LWT - Food Science and Technology 153 (2022) 112562

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LWT
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Improvement in quality of fast-frozen steamed bread by different gluten

content and glutenin/gliadin ratio and its mechanism
Xiaojie Qian , Yujuan Gu , Binghua Sun *, Sen Ma , Xiaoling Tian , Xiaoxi Wang **
College of Food Science and Engineering, Henan University of Technology, Zhengzhou, Henan Province, PR China

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

Keywords: The effect of frozen storage on the quality of fast-frozen steamed bread has not been explored. Meanwhile,
Fast-frozen steamed bread gluten, as a key component of wheat, can be used as a quality improver for fast-frozen steamed bread due to its
Gluten functional characteristics. Therefore, the effects of different gluten contents (0%, 0.5%, 1.0%, 1.5% on flour
Glutenin-gliadin
weight) and glutenin/gliadin (glu/gli) ratios (1:1, 1:2, and 2:1) on the quality of fast-frozen steamed bread were
Frozen storage
Quality
investigated, and the influence mechanism on its quality was also analyzed. Frozen storage induced the amount
of SDS-soluble protein and freezable water increased, which further resulted in the reduction of specific volume,
the increase of hardness and the destruction of microstructure. The results further indicated that 1.0%-1:1 gluten
addition resulted in steamed bread with supreme specific volume and minimum hardness during frozen storage.
By analyzing the mechanism of quality improvement, we determined that the SDS-soluble protein and freezable
water content of steamed bread frozen for 60 d decreased by 8.45% and 9.63% following 1%-1:1 gluten addition,
respectively. Adequate elasticity and extensibility of dough (specific alveograph index: P = 62, L = 55, P/L =
1.08) with 1.0%-1:1 of gluten addition resulted in the best quality and freeze stability of the fast-frozen steamed
bread.

1. Introduction quality deterioration of the final products is related to the changes of

Chinese steamed bread is a wheat-based traditional staple food that
accounts for about 40% of the wheat consumption in China (Kim,
Huang, Zhu, & Rayas-Duarteb, 2009). With the rapid development of the
freezing preservation technology and demand of ready-to-eat frozen
food products from the market, fast-frozen steamed bread was designed
as a partially ready meal to save time and energy for consumers or re­
tailers (Gaikwad & Arya, 2018; Xu, Zhang, Mujumdar, & Adhikari,
2015). Generally, fast-frozen steamed bread was made through steam­
ing, cooling and freezing process, and it can be eaten after simple
re-steaming (Zhu, 2021).
It is necessary to emphasize that the freezing and frozen storage lead
to quality loss of the frozen products (He et al., 2020). From the existing
studies on frozen gluten (Wang, Chen, Mohanad, Xu, Ning & Xu, 2014),
frozen starch (Silvas-García et al., 2016; Wang et al., 2021), frozen
dough (Yu et al., 2020), frozen dough bread (He et al., 2020; Wang,
Yang, Gu, Xu, & Jin, 2017) and frozen cooked noodles (Liu, Guo, & Zhu,
2019; Obadi, Zhang, Yanan, & Xu, 2021), it can be concluded that the
water characteristics and protein network during frozen storage.
Throughout the frozen storage process, the redistribution of the freez­
able water was accompanied by progressive recrystallization of the ice
crystals (Chen et al., 2013). Crystals growing inside the pores caused
serious destroy on the gluten-starch structure, which further resulted in
the poor baking quality of the frozen dough (Esselink, Van Aalst,
Maliepaard, & Van Duynhoven, 2003). Zhao et al. (2013) indicated that
the gas retention ability and viscoelasticity of dough decreased due to
the glutenin macropolymer (GMP) depolymerized during frozen storage.
Furthermore, Liu et al. (2019) found that frozen storage induced the
increase of freezable water content, the destruction of microstructure
and the depolymerization of protein in frozen cooked noodles. To sum
up, the changes of water characteristics and protein network during
frozen storage had major effects on the quality deterioration of frozen
products.
The addition of improvers in steamed bread might overcome above
quality deterioration associated with frozen storage. While, for the sake
of product safety, it is recommended to adjust the quality of fast-frozen

* Corresponding author. College of Food Science and Engineering, Henan University of Technology, Zhengzhou, Henan Province, PR China.
** Corresponding author.
E-mail addresses: sbhfood@126.com (B. Sun), xxwanghaut@126.com (X. Wang).

https://doi.org/10.1016/j.lwt.2021.112562
Received 2 June 2021; Received in revised form 27 September 2021; Accepted 29 September 2021
Available online 30 September 2021
0023-6438/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Qian et al.
steamed bread from the perspective of raw material composition rather
than using other types of additives. Gluten, as a key component of
wheat, has strong water holding capacity, gelation property and could
change the dough viscoelasticity. Consequently, it has certain theoret­
ical basis and feasibility to use gluten protein as a kind of quality
improver of fast-frozen steamed bread. Glutenin and gliadin are the
main components of wheat gluten with a ratio of 4:3, accounting for
more than 85% of the total wheat protein. Glutenin are interchain di­
sulfide bonds-linked polymers with a wide molecular weight (Mw)
ranging from 105 to 107 Da, whereas gliadins are monomeric proteins
with the Mw distribution from 3 to 8 × 104 Da (Wahlund, Gustavsson,
MacRitchie, Nylander, & Wannerberger, 1996). Moreover, it has been
suggested that the glutenin impart strength and elasticity to dough, and
gliadin contribution to dough viscosity (Uthayakumaran, Gras, Stod­
dard, & Bekes, 1999). In early studies, researchers in the field of steamed
bread relatively agree that the key quality parameters of wheat flour are
protein and/or wet gluten content, and the suitable protein content for
steamed bread making varies from 10% to 13%. While, the above rec­
ommended protein content is not suitable for fast-frozen steamed bread,
because it involves the frozen storage process. In addition, Yue, Guo, and
Zhu (2017) studied on the effect of different wheat flours on the quality
of noodles, and found that flour with high-gluten strength was better for
making frozen cooked noodles. Whereas, above studies also pointed out
that the rheological properties of gluten are more important than protein
content in evaluating the quality of wheat flour. Selaković et al. (2021)
suggested that the rheological properties of gluten are far more signifi­
cant than the amount of protein as well.
However, for the emerging fast-frozen steamed bread, the effect of
frozen storage on the quality of fast-frozen steamed bread has not been
explored, not to mention the improvement of the fast-frozen steamed
bread quality by the key quality parameters of wheat flour: gluten
protein and its components. In this study, the quality changes of fast-
frozen steamed bread during frozen storage were investigated and the
effect of different gluten content and glutenin/gliadin (glu/gli) ratio on
the rheological properties of the dough and quality of fast-frozen
steamed bread were also studied. In addition, the protective mecha­
nism of gluten to the deterioration of gluten network structure and water
state during frozen storage was discussed. Furthermore, the optimal
amount of gluten that would provide the desired dough rheological
properties and achieve better fast-frozen steamed bread quality was
determined.
2. Materials and methods
2.1. Materials
Commercial steamed bread flour (brand: wonder farm; batches
number, 20181117B6) was purchased from Yihai Kerry Grain and Oil
Industry Co., Ltd (Zhengzhou, China) and active dry yeast was obtained
from Angel Yeast Industry Co., Ltd (Yichang, China). The protein (N ×
5.7), starch, ash, moisture and wet gluten content of the steamed bread
flour was 11.1%, 73.5%, 0.4%, 13.3% and 29.7%. The flour was free of
additives.
2.2. Extraction of gliadin and glutenin
Gluten fraction was extracted from wheat flour based on the method
described by Kieffer, Schurer, Köhler, and Wieser (2007) with slight
modification. Briefly, 300 g of wheat flour was mixed with 160 mL NaCl
solution (0.4 mol/L) in a mixer for 6 min and then resting for 20 min.
Next, the dough was first washed with a NaCl solution until a crude
gluten was formed, then washed with deionized water to remove NaCl,
and the wet gluten was lyophilized. After that, dried gluten (100 g) was
gently shaken with 200 mL chloroform for 1 h at room temperature and
then filtered through filter paper to obtain defatted gluten. The defatted
gluten was then allowed to dry at room temperature and grounded to
2
LWT 153 (2022) 112562
pass through a 100-mesh sieve.
Above defatted gluten (100 g) was dispersed in 300 mL ethanol:
water (7:3 v/v), and stirred on a magnetic stirrer for 3 h at 25 ◦ C. After
centrifugation for 20 min (3000 g) at 4 ◦ C, the supernatant was collected
and the lower precipitate was again extracted with ethanol: water (7:3
v/v) for three times. The supernatants were pooled and the ethanol was
removed using a rotary evaporator at 30 ◦ C to obtain gliadin. The pre­
cipitate after repeated extraction of ethanol: water (7:3 v/v) was glu­
tenin. Then the obtained gliadin and glutenin were freeze-dried and
grounded to pass through a 100-mesh sieve.
2.3. Dough formulation
The dough was prepared based on the method described in Chang
et al. (2020). The unleavened dough formula was consisted of 100 g
wheat flour and a certain amount of water which was determined on the
basis of farinograph water absorption test. Flour was replaced by gluten
at the concentrations of 0%, 0.5%, 1.0%, and 1.5% of wheat flour dry
basis (w/w), respectively, and gluten was composed of three different
glutenin/gliadin ratios (1:1, 1:2, and 2:1). All materials of each formula
were mixed in a kneader (HM740, Hanshang Co., Ltd., China) for 5 min
(4 min at low speed, then 1 min at medium speed). The dough was
divided into pieces and kneaded to smooth for further analysis. The
leavened dough was made according to the above procedure for un­
leavened dough, with some modification, in which 0.7% w/w active dry
yeast was added to the recipe and the dough was leavened at 30 ◦ C with
85% ± 1% relative humidity for 40 min.
According to the difference of gluten protein addition and glutenin to
gliadin ratio, the 10 samples were named BF, A1-P1, A1-P2, A1-P3, A2-
P1, A2-P2, A2-P3, A3-P1, A3-P2, and A3-P3, respectively, where BF
represents control sample without gluten protein addition, A1-A3 rep­
resents gluten protein addition level of 0.5%, 1.0%, and 1.5%, and
P1–P3 represents the ratio of glutenin to gliadin of 1:1, 1:2, and 2:1.
2.4. Fast-frozen steamed bread production
The leavened dough made by the above steps was divided into 80 g
pieces, rounded, molded and continued fermentation for 10 min.
Finally, the fermented dough balls were steamed for 25 min in a steamer.
After steaming, the steamed bread was cooled at room temperature for 1
h and placed into a freezer at − 35 ◦ C for fast cooling. And then, frozen
steamed breads were packed in polypropylene bags and stored at − 18
◦
C. After fixed days of frozen storage (0, 10, 20, 30, 40, 50 and 60 d),
part of the frozen steamed bread was freeze-dried for further analysis,
and the other part was re-steamed in steamer for 20 min to determine
the quality properties.
2.5. Dough rheological characterization
2.5.1. Alveograph analysis
Alveograph parameters were performed by the Alveograph NG
Consistograph (Chopin Co., Ltd., France) with a few modifications. The
250 g of the reconstituted flour sample was poured into the Alveograph
mixer, and a solution of sodium chloride (2.5% w/w) equal to 75%
water absorption was dispensed from the burette for mixing. Dough
resistance to extension (P), extensibility (L), strength (W), inflation
index (G) and configuration ratio (P/L) were determined (Agyare, Addo,
Xiong, & Akoh, 2005).
2.5.2. Frequency sweeping test
Small-amplitude oscillatory shear test of the dough samples were
carried out using Haake RheoStress 6000 (Thermo Scientific, Karlsruhe
Co., Ltd., Germany) with a parallel stainless plate (35 mm diameter).
The unleavened and leavened dough were placed on the plate of the
rheometer, the measuring gap between the plates was fixed at 1 mm, and
the edge of the sample was covered with silicone oil to minimize the
X. Qian et al. LWT 153 (2022) 112562

water evaporation during testing. Before frequency sweep measure­ placed over the SEM microscope stage. The micrographs were taken at
ment, the strain sweep was performed to determine the range of the 300× magnification.
linear viscoelastic region of dough. Then, the sample was allowed to rest
for 5 min and the frequency sweep measurement was conducted at 25 ◦ C 2.8. Statistical analysis
by frequency ranging from 0.1 to 10 Hz and strain amplitude of 0.5% in
the linear viscoelastic region. All analyses were performed at least three times. The results obtained
were subjected to the one-way analysis of variance (ANOVA) and eval­
2.6. Quality of fast-frozen steamed bread uated by Duncan’s method. The significant difference was set at p <
0.05.
2.6.1. Determination of specific volume
The steamed bread after 1 h of re-steaming were weighed and their 3. Results and discussion
volumes were determined by the millet replacement method, and the
specific volume was expressed at the ratio of the steamed bread volume 3.1. Dough alveograph properties
to its weight (cm3/g) (He et al., 2020).
The alveograph parameters (Table 1) showed that the addition of
2.6.2. Crumb hardness measurement gluten protein resulted in significant changes in the dough alveograph
The effects of gluten protein addition on the hardness properties of properties, as evidenced by the increase of the resistance to deformation
fast-frozen steamed bread were investigated using TA-XT2i Texture (P), baking strength (W) and configuration ratio (P/L), and the re­
Analyzer (Stable Microsystems Co., Ltd., UK) equipped with a 25 mm ductions of the extensibility (L). The highest values of P and P/L were
diameter cylindrical probe. Each steamed bread after 1 h of re-steaming observed in the 1.5%-2:1 dough sample, up from 55.50 mmH2O to 70.25
was sliced equally into 15 mm thickness and the central slice was mmH2O and 0.83 to 1.22, respectively. When the gluten addition level
selected for texture measurement. Meanwhile, measurement parameter was 0.5%, the difference of glutenin and gliadin content did not cause
setting was: pre-test speed 3.0 mm/s, test speed 1.0 mm/s, post-test significant changes of dough alveograph index. It is obvious that the
speed 3 mm/s and strain 60%. influence of glutenin/gliadin ratio on the values of dough alveograph
index is not pronounced at a low amount of gluten addition. Meanwhile,
2.7. Mechanism of quality changes of fast-frozen steamed bread when the amount of gluten protein added was 1.0% and 1.5%, the
maximum proportion of glutenin added gave the dough the largest P, W
2.7.1. SDS-soluble protein content analysis and P/L values. The increase of P and P/L values was accompanied by
The SDS-soluble protein of samples was extracted according to the the increase of dough elasticity and the decrease of dough extensibility.
method of Lagrain, Brijs, Veraverbeke, and Delcour (2005) and then the The above results confirmed that the glutenin impart strength and
SDS-soluble protein content was measured by size-exclusion high per­ elasticity to dough. Generally, the glutenin subunit formed ordered
formance liquid chromatography (SE-HPLC). Each sample (containing fibrous macromolecular polymers through the exchange reaction of
1.7 mg of protein) were suspended in 1 mL sodium phosphate buffer sulfur and disulfide bond in intramolecular and intermolecular, which
(0.1 mol/L, pH 6.9) containing 0.5% (w/v) SDS, mixed for 30 s and then showed that the dough gradually became elastic from macroscopically
shaken for 2 h at room temperature. The supernatant was collected after (Uthayakumaran et al., 1999). Therefore, the addition of more glutenin
centrifugation at 10,000 g for 10 min and then filtered through 0.45 μm given the dough more strength and elasticity. In particular, the highest
filters before SE-HPLC analysis. Under reducing conditions, the value of W was distinctly observed in the 1.0%-2:1 dough sample,
freeze-dried samples were dissolved in 1.0 mL SDS buffer with 1.0%
(w/v) dithiothreitol (DTT) and the remaining extraction steps were the Table 1
same as above. Effect of gluten protein addition and glutenin/gliadin ratio on the dough
SE-HPLC was performed on Agilent 1260 HPLC system (Agilent alveograph properties.
Technologies Co., Ltd., USA) analysis. Then, 20 μL of the SDS-soluble Sample P (mmH2O) L (mm) G W (mJ) P/L
protein extracts were injected to the Biosep-SEC-4000 column (300
BF 55.50 ± 75.33 ± 18.28 ± 111.67 0.83 ±
mm × 7.8 mm, Phenomenex Co., Ltd., USA). The elution solvent was
±
0.58f 3.51a 1.29a 2.31c 0.07f
acetonitrile: water (1:1 v/v) containing 0.1% (v/v) trifluoroacetic acid A1-P1 59.00 ± 64.67 ± 17.90 ± 113.33 ± 0.92 ±
(TFA) with a flow rate of 0.5 mL/min. The column temperature was 30 0.82e 2.52bc 0.72a 2.08c 0.06ef
◦
C and the eluted proteins were monitored by UV absorbance at 214 nm. A1-P2 57.00 ± 66.00 ± 17.62 ± 110.00 ± 0.91 ±
2.16ef 1.73bc 0.64ab 3.61c 0.06ef
The SDS-soluble protein content in non-reducing conditions was calcu­
A1-P3 58.75 ± 58.33 ± 17.20 ± 112.00 ± 0.99 ±
lated from the corresponding peak area and expressed as percentage of 0.96e 0.58de 0.40ab 2.65c 0.06de
peak area of sample under reducing conditions. A2-P1 61.75 ± 55.33 ± 16.84 ± 124.67 ± 1.08 ±
2.87cd 1.53e 0.59ab 1.53b 0.04cd
2.7.2. Freezable water changes A2-P2 59.50 ± 63.33 ± 17.56 ± 104.00 ± 1.04 ±
2.38de 1.15bcd 1.31ab 3.00d 0.09cd
The freezable water content was investigated by a differential
A2-P3 65.25 ± 60.33 ± 17.30 ± 137.00 ± 1.18 ±
scanning calorimeter (Q20, TA Co., Ltd., USA) according to Xin, Nie, and 1.26b 6.60cde 1.08ab 3.56a 0.07ab
Chen (2018) with a slight modification. Approximately 10 mg of A3-P1 62.25 ± 54.33 ± 16.68 ± 112.00 ± 1.11 ±
fast-frozen steamed bread was cut out then placed into the aluminum 0.50c 2.89e 0.61ab 1.41c 0.01bc
pans and hermetically sealed. All pan was cooled to − 30 ◦ C and equil­ A3-P2 62.25 ± 66.67 ± 15.78 ± 109.67 ± 1.11 ±
1.50c 3.51b 3.06b 2.08c 0.01bc
ibrated for 5 min at this temperature and then subsequently heated to A3-P3 70.25 ± 58.00 ± 16.98 ± 126.33 ± 1.22 ±
10 ◦ C at the rate of 1 C/min. Enthalpy (ΔH) of ice melting was calcu­
◦
3.06a 1.00de 0.37ab 2.89b 0.03a
lated by Universal Analysis software provided by the DSC manufacturer.
P - dough resistance to extension, L - extensibility, G - inflation index, W -
strength, and P/L - configuration ratio.
2.7.3. Microstructure Data were means ± standard deviations (n = 3 for dough production, n = 6 for P,
The internal microstructure of steamed bread samples were observed L, G, W and P/L). Different letters in the column represent significant differences
by scanning electron microscope (Co.S–3400N II, HITACHI Co., Ltd., among mean values (p < 0.05). BF represents control sample without gluten
Japan). The freeze-dried steamed bread samples were fractured to protein addition; A1-A3 represents gluten protein addition level of 0.5%, 1.0%,
expose interior structure (1 cm × 1 cm × 1 cm), coated with gold, and and 1.5%; P1–P3 represents the ratio of glutenin to gliadin of 1:1, 1:2, and 2:1.

3
X. Qian et al. LWT 153 (2022) 112562

followed by 1.5%–2:1 and 1.0%-1:1, and the corresponding values of W and maintain the balance between elasticity and extensibility. More­
were 137.00 mJ, 126.33 mJ and 124.67 mJ, respectively. In the over, combining with the changes of specific volume (Fig. 2a) and
1.5%-2:1 dough sample, although the proportion of glutenin was high­ hardness (Fig. 2b), the alveograph parameters of ideal-quality flour for
est, the amount of gliadin was also higher, which increased the dough fast-frozen steamed bread could be determined.
viscosity and thus weakened its strength. For the production of
fast-frozen steamed bread, the dough strength should not be too low or
3.2. Dough frequency sweeping properties
too high. If the gluten strength is too weak, the dough would be too soft
to maintain a stable internal network structure, further causing the
It is widely acknowledged that the viscoelastic behavior of dough is
shrinkage of the steamed bread, like biscuit flour (Li, Hou, Chen, Chung,
dependent on its three-dimensional network formed by the interaction
& Gehring, 2014). It was reported that the wheat flour with high-gluten
between gluten and starch or non-starch polysaccharides (Pedersen,
strength was better for making frozen cooked noodles (Yue et al., 2017).
Kaack, Bergsøe, & Adler-Nissenb, 2004). Moreover, the viscoelastic
Specifically, in order to avoided the quality deterioration of fast-frozen
properties of dough is highly affected by the gluten quality that depends
steamed bread, it is necessary to strengthen the gluten network structure
on the glutenin and gliadin content, ratio and subunit composition

Fig. 1. Effect of gluten protein addition and glutenin/gliadin ratio on viscoelastic properties of unleavened doughs (G′ (a), G′ ′ (b) and tanδ (c)) and leavened doughs
(G′ (d), G′ ′ (e) and tanδ (f)). □BF, ○A1-P1, △A1-P2, ▽A1-P3, ◇A2-P1, ◃A2-P2, ▹A2-P3, A3-P1, ☆A3-P2, A3-P3. BF represents control dough sample without
gluten protein addition; A1-A3 represents gluten protein addition level of 0.5%, 1.0%, and 1.5%; P1–P3 represents the ratio of glutenin to gliadin of 1:1, 1:2, and 2:1.

4
X. Qian et al.
(Lefebvre & Mahmoudi, 2007). Therefore, the frequency sweep was
implemented to evaluate how the dough structure and viscoelasticity
change with the different gluten contents and glu/gli ratios. The visco­
elastic properties of unleavened doughs and leavened doughs with
different gluten contents and glu/gli ratios are shown in Fig. 1, which
displays the storage moduli (G′ ), loss moduli (G′′ ) and loss factor (tanδ)
of the dough samples as a function of frequency. Specifically, the above
three parameters represent the elasticity, viscosity and viscoelasticity of
the dough, respectively. When tanδ<1, it means that the sample show
more elastic and lesser viscous behavior (Berski, Krystyjan, Buksa, Zięć,
& Gambu).
For all of the dough samples, the values of G′ and G′′ had a tendency
to increase with increasing angular frequency, and all samples exhibited
higher values of G′ than those of G′′ (corresponding to the tanδ<1),
suggesting a tendency toward solid and elastic-like behavior for all
samples (Federici, Jones, Selling, Tagliascod, & Campanella, 2020).
According to the obtained results, it was evident that the addition of
gluten to the dough led to the changes of G′ , G′′ and tanδ compared to
the control dough sample. This result was in accord with Selaković et al.
Fig. 2. Effect of gluten protein addition and glutenin/gliadin ratio on the
specific volume (a) and hardness (b) of steamed bread during frozen storage.
BF, A1-P1, A1-P2, A1-P3, A2-P1, A2-P2, A2-P3, A3-P1,
A3-P2, A3-P3. BF represents control sample without gluten protein addi­
tion; A1-A3 represents gluten protein addition level of 0.5%, 1.0%, and 1.5%;
P1–P3 represents the ratio of glutenin to gliadin of 1:1, 1:2, and 2:1. Errors bars
represent ± standard deviation (n = 3 for fast-frozen steamed bread production,
n = 6 for specific volume and hardness analysis).
5
LWT 153 (2022) 112562
(2021), who reported that the viscoelasticity and adaptability to applied
stress of laminated dough were obviously affected by the addition of
vital gluten.
For unleavened dough, as can be seen from Fig. 1a, the highest value
of G′ (strongest dough elasticity) was distinctly observed in the dough
when the amount of gluten protein was 1.5% and glu/gli ratio was 2:1,
followed by 1.0%–2:1, While, the control sample had the lowest elastic
properties. Specifically, with the same amount of gluten added, the
higher the glutenin content, the more elastic the dough will be. At the
same time, in Fig. 1b, the highest value of G′′ (strongest dough viscosity)
was clearly observed in the 1.5%-1:2 dough sample, and the control
sample exhibited lowest viscosity. This demonstrated that the addition
of gliadin could significantly change the viscosity characteristic of the
dough and provide the adhesive force needed for the dough fluidity and
extension. As presented in Fig. 1c, the values of tanδ had apparently
changed, due to the different variations of G′ and G′′ , suggesting that the
addition of gluten protein substantially altered the three-dimensional
network structure inside the dough samples. In general comparison,
the dough sample added with 1.0%–2:1 had the lowest tanδ value and
preferred to show elasticity behavior, while the dough sample added
with 1.0%-1:2 possessed the highest tanδ value and tended to display
viscosity-like.
In fact, fermentation process is indispensable in steamed bread
making, and the state and rheological properties of dough changed
during fermentation. Compared to unleavened dough samples, the
leavened dough samples behaved more like a liquid and viscous, which
can be 2confirmed by the significant increase in the tanδ value after
dough was fermented (Fig. 1f). This phenomenon was mainly due to a
series of phyco-chemical changes in the fermentation process: dough
volume expanded obviously, the internal gas chamber formed, and the
gluten structure stretched (Yue et al., 2020). For leavened dough, the
control sample also had the lowest elasticity and viscosity. Meanwhile,
like unleavened dough, the highest value of G′ and G′′ were also
distinctly observed in the 1.5%-2:1 dough sample and 1.5%-1:2 dough
sample, respectively. However, it should be noted that only a good
synergy between gluten and gliadin can form the ideal structure of
gluten protein network, further resulting in good dough characteristics
and steamed bread quality.
3.3. Fast-frozen steamed bread quality
One of the key indicators of steamed bread quality is the specific
volume. As presented in Fig. 2a, the specific volume of steamed bread
decreased significantly with the increase of frozen storage time, and
further indicated that frozen storage induced the decrease of specific
volume. The specific volume of the control fast-frozen steamed bread
after 60 d storage were decreased from 2.72 cm3/g to 2.38 cm3/g.
Notably, combined with the previous studies and our existing study, it
was found that the quality deterioration of fast-frozen steamed bread
(including the reduction of specific volume and the increase of hardness)
was mainly attributed to the water recrystallization and protein depo­
lymerization induced by frozen storage, in which protein depolymer­
ization was mainly caused by the breakdown of heat-induced glutenin-
gliadin disulfide cross-linking (Esselink et al., 2003; Qian, Gu, Sun, &
Wang, 2021). The addition of gluten protein has remarkable effects on
the specific volume of fast-frozen steamed bread. To be specific, for
frozen storage 0 d, adding appropriate amount of gluten protein could
significantly increase the specific volume of steamed bread. While,
adding excessive amount of gluten protein (more than 1.0%) could have
a negative impact on the specific volume of steamed bread, leading to
the decrease of specific volume and the shrinkage of steamed bread. This
is due to the breakdown of the balance between the gas capacity and
retractive force of dough. At the same time, a suitable ratio of glutenin to
gliadin in wheat flour could greatly support the gluten structure, and
obtain the steamed bread with better quality. Moreover, in the process of
frozen storage, adding appropriate amount of gluten protein delayed the
X. Qian et al.
decrease of the specific volume, indicating that the internal network
structure was not easily damaged by ice crystals. In addition, according
to Fig. 2a, when gluten protein added at 1.0% and glu/gli ratio was 1:1,
the specific volume of steamed bread could be significantly increased,
and the deterioration of the specific volume of steamed bread could be
better delayed during the frozen storage.
The hardness properties of fast-frozen steamed bread were
researched depending on different gluten protein added. As shown in
Fig. 2b, the hardness of fast-frozen steamed bread decreased signifi­
cantly with the increase of frozen storage time. In particular, the hard­
ness values of the control fast-frozen steamed bread after frozen for 0 d,
30 d and 60 d were 23.10 N, 23.59 N and 31.39 N, while the hardness
values of the fast-frozen steamed bread significantly decreased to 18.43
N, 20.93 N and 25.46 N after 1.0%-1:1 gluten protein addition,
respectively. It was likely that appropriate addition of gluten protein
restricted the movement of freezable water molecules in the frozen
steamed bread, thereby reducing the number and volume of ice crystals.
Meanwhile, the results showed that the steamed bread sample added
with 1.0%-1:1 had the strongest internal structure stability, which was
further confirmed by the obvious delay of protein depolymerization and
water loss during frozen storage. At the same time, the hardness of
steamed breads exhibited the exact opposite trends to their specific
volumes.
Combining with the changes of specific volume (Fig. 2a) and hard­
ness (Fig. 2b), it can be assumed that when gluten protein added at 1.0%
and glu/gli ratio was 1:1, fast-frozen steamed bread had the maximum
specific volume and the minimum hardness, indicating that the internal
network structure of steamed bread had perfect freeze stability under
the gluten addition level of 1.0%-1:1. Meanwhile, it can be further
inferred that the viscoelasticity of dough formed under 1.0%-1:1 gluten
added was more suitable for the production of fast-frozen steamed
bread, and the alveograph parameters of ideal-quality flour (specific
alveograph index: P = 62, L = 55, P/L = 1.08) for fast-frozen steamed
bread could be determined.
3.4. SDS-soluble content changes
The protein solubility in SDS solutions was a well indication of de­
gree of crosslinking (Hayta & Schofield, 2004). In general, the protein
depolymerizes, resulting in a decrease in relative molecular weight,
which in turn leads to an increase in SDS-soluble protein content.
Moreover, the aggregation behavior and solubility of proteins are
closely related to the quality of the final product (Liu et al., 2019). Ac­
cording to the results of specific volume and hardness, the effect of
gluten protein addition on the protein solubility of steamed bread during
frozen storage when the ratio of glutenin to gliadin was fixed at 1:1 was
compared in this part.
It could be seen in Table 2, the protein solubility of fast-frozen
Table 2
Effect of gluten protein addition and glutenin/gliadin ratio on SDS-soluble
protein content of steamed bread during frozen storage.
Sample SDS-soluble protein content (%)
0d 30 d 60 d
cA bA
BF 10.28 ± 0.04 11.65 ± 0.08 12.54 ± 0.02aA
A1-P1 9.84 ± 0.45bAB 11.42 ± 0.30aAB 11.69 ± 0.23aB
A2-P1 9.55 ± 0.14bB 11.17 ± 0.12aBC 11.48 ± 0.16aBC
A3-P1 9.23 ± 0.15bB 10.83 ± 0.07aC 11.18 ± 0.18aC
Data were means ± standard deviations (n = 3 for fast-frozen steamed bread
production, n = 3 for SDS-soluble protein content analysis). Different small
letters in the same line and different capital letters in the same column represent
significant differences among mean values (p < 0.05). BF represents control
sample without gluten protein addition; A1-A3 represents gluten protein addi­
tion level of 0.5%, 1.0%, and 1.5%; P1 represents the ratio of glutenin to gliadin
of 1:1.
6
LWT 153 (2022) 112562
steamed bread increased as storage time prolonged, suggesting that
frozen storage induced the depolymerization of protein. Specifically, our
previous research on the change of gluten aggregation properties in fast-
frozen steamed bread found that the proteins depolymerization induced
by frozen storage was mainly involved in the breakdown of heat-induced
glutenin-gliadin disulfide cross-linking (Qian et al., 2021). Similarly, the
increase in protein solubility of frozen cooked noodles was observed
during frozen storage (Liu et al., 2019). The addition of gluten protein
significantly reduced the amount of SDS-soluble protein, which was
mainly attributed to the tighter structure of gluten network and
enhanced interaction between proteins caused by the addition of gluten
protein. It can be speculated that the fast-frozen steamed bread with
more stable internal structure had better freeze stability. After frozen for
60 d, the protein solubility of steamed bread without adding gluten was
increased by 2.26%, while that with gluten added was increased by
1.85%, 1.93% and 1.95%, respectively. The addition of gluten not only
enhanced the protein aggregation behavior, but also delayed the
migration of water and the formation of ice crystals, thus reducing the
destruction of internal structure of steamed bread in the process of
frozen storage.
3.5. Freezable water changes
In the DSC measurement, the enthalpy of ice melting (ΔH) could be
used to reflect the freezable water content. A smaller melting enthalpy of
fast-frozen steamed bread represent a lower freezable water content.
The ice melting enthalpy (ΔH) of fast-frozen steamed bread under
different gluten addition was shown in Fig. 3. For steamed bread without
adding gluten, with the increment of frozen storage, the ΔH obviously
increased from 27.54 J/g to 33.86 J/g. The above results indicated that
the frozen storage could lead to the amount of freezable water content
increased, which might further result in the decline of sensory and
texture of steamed bread. For fast-frozen steamed bread, the amount of
freezable water content increased which may be caused by the weak­
ened binding between protein or starch to water during frozen storage.
This result was in agreement with that of Liang et al. (2021), who also
demonstrated that the increment of freezable water might be ascribed to
the ice growth and recrystallization, which further lead to the deterio­
ration of product quality.
When the ratio of glutenin to gliadin was fixed at 1:1 (Fig. 3a), the
freezable water content decreased significantly with the increase of
gluten. After frozen for 0 d, the ΔH was 27.54 J/g without adding gluten
protein, while the ΔH of steamed bread enriched with gluten protein
was 27.31 J/g, 25.12 J/g and 24.98 J/g, respectively. This was mainly
attributed to the strong hydrophilicity of gluten, which leads to the
weakening of water mobility and the reduction of freezable water con­
tent. When frozen for 60 d, the ΔH of steamed bread without adding
gluten was increased by 6.32 J/g, while that of steamed bread with 1.5%
gluten was increased by 5.01 J/g. The addition of gluten effectively
strengthened the binding between protein and water, and inhibited the
migration of water during whole frozen storage. This further reduced
physical damage of ice crystals to the gluten network, thereby improving
stability of steamed bread during frozen storage.
When the gluten content was fixed at 1.0% (Fig. 3b), the effect of the
ratio of glutenin to gliadin on the freezable water content of fast-frozen
steamed bread was investigated. In the whole frozen storage, ΔH of fast-
frozen steamed bread supplemented with 1:2 of gluten was lower than
that of the other three groups, followed by those with 1:1 of gluten.
These results demonstrated that compared with glutenin, the addition of
gliadin distinctly reduced the freezable water content, indicating that
the good water-holding characteristics of steamed bread were signifi­
cantly related to the gliadin content. On the other hand, the addition of
gliadin enhanced the viscosity of the dough, which in turn changed the
rheological properties of the dough. Similarly, Wang, Xu, Nikoo, Ocen,
Wu, and Yang (2014) compared the effect of frozen storage on the
freezable water content of gluten, glutenin and gliadin, and concluded
X. Qian et al. LWT 153 (2022) 112562

Fig. 3. Effect of gluten protein addition and glutenin/gliadin ratio on the melting enthalpy (ΔH) of freezable water in steamed bread during frozen storage. (a): the
ratio of glutenin to gliadin was fixed at 1:1, and the amount of gluten protein addition was changed; (b): the amount of gluten protein addition was fixed at 1.0%, and
the ratio of glutenin to gliadin was changed. BF represents control sample without gluten protein addition; A1-A3 represents gluten protein addition level of 0.5%,
1.0%, and 1.5%; P1–P3 represents the ratio of glutenin to gliadin of 1:1, 1:2, and 2:1. Errors bars represent ± standard deviation (n = 3 for fast-frozen steamed bread
production, n = 3 for freezable water analysis.

Fig. 4. Effect of gluten addition on microstructure of steamed bread during frozen storage; (a): BF, (b): A1-P1, (c): A2-P1, (d): A3-P1. BF represents control sample
without gluten protein addition; A1-A3 represents gluten protein addition level of 0.5%, 1.0%, and 1.5%; P1 represents the ratio of glutenin to gliadin of 1:1.

7
X. Qian et al.
that gliadin had the best water holding capacity and could stabilize the
structure of gluten network.
3.6. Microstructure changes
The internal network structures of steamed bread with different
frozen storage time (0 d, 30 d and 60 d) and gluten addition (0%, 0.5%,
1.0% and 1.5%) were exhibited in Fig. 4. As shown in Fig. 4a, the in­
ternal structure of fast-frozen steamed bread frozen stored for 0 d dis­
played a clear and continuous pore structure. While, an irregular holes
and loose structure were gradually observed when it frozen stored for
extended time, indicating that the gluten matrix structure of fast-frozen
steamed bread suffered from destruction of the mechanical damage
during frozen storage. Similar destruction phenomena were observed in
the microstructure of frozen cooked noodles by Liu et al. (2019).
Meanwhile, during the whole process of frozen dough, including
freezing, frozen storage and thawing, the dough was exposed to the
stresses leading to the destroy of internal structure (Gaikwad & Arya,
2018). The mechanical damage of fast-frozen steamed bread may be
related to water migration and ice crystallization, which further lead to
the depolymerization of gluten polymers. These results were consistent
with the changes of freezable water (Fig. 3) and protein solubility
(Fig. 2). Therefore, it is critical to add gluten to prevent water migration
and gluten depolymerization, which could effectively improve
fast-frozen steamed bread quality. Next, the effects of different protein
addition (the ratio of glutenin to gliadin was fixed at 1:1) on the
microstructure of steamed bread during frozen storage were discussed.
After comparison, it was found that when the protein addition con­
tent was 1.0% (Fig. 4c), a relatively regular gluten network structure
could be observed after frozen storage for 60 d. At this time, the fast-
frozen steamed bread had the maximum specific volume and the mini­
mum hardness (Fig. 2). This suggested that the addition of 1.0%-1:1 of
gluten provides adequate gluten structural stability and hydrophilicity,
which help to counteract the negative effects of frozen storage on the
fast-frozen steamed bread quality. Observing microstructure of fast-
frozen steamed bread with 1.5%-1:1 gluten, it was noticed that the
apparent shrinkage of the internal structure of steamed bread is mainly
attributed to the higher elasticity and the lower extensibility. Adding
excessive amount of gluten protein (more than 1.0%) could have a
negative impact on the microstructure of steamed bread. Meanwhile, the
improvement of microstructure of fast-frozen steamed bread was not
obvious when adding less gluten protein. Those results confirmed that
the addition of more than 1.0% of gluten was not technologically
justified.
4. Conclusion
The present study demonstrated that the specific volume and hard­
ness qualities of fast-frozen steamed bread were deteriorated during
frozen storage. An irregular holes and loose structure with small frag­
ments were gradually observed when it frozen stored for extended time.
The freezable water content and protein solubility increased during
frozen storage, suggesting that the water characteristics and protein
network during frozen storage had major effects on the quality of fast-
frozen steamed bread. The appropriate addition of gluten alleviated
the deterioration of specific volume, hardness, and microstructure
qualities of fast-frozen steamed bread during frozen storage. By
analyzing the mechanism of quality improvement, we found that the
protein solubility and melting enthalpy (ΔH) of fast-frozen steamed
bread were decreased after gluten added. This was closely associated
with the fact that the addition of gluten minimized freezable water
content, inhibited protein depolymerization, and enhanced gluten
structure stability. The results further indicated that 1.0%-1:1 gluten
addition resulted in steamed bread with supreme specific volume and
minimum hardness during frozen storage.
From the technological aspect the addition of 0.5% and 1.5% of
8
LWT 153 (2022) 112562
gluten was not justified. Based on the obtained results, 1.0%-1:1 of
gluten was considered to be the optimal amount for the production of
fast-frozen steamed bread. Adequate elasticity and extensibility of
dough (specific alveograph index: P = 62, L = 55, P/L = 1.08) with
1.0%-1:1 of gluten addition resulted in the best quality and freeze sta­
bility of the fast-frozen steamed bread. This work also contributes to a
better understanding of the relationship between the rheological per­
formance of dough and fast-frozen steamed bread quality from the
perspective of protein quality.
CRediT authorship contribution statement
Xiaojie Qian: Conceptualization, Software, Validation, Investiga­
tion, Data curation, Methodology, Writing – original draft, Writing –
review & editing. Yujuan Gu: Software, Visualization, Writing – review
& editing. Binghua Sun: Conceptualization, Writing – review & editing,
Supervision. Sen Ma: Conceptualization, Software, Visualization.
Xiaoling Tian: Conceptualization, Visualization. Xiaoxi Wang: Re­
sources, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The present research was conducted by the research fund of Henan
University of Technology and Key scientific and technological project of
Henan Province (No.212102110350).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2021.112562.
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