3.7. Protein network microstructure of steamed bread made from frozen
After 4 freeze–thaw treatment, the absolute value of zeta potential
dough
decreased slightly in all samples (Fig. 4D), indicating the decrease of
electrostatic interaction after freeze–thaw treatment. The change of
To further analyze the protein network, CLSM was used to observe
electrostatic interaction (such as hydrogen bonding and to the hydro­
changes in the microstructure of the protein network as shown in Fig. 5.
phobic interaction) could be related to the interaction between gluten-
Meanwhile, a PNA method was used for quantitative analysis of the
gluten, and to the reduced change of pH after freeze–thaw cycles. As
protein network based on the CLSM images. This analysis method was
shown in Table S2, the pH values of the frozen dough with NaHCO3
first reported by Zudaire, Gambardella, Kurcz, and Vermeren (2011) to

Fig. 5. (A) Original CLSM image of steamed bread. (B) Corresponding AngioTool analysis image. The 0 and 0.6% mean 0 and 0.6% addition of NaHCO3.

8
L. Lu et al.
Fig. 5. (continued).
overcome the difficulties in quantification and description of CLSM
images. In this study, CLSM images and corresponding PNA images
showed that the microstructure of the protein network was compact and
regular after 0.2% and 0.6% addition of NaHCO3 (Fig. 5A and B). With
the addition of NaHCO3, the values of protein area, junction number,
total protein length, and end points number were all increased (Table 1).
After freeze–thaw treatment, the protein network was damaged.
Without addition of NaHCO3, the microstructure of the protein network
became loose and in the form of small fragments after 4 freeze–thaw
treatments (Fig. 5A). In contrast, a compact and regular microstructure
of the protein network was observed in 0.2% and 0.6% NaHCO3 addition
groups after 4 freeze–thaw treatments. Moreover, the protection effect
of 0.6% NaHCO3 on the protein network was better than 0.2% as shown
in Fig. 5. Correspondingly, the quantitative analysis also demonstrated
the similar result (Table 1). After 4 freeze–thaw treatments, the
9
Food Chemistry 358 (2021) 129869
protective effect of high concentration group (0.6%) on protein network
was better than that of low concentration group (0.2%). Specifically,
after 4 freeze–thaw treatments, the protein area, junction number, total
protein length, and end points number of the steamed bread with the
addition of 0.6% NaHCO3 (26.0%, 472, 23.6 mm, and 944, respectively)
were all higher than the steamed bread without NaHCO3 (20.4%, 313,
17.3 mm, and 714, respectively) and 0.2% addition NaHCO3 (21.0%,
349, 18.7 mm, and 808, respectively). These results showed that the
microstructure of the protein network in frozen dough with the addition
of NaHCO3 remained compact and regular after 0, 2, and 4 freeze–thaw
treatments.
4. Conclusion
This study demonstrated that the freeze–thaw tolerance of frozen
L. Lu et al. Food Chemistry 358 (2021) 129869

Table 1 Appendix A. Supplementary data

Effect of NaHCO3 on the protein network analysis result data of CLSM images.
NaHCO3 Protein Junction Total Average End Supplementary data to this article can be found online at https://doi.
area (%) number protein protein points org/10.1016/j.foodchem.2021.129869.
length length number
(mm) (μm)
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