Food Hydrocolloids 106 (2020) 105885

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Food Hydrocolloids
journal homepage: http://www.elsevier.com/locate/foodhyd

Influence of gluten and starch granules interactions on dough mixing

properties in wheat (Triticum aestivum L.)
Xin Gao a, 1, Jingyang Tong a, 1, Lei Guo a, Liwei Yu a, Shaopeng Li a, Bingpeng Yang a,
Libin Wang b, Yang Liu b, Faji Li b, Jun Guo b, Shengnan Zhai b, Cheng Liu b, Ata-ur Rehman c,
Asgar Farahnaky d, Pei Wang e, Zhonghua Wang a, **, Xinyou Cao b, *
a
State Key Laboratory of Crop Stress Biology in Arid Areas and College of Agronomy, Northwest A&F University, Yangling, Shaanxi, 712100, China
b
Crop Research Institute, Shandong Academy of Agricultural Sciences/National Engineering Laboratory for Wheat and Maize/Key Laboratory of Wheat Biology and
Genetic Improvement in North Yellow and Huai River Valley, Ministry of Agriculture, Jinan, 250100, China
c
E H Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia
d
Biosciences and Food Technology, School of Science, RMIT University, Bundoora West Campus, Melbourne, VIC, 3083, Australia
e
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China

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

Keywords: Wheat flour dough can be regarded as a complicated mixture in which gluten forms a continuous three-
Wheat dough dimensional network filled with starch particles. Previous studies have concentrated on the influences of
Gluten-starch interactions either gluten or starch alone on the rheological properties of wheat dough and found the components of gluten
Dough mixing properties
and starch responsible for the dough behavior, including use of parameters, such as the content of protein, wet
Quantitative analysis
gluten and amylose, for the evaluation of wheat quality. Earlier, in our study of six wheat materials, significant
differences were observed in size distribution of starch granules affecting the dough mixing behavior. However,
to our knowledge little research has been undertaken on the association of starch granules and protein networks
on dough formation. In this study, the six wheat lines were further explored for the effect of gluten-starch in­
teractions on dough properties. In order to achieve this, the wheat flour samples were quantified for gluten
secondary structures, percentage of unextractable polymeric protein, high- to low-molecular-weight glutenin
ratio, gluten micro-structure, number distribution of starch granules and moisture distribution in dough samples.
A/Lacunarity (A-type starch granule content divided by lacunarity of gluten network) and B/A/Lacunarity (B- to
A-type starch granule ratio divided by lacunarity) were proposed to characterize the level of interactions of
differently sized starch granules to gluten protein networks in the dough. Results showed that dough stability
time increased with the increase of B/A/Lacunarity, as closely packed dough, due to close interaction between
starch granules and gluten, exhibited greater dough stability. Application of A/Lacunarity and B/A/Lacunarity as
novel indicators is proposed to further elucidate precise influences between starch and gluten for better evalu­
ation of dough properties.

1. Introduction subsequent mechanical mixing, the gluten, as a natural hydrocolloid,

Bread wheat (Triticum aestivum L.) is one of the most important cereal
crops in the world and wheat-based food products are the main con­
stituents of human diets in many countries (Bakke & Vickers, 2007).
Wheat flour is mainly composed of starch, protein and lipid (Guo, Yu,
Copeland, Wang, & Wang, 2018). Upon addition of water and
interacts with water molecules to create wheat dough, embracing a
specific three-dimensional network filled with starch granules. The
rheological and mixing properties of wheat dough are responsible for the
quality of the final product and thus can be used as important indicators
of wheat quality (Safariardi & Phanthien, 1998; Tronsmo et al., 2003).
Gluten proteins constitute 80%–85% of wheat flour proteins and are

* Corresponding author. Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan, Shandong Province, 250100, China.
** Corresponding author. Northwest A&F University, 3 Taicheng Rd, Yangling, Shaanxi Province, 712100, China.
E-mail addresses: zhwangnew@126.com (Z. Wang), caoxinyou@126.com (X. Cao).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.foodhyd.2020.105885
Received 1 August 2019; Received in revised form 22 March 2020; Accepted 24 March 2020
Available online 25 March 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
X. Gao et al.
recognized as crucial components determining wheat dough function­
ality (Goesaert et al., 2005). The gluten proteins mainly contain
high-molecular-weight glutenin subunits (HMW-GSs),
low-molecular-weight glutenin subunits (LMW-GSs) and gliadins.
HMW-GSs and LMW-GSs form glutenin macropolymers (GMPs) through
intermolecular disulfide bonds within the gluten network (MacRitchie,
2014). The GMPs, according to their different solubility in 0.5% sodium
dodecyl sulfate (SDS) solution, can be classified into SDS-unextractable
polymeric protein (UPP) comprising of larger GMP complexes, and
extractable polymeric protein (EPP) comprising of smaller GMP com­
plexes (Vensel, Tanaka, & Altenbach, 2014). The UPP% is the percent­
age of UPP relative to total polymeric protein, and previous studies have
reported positive correlations between UPP% and dough rheological
properties (Ferrise, Bindi, & Martre, 2015; Gupta, Khan, & MacRitchie,
1993; Liu et al., 2016). Several studies have reported that a greater high-
to low-molecular-weight glutenin (H/L) ratio can improve dough
behavior and wheat quality (Bouacha, Rhazi, Aussenac, Rezgui, &
Nouaigui, 2015; Visioli et al., 2018; Zhu & Khan, 2004). Recently, the
micro-structure of gluten was quantitatively analyzed by several pa­
rameters for their putative link to dough mixing behavior and wheat
quality (Gao et al., 2018). These parameters included gluten area,
number of gluten junctions, junction density, gluten length, gluten
end-points and end-point rate, branching rate and lacunarity. The spe­
cific interactions between gluten and starch in the micro-structure of
wheat dough, where gluten forms a continuous reticular skeleton in
which starch granules act as filling particles, has been shown to influ­
ence dough behavior (Jekle, Mühlberger, & Becker, 2016). Therefore,
determining the properties of separated gluten and starch independently
may not fully explain the behavior of wheat dough. It is therefore
pertinent to analyze gluten-starch matrix by investigating all the
aforementioned parameters individually for their contribution in the
formation of a quality and functional dough.
Starch accounts for approximately 70%–80% of wheat flour and also
plays a crucial role in determining dough behavior (Martínez & Go �mez,
2017; Shewry, 2009). Wheat starch granules exhibit different sizes and
shapes during the development of grain structure, and thus have been
classified into three types as, A (23–28 μm), B (9–11 μm) and C (2–3 μm).
However, B- and C-type granules are sometimes considered together as
small B-granules (Bancel, Rogniaux, Debiton, Chambon, & Branlard,
2010; Cao et al., 2015). The relative distribution of A- and B-granules
often varies among different varieties (Shevkani, Singh, Bajaj, & Kaur,
2017). Some studies have reported that size of starch granules affects
dough rheological properties, and that smaller particles increase dough
elastic modulus (Roman, Cal, Go �mez, & Martínez, 2018; Tao, Wang, Wu,
Jin, & Xu, 2016; Zi et al., 2019). Several studies have explored the in­
fluences of starch properties on dough behavior when the wheat flour
was supplemented with various starches (Edwards, Dexter, & Scanlon,
2002; Huang & Lai, 2010; Roman et al., 2018; Tao et al., 2016). How­
ever, there are few studies reporting on the effects of starch character­
istics on dough behavior using natural wheat flour prepared with
different varieties (lines) with distinct genetic background. A study
undertaken in our lab established that the starch granules from six tested
wheat lines: 954072, Jinan 17, Jimai 44, P13, Jimai 229, and Xinmai 26,
had significant variations in number and volume distribution within
different granule size ranges. Additionally, the morphological properties
were considerably different among the six lines. Furthermore, the
micro-properties of starch granules exhibited different dough mixing
properties. Jimai 44 and Xinmai 26 had long dough stability time and
strong dough, whereas P13 and 954072 performed poorly based on the
evaluation of bread-making quality (Cao et al., 2019). Several studies
have reported the influence of either gluten or starch alone on the
rheological properties of wheat dough indicating several parameters
responsible for the wheat dough quality (Gao et al., 2016, 2018; Goe­
saert et al., 2005; Huang & Lai, 2010; Roman et al., 2018; Zhang, Mu, &
Sun, 2018). Previous studies using near-isogenic lines also demonstrated
significant influence of gluten secondary and micro-structure on dough
2
Food Hydrocolloids 106 (2020) 105885
rheological properties. The protein and starch interaction affecting
dough properties is a complex phenomenon that has been seldom
studied because of its intricate multidimensional nature (Yang, Song, &
Zheng, 2011). The quantitative analytical method using CLSM images
analyzed by AngioTool software reported recently has enabled explo­
ration of new indicators to demonstrate the gluten network (Bernklau,
Lucas, Jekle, & Becker, 2016; Gao et al., 2018). Together with the par­
ticle size distribution of starch, these quantitatively analyzed parameters
are supposed to be associated on the micro-structure level to indicate the
complex nature of the gluten network and starch granules interface.
Notably, water plays an important role in the dough formation
exhibiting close interaction with the flour polymers. Its distribution in
the dough system can be explained in the form of bond water, bulk water
and expelled water. The attribute of water mobility varies among
different dough samples, which can be determined with proton relaxa­
tion nuclear magnetic resonance (1H NMR) (Bosmans et al., 2012; Wang
et al., 2014; Zhang et al., 2018). Water distribution indirectly reflects the
structural properties between gluten and starch in the dough matrix. It is
of interest to find out the relationship between water distribution and
the interaction of gluten and starch in the natural wheat dough systems
rather than in model dough systems.
The objective of this study was to analyze the relationships between
starch particles and gluten network. A set of six wheat lines were used as
materials. Grain quality, dough mixing properties, gluten secondary
structures, H/L ratio, UPP%, moisture distribution in dough and pa­
rameters derived from gluten micro-structure analysis along with the
size distribution of starch granules in the dough matrix were quantita­
tively determined. The relationships between starch granules and gluten
network were evaluated to discover new indicators reflecting the level of
protein-starch interactions which are suggested to influence dough
mixing properties. This study provides helpful insight to further eluci­
date the complex gluten-starch interactions influencing dough
development.
2. Materials and methods
2.1. Materials
Six wheat genotypes Jimai 44, P13, 954072, Jinan 17, Jimai 229,
and Xinmai 26 were obtained from Shandong Academy of Agricultural
Sciences, Jinan, Shandong province. Both Jimai 44 and P13 are the
offspring of a cross between the two parental lines: 954072 and Jinan
17, with contrasting dough rheological properties in dough develop­
ment time, dough stability and dough adhesiveness and cohesiveness
(Cao et al., 2019). Jimai 44 and Jimai 229 are two recent released wheat
varieties with strong gluten whereas Xinmai 26 with strong gluten is
used as the control for quality test. Field experiments and wheat flour
sample preparations were carried out as reported previously (Cao et al.,
2019). Field experiments were carried out at Jinan (36� 420 N, 117� 4’ E)
in 2016–2017. The harvested wheat grains were sun-dried and stored for
two months. The grain samples were tempered overnight to achieve
15% moisture before grinding using Brabender Quadrumat mill (Bra­
bender Instruments, Hackensack, NJ, U.S.A.). The resultant flour sam­
ples were sifted through a 100-mesh sieve with an extraction rate of 65%
and stored in airtight containers at 4 � C for further analysis.
2.2. Determination of grain quality-related properties by near-infrared
spectroscopy (NIR)
A Diode Array 7250 NIR spectrometer instrument (Perten Instrument
AB, Sweden) was used to determine the grain quality-related properties:
moisture, protein content, starch content, wet gluten content, Zeleny
sedimentation volume and flour yield, by analyzing the hand-cleaned
wheat grains of the six wheat lines, as described by Zhao, Guo, Wei,
and Zhang (2013). With the wavelength ranging from 950 nm to 1650
nm, the data collection mode was set up as reflectance. A mean
X. Gao et al.
evaluation was generated based on the internal Perten calibration for
each replicate and each sample was measured twice. The mean value for
each sample was calculated based on the two duplicates.
2.3. Determination of H/L ratio, UPP% and dough development time
The glutenins were extracted according to the method described by
Gao, Appelbee, Mekuria, Chalmers, and Mather (2012). Reversed phase
high-performance liquid chromatography (HPLC) was conducted to
separate the glutenin subunits according to the method described by Li
et al. (2016). The H/L ratio was calculated by the peak area of HMW-GS
and LMW-GS. Size-exclusion HPLC was conducted to determine UPP%
in the mature grains, and the SDS extractable and unextractable proteins
were prepared and separated according to the method reported by Gao
et al. (2012). UPP% was determined as the percentage of the peak area
for unextractable polymeric protein relative to the total area for
extractable and unextractable polymeric protein. The above calculation
was based on the results with two replicates for each sample. Dough
development time of the six wheat lines was determined by Mixolab
(Mixolab 2, Chopin, France), which are cited from the recent study
undertaken in our lab (Cao et al., 2019).
2.4. Determination of the secondary structures of gluten
Secondary structures of gluten proteins: β-sheets, intermolecular
β-sheets, α-helixes, β-turns and β-antiparallels were determined in
duplicate for each sample by a Fourier Transform Infrared (FTIR)
spectrometer (Vetex70, Bruker Optics, Germany), as reported by Gao
et al. (2016). Relative data points were exported by Omnic software
(Omnic Specta 7.1, Thermo Scientific, USA) and analyzed by PeakFit
software (PeakFit v4.12, SeaSolve Software Inc., USA). The protein
amide I region (1600-1700 cm 1) was adjusted by baseline and
smoothed by Svatizky-Golay, respectively, followed by the Fourier
deconvolution and second order derivation. Gauss algorithm was used to
fit the sub-peaks. The secondary structures were assigned referring to
the previous studies (Kaur, Singh, Kaur, Ahlawat, & Singh, 2014; Wang
et al., 2014), and the targeted structures were intermolecular β-sheets:
1613–1620 cm 1; intramolecular β-sheets: 1627–1635 cm 1; α-helices:
1650–1660 cm 1; β-turns: 1670–1680 cm 1 and antiparallel β-sheets:
1680–1695 cm 1. The area under each sub-peak was calculated and the
corresponding secondary structure content was obtained as follows:
Secondary structure content ¼ Secondary structure sub-peak area/
total area of amide I region.
2.5. Micro-structure of gluten and starch observed by confocal laser
scanning microscopy (CLSM)
Micro-structure of dough samples was captured by CLSM according
to the reported method (Gao et al., 2018) with some modifications.
Dough samples of each wheat line were freshly prepared by mixing 10 g
well-ground flour with 6 mL Rhodamin B solution (0.1 mg/mL), fol­
lowed by 10-min recovery. The samples were observed with an Olympus
IX83 inverted microscope with an FV1000 biological confocal laser
scanning system (Olympus, Tokyo, Japan), with a semiconductor laser
LD559 and an objective lens (40 � , NA ¼ 0.95). Five different captures
(512 � 512 pixels for the resolution and 317 � 317 μm for the size) were
obtained for each dough sample. The CLSM images of gluten were
analyzed with the software AngioTool64 version 0.6a (National Cancer
Institute, National Institute of Health, Maryland, USA). The software is
regularly used to analyze blood vessels in medical science (Zudaire,
Gambardella, Kurcz, & Vermeren, 2011). Referring to our previous
study (Gao et al., 2018), the micro-structure of gluten was processed
from the CLSM images analyzed by AngioTool64. Several parameters
were calculated, including gluten area, gluten percentage area, gluten
junctions, junction density, total gluten length, gluten width, gluten
end-points, lacunarity, branching rate and end-point rate.
3
Food Hydrocolloids 106 (2020) 105885
The CLSM images of starches and the overlapped illustrations of
gluten and starch granules in dough were taken by an Olympus FV1000
(Olympus, Tokyo, Japan). Images with the same magnification of the
dough were taken for each wheat line, respectively, with the resolution
of 512 � 512 pixels and the size of 212 � 212 μm or 106 � 106 μm.
2.6. Particle distribution of flour and starch granules
To exclude the effect of flour particle size on dough properties, the
sieved ground flour samples of the six wheat lines were used to deter­
mine their size distribution using Microtrac S3500 laser diffraction
analyzer (Microtrac S3500 SI, Microtrac Inc., USA) following the
method of Cao et al. (2019). The measured values were exported to draw
the distribution curves using Microsoft Excel 2016. The number distri­
bution of flour samples was measured in triplicate.
To remove proteins, wheat flour (50 g) was added into 0.45% sodium
metabisulfite solution (500 mL) and soaked for 1 day at room temper­
ature. Each flour sample was washed with deionized water and stirred
for 3 min to obtain the homogenate, which was further filtered through
ten layers of cloth and 100-mesh filter. After centrifuging at 3000�g for
6 min, the collected pellet was transferred to a 0.2% NaOH solution,
sonicated for 10 min and centrifuged at 3000�g for 6 min. The pure
starch sample was obtained by scrapping off the coloured layer on the
surface of the pellet. The pelleted starch sample was dried for 48 h at 40
�
C, ground and sieved with a 200-mesh sieve. Similarly, the number
distribution of starch granules was also measured as described by Cao
et al. (2019). The A-type starch granule content is the percentage of
large starch granules (with a diameter more than 10 μm) number to total
starch granule number. The B-type starch granule content is the per­
centage of small starch granules (with diameter less than 10 μm) number
to total starch granule number. The number distribution of starch
samples from each wheat line was analyzed in triplicate and the number
distribution of B-type starch to that of A-type starch (B/A ratio) was
calculated.
2.7. Water distribution analysis of the dough samples
To determine the water distribution in the dough samples with a
water content of 47%, the low-resolution 1H NMR (Low-resolution
MesoMR Spectrometer, Niumag, Shanghai, China) was applied with an
operating resonance frequency of 23 MHz for 1H according to the
method of Wang et al. (2014). The spin-spin relaxation times (T2) of
protons in wheat dough were analyzed using the
Carr-Purcell-Meiboom-Gill sequences according to Bosmans et al.
(2012). The T2-magnitude profile demonstrates three distinguished
peaks: T21 peak ranging from 0.1 to 1 ms, corresponding to the water
that are tightly bound to gluten proteins, also termed as bond water; T22
ranging from 1 to 100 ms, corresponding to the water on the gluten
surface, regarded as bulk water; T23 ranging from 100 to 1000 ms, which
is assigned as the expelled water due to gluten polymerization with a
higher mobility (Bosmans et al., 2012; Wang et al., 2014). The relative
amount of bond water and bulk water in each sample can be quantita­
tively indicated by the amplitude of their corresponding peaks, A21 and
A22, respectively. The determination of moisture distribution was car­
ried out in duplicate for each sample.
2.8. Statistical analysis
The data is shown as a mean of the replicated samples � standard
deviation. All data were analyzed by one-way analysis of variance
(ANOVA) using SAS software (version 9.1, Institute Inc., Cary, NC, USA),
and significant differences (P < 0.05) between parameters were deter­
mined by Student’s t-test.
X. Gao et al. Food Hydrocolloids 106 (2020) 105885

3. Results and discussion
3.1. Grain quality and dough mixing properties
Grain quality of the six wheat lines showed significant differences in
moisture, protein content, starch content, wet gluten content, Zeleny
sedimentation volume and flour yield (Table 1) determined by NIR. The
HMW-GSs have been considered to be the most crucial determinant of
gluten strength and bread-making quality of wheat (Wang, Zhang et al.,
2018). Earlier, a study undertaken in our laboratory on the evaluation of
the quality based on HMW-GS composition indicated Jimai 44, Jimai
229 and Xinmai 26 having the highest quality score, followed by
954072, Jinan 17 and P13 (Cao et al., 2019). However, the parameters
of grain quality in Table 1 do not match the quality score, indicating
that, besides HMW-GSs, there are other factors affecting the grain
quality. It is therefore important to evaluate dough quality as a complex
system that needs to be studied using multi-dimensional approach. Ac­
cording to Cao et al. (2019), the six wheat lines showed a range of dough
stability times from 12.20 min (P13) to 14.46 min (Xinmai 26).
Compared with the above grain quality parameters, the dough stability
time has been accepted to be a more reliable indicator in predicting the
processing quality of the wheat dough (MacRitchie, 2016). In this study
we aim to use dough stability time to understand the influence of gluten
and starch in the development of dough quality.
H/L ratio has been confirmed to be positively associated with dough
rheological properties. Dough samples with higher HMW-GSs content
exhibit closer protein network connections (Visioli et al., 2018). H/L
ratio of the four wheat lines ranged between the two extremes exhibited
by the parents, Jinan 17 (63.69 � 0.22) and 954072 (46.68 � 2.57).
Significant differences were observed in UPP% among the six tested
lines. Jimai 44 has the highest UPP% (44.35%) and P13 has the lowest
UPP% (23.67%). Given that H/L ratio and UPP% have consistently been
reported as having positive impact on dough and gluten properties
(Ferrise et al., 2015; Gupta et al., 1993; Rasheed et al., 2015; Visioli
et al., 2018), these results indicate that Jimai 44 has the strongest dough,
while P13 performs the weakest. H/L ratio of gluten and UPP% are both
important quantitative indicators for wheat dough rheological proper­
ties and processing quality (Wang, Zhang et al., 2018). The
above-mentioned parameters reflect the size distribution of gluten
components, which have been accepted to be associated with dough
rheological properties. Compared with LMW-GSs, the HMW-GSs tend to
form larger polymers (Delcour et al., 2012). The various size distribu­
tions of glutenins can further result in different gluten networks within
the gluten-starch dough matrix (Lefebvre, Popineau, Deshayes, & Lav­
enant, 2000). Nonetheless, dough behavior is obviously not only
determined by gluten components, but also by the size distribution,
morphology and surface functionality of starch granules (Roman et al.,
2018; Wang et al., 2017). The effect of gluten and starch granule in­
teractions on dough rheology need to be further studied by quantitative
analysis.
3.2. Secondary structures of gluten
The secondary structure of gluten was determined based on the
amide I band, containing intermolecular β-sheet, β-sheet, α-helix, β-turn
and β-antiparallel. Significant structural variations were observed
among the test wheat lines (Table 2). The α-helix content ranged from
30.31% to 39.22% across the six lines. The parental line 954072 had the
highest α-helix content while Jimai 44 had the lowest. Jimai 229 had the
highest content of β-sheet structure (29.64%) and P13 showed the
lowest β-sheet content (23.75%). The α-helix structure is more hydro­
phobic, rigid and less accessible to hydrating water, compared to
β-sheet, and higher α-helix content in gluten indicates greater water
mobility and results in less dough stability (Wang, Yang et al., 2018).
The α-helix content has been accepted to be negatively correlated to the
viscoelasticity of dough (Mondher, Barbara, Abderfettah, Franck, &
Mohamed, 2005). High β-sheet content can enhance the intermolecular
interactions by hydrogen bonds, contributing to protein aggregation,
which is positively correlated to dough viscoelasticity (Bock, Connelly,
& Damodaran, 2013; Georget & Belton, 2006). Based on the correlation
between the secondary structures and dough rheological properties,
both Jimai 44 and Jimai 229 revealed superior dough properties
compared to the other four whereas P13 showed inferior dough char­
acteristics based on the evaluation of bread-making quality.
3.3. The micro-structure of gluten network
The CLSM images captured from the freshly prepared dough samples
were quantitatively analyzed with ‘AngioTool’ (Fig. 1), enabling the
study of the micro-structure of gluten network with 10 parameters
(Table 3). Among them, gluten percentage area is the area occupied by
the gluten network in the range of the selected images, reflecting protein
content in the dough sample. Jimai 44 had the highest gluten percentage
area (56.94%) while Jinan 17 had the lowest (48.10%) (Table 3), indi­
cating high protein content in Jimai 44, which is consistent with the
values for protein content in grains estimated by NIR. Jimai 44 also had
the highest level of gluten junctions (1476.5), while 954072 showed the
lowest (1081.0). This indicates that more protein junctions are gener­
ated in the reticular gluten thereby forming a compact protein crosslink
contributing better gluten and dough stability in Jimai 44.

Table 1
Parameters related to grain quality and dough mixing properties of the six wheat lines.
Line Grain quality determined by near-infrared reflectance Dough stability H/L ratio UPP (%)
time (min)a (%)
Moisture Protein Starch Wet gluten Zeleny sedimentation Flour yield
(%) content (%) content (%) content (%) volume (mL) (%)

954072 9.65 � 14.07 � 0.03d 64.82 � 34.39 � 0.19c 36.76 � 0.21a 72.50 � 13.30 � 0.13d 46.68 � 34.68 �
0.01d 0.25b 0.50a 2.57e 1.12c
Jinan 17 10.39 � 14.03 � 0.02d 60.33 � 30.81 � 0.64e 29.65 � 0.25c 70.50 � 13.86 � 0.11c 63.69 � 34.00 �
0.01a 0.05d 0.50bc 0.22a 0.55c
Jimai 44 9.73 � 16.22 � 0.03a 65.46 � 35.28 � 0.09b 36.53 � 0.22a 70.50 � 14.14 � 0.12b 47.90 � 44.35 �
0.02d 0.20b 0.50b 1.14d 0.18a
P13 10.15 � 14.66 � 0.10c 63.03 � 32.42 � 0.04d 31.60 � 0.39b 72.00 � 12.20 � 0.11e 46.81 � 23.67 �
0.04c 0.15c 0.00a 4.68e 0.22d
Jimai 10.19 � 14.51 � 0.01c 66.13 � 36.20 � 0.06a 36.58 � 0.13a 69.50 � 13.90 � 0.10bc 58.56 � 41.46 �
229 0.01bc 0.14a 0.50c 1.30b 0.26b
Xinmai 10.25 � 15.33 � 0.04b 60.72 � 30.86 � 0.43e 33.12 � 0.54b 71.00 � 14.46 � 0.12a 54.63 � 42.40 �
26 0.01b 0.12d 0.00b 2.30c 0.56 ab

H/L ratio: high- to low-molecular-weight glutenin ratio; UPP (%): the percentage of unextractable polymeric protein to total polymeric protein. Different lower-case
letters in the same column indicate significant difference (P < 0.05).
a
Dough stability time of the six wheat lines have been measured and reported previously (Cao et al., 2019). Here we use the parameter for further analysis of the
relationship between dough mixing property and the gluten-starch interaction.

4
X. Gao et al. Food Hydrocolloids 106 (2020) 105885

Table 2
Determination of secondary structure contents in the six wheat lines using FT infrared spectroscopy.
Line Intermolecular β-sheet (%) β-sheet (%) α-helix (%) β-turn (%) β-antiparallel (%) α-helix/β-sheet ratio
954072 21.58 � 0.30a 27.75 � 0.20c 39.22 � 0.28a 17.65 � 0.58c 12.51 � 0.39b 1.41 � 0.02a
Jinan 17 20.16 � 0.36b 28.64 � 0.51a 31.38 � 0.43bc 20.58 � 0.47 ab 15.36 � 0.53a 1.10 � 0.01c
Jimai 44 20.02 � 0.07b 29.44 � 0.04a 30.31 � 0.70b 19.13 � 0.55b 13.52 � 0.22b 1.03 � 0.03c
P13 18.76 � 0.32c 23.75 � 0.28d 30.89 � 1.05c 15.76 � 0.93d 10.84 � 0.72c 1.30 � 0.03b
Jimai 229 20.26 � 0.07b 29.64 � 0.04a 33.31 � 0.70b 19.47 � 0.55b 13.63 � 0.22b 1.12 � 0.03c
Xinmai 26 17.96 � 0.25c 28.22 � 0.41bc 31.60 � 0.17bc 21.83 � 0.26a 15.34 � 0.04a 1.12 � 0.02c

Different lower-case letters in the same column indicate significant difference (P < 0.05).

Fig. 1. Protein network analysis of dough sam­

ples prepared from the six wheat lines. The
dough samples were stained with Rhodamine B
to visualize proteins and analyzed by confocal
laser scanning microscopy (CLSM). The images at
the top of each sample are the original CLSM
captures, with the scale bar of 50 μm. The images
at the bottom of each sample are processed by
AngioTool, with junctions shown in white, gluten
skeleton shown in green and protein outline/area
shown in yellow. The red arrows point out the
irregular and larger voids embedded with several
starch granules close to each other. (For inter­
pretation of the references to colour in this figure
legend, the reader is referred to the Web version
of this article.)

5
X. Gao et al. Food Hydrocolloids 106 (2020) 105885

Table 3
Quantitative analysis of the gluten network in the six wheat lines determined by AngioTool software.
Line Gluten area Gluten Gluten Junction Total gluten Gluten Gluten end- Lacunarity ( Branching rate End-point
( � 104 percentage area junctions density ( � length ( � width points ( � � 10 2) ( � 10 2) rate ( �
μm2) (%) 10 3) 103 μm) (μm) 102) 10 3)

954072 12.74 � 49.10 � 1.13c 1081.0 � 4.2 � 0.4c 22.28 � 0.14c 5.73 � 4.98 � 0.72a 4.57 � 0.13 0.85 � 0.08b 3.92 �
0.32c 119.0c 0.23a ab 0.64a
Jinan 17 12.50 � 48.10 � 1.49c 1187.3 � 4.6 � 0.5c 23.19 � 0.10c 5.39 � 5.08 � 0.23a 4.98 � 0.48a 0.95 � 0.09a 3.36 �
0.38c 130.4b 0.17c 0.34 ab
Jimai 44 14.81 � 56.94 � 0.54a 1476.5 � 5.7 � 0.1a 26.65 � 5.56 � 4.44 � 2.95 � 0.08d 1.00 � 0.01a 2.87 �
0.16a 27.8a 0.09a 0.08abc 0.49b 0.28b
P13 13.66 � 52.56 � 0.58b 1375.6 � 5.3 � 0.2 ab 25.02 � 5.46 � 4.56 � 3.83 � 0.10c 1.01 � 0.03a 3.34 �
0.17b 55.9 ab 0.03b 0.07bc 0.37b 0.24 ab
Jimai 12.38 � 51.56 � 0.51b 1128.0 � 5.3 � 0.1 ab 22.55 � 5.50 � 4.95 � 4.03 � 0.10c 0.92 � 0.02a 3.52 �
229 0.11b 52.1bc 0.24b 0.07bc 0.40b 0.24 ab
Xinmai 14.47 � 55.62 � 1.93a 1350.3 � 5.2 � 0.2b 25.29 � 5.72 � 4.85 � 0.32a 3.21 � 0.12d 0.93 � 0.01a 3.36 �
26 0.49a 48.0 ab 0.09b 0.02a 0.34 ab

Different lower-case letters in the same column indicate significant difference (P < 0.05).

Gluten end-points are the open-ended protein threads, which are
believed to be a useful indicator for the network cohesiveness (Bernklau
et al., 2016). No significant difference was found between P13, Jimai 44
and Jimai 229. Furthermore, 954072, Jinan 17 and Xinmai 26 showed
no significant difference in this characteristic. However, lacunarity,
which was suggested as an indicator for dough quality (Gao et al., 2018),
showed significant differences among the six wheat lines, ranging from
2.95 � 10 2 (Jimai 44) to 4.98 � 10 2 (Jinan 17). Higher lacunarity
indicates the presence of more irregular gaps within the gluten
micro-structure, which is determined by both the length and structure of
gluten protein subunits and different-sized starch granules incorporated
in the gaps. A gluten network with a higher lacunarity value means that
the gluten would have more differently sized gaps and that each gap
would have greater possibility of filling with more than one tightly
adhered starch granules, thereby making the network structure of the
gluten irregular and less compact. Thus, we can predict that the gluten
and dough properties of Jimai 44 and Jinan 17 will show significant
differences. Gluten network with different strength has been shown to
have number of heterogeneously distributed, diversely sized holes or
gaps (Gao et al., 2018; Liu et al., 2016), embedded with starch granules
with different size distribution (Shevkani et al., 2017). Lacunarity is an
attribute for gluten network, which indirectly reflects the size distribu­
tions of starch granules, and it can be seen that besides the structure of
individual gluten protein and their cross-linkage, the structure of gluten
network seems to partially depend on the size distribution of the starch
granules. These studies have provided novel ways for us to study specific
relationships between gluten networks and starch granules on the basis
of their micro-structures. These micro-structures have not been studied
to date and thus may be an important investigation in gaining accurate
understanding of gluten and starch multi-dimensional interactions
influencing dough mixing behavior and processing qualities.
3.4. Particle distribution of flour and starch granules
Since the granulation during preparation of wheat flour can signifi­
cantly affect the dough behavior and bread characteristics (Lap�cíkov� a,
Bure�sova�, Lap�cík, Dabash, & Valenta, 2019), the seeds of the six wheat
lines were individually ground to determine the particle size distribution
of flour samples (Table 4 and Fig. S1). Compared with the one-peak
pattern of starch number distribution (Cao et al., 2019), all six wheat
lines exhibited a two-peak pattern (Fig. S1). The diameter of flour par­
ticles ranged from 0 to 167.2 μm, and the two main peaks ranged from
0 to 4.5 μm and from 4.5 to 74.8 μm, respectively. Over all 99% of the
particles had the diameter less than 74.8 μm. Contrary to the one-peak
pattern of starch number distribution (Cao et al., 2019), the traces for
flour exhibited one more peak and the peaks differ from each other in
their height. The number of particles with diameter more than 4.5 μm in
flour samples were significantly greater than that in starch samples
alone, indicating the contribution of flour particles other than starch on
dough properties, eliminating the potential effect of flour granulation.
The above result of flour size distribution in the six samples enabled us
to study the effect of starch size distribution on dough formation and
development.
All six wheat samples exhibited varying number of A- and B-type
starch granules, ranging from 4.34% to 18.13% for A-type starch and
from 81.87% to 95.66% for B-type starch, respectively. B-type starch
granule content in Jimai 44 (93.76%) was much higher than that in both
of the parents (86.35% for 954072 and 87.23% for Jinan 17). On the

Table 4
Number distribution of flour and starch granules and parameters derived from the structure of starch granules and gluten network reflecting their interaction in the six
wheat lines.
Line Flour granules (%) Starch granules (%) B/A B/ A/ B/A/Lacunarity ( �
ratio Lacunarity Lacunarity 102)
0–4.5 μm 4.5–74.8 μm 74.8–167.2 BG AG
μm
954072 58.76 � 3.94b 40.99 � 4.05a 0.26 � 0.11b 86.35 � 1.36c 13.65 � 6.33 18.89 2.99 1.38
Jinan 17 59.64 � 3.32b 39.68 � 3.46a 0.68 � 0.14a 87.23 � 1.12c 1.36b 6.83 17.52 2.56 1.37
Jimai 44 64.12 � 6.75b 35.12 � 6.78b 0.77 � 0.04a 93.76 � 12.77 � 15.03 31.78 2.12 5.09
P13 53.00 � 46.86 � 6.05a 0.16 � 0.10b 0.86b 1.12b 4.52 21.38 4.73 1.18
Jimai 229 6.14bc 28.17 � 4.43b 0.63 � 0.12a 81.87 � 6.24 � 0.86c 15.23 23.29 1.53 3.78
Xinmai 26 71.21 � 4.54a 27.32 � 0.77 � 0.01a 1.33d 18.13 � 22.04 29.80 1.35 6.87
71.96 � 1.56a 1.47bc 93.84 � 1.33a
0.45b 6.16 � 0.45c
95.66 � 4.34 � 0.52d
0.52a

BG: B-type starch granule content; AG: A-type starch granule content; B/A ratio: number distribution of B-type starch to that of A-type starch; B/Lacunarity: the ratio of
B-type starch content to lacunarity; A/Lacunarity: the ratio of A-type starch content to lacunarity; B/A/Lacunarity: the ratio of B/A ratio to lacunarity. Different lower-
case letters in the same column indicate significant difference (P < 0.05).

6
X. Gao et al.
contrary, B-type starch granule content in P13 (81.87%) was signifi­
cantly lower than that in both of the parents. The B/A ratio of the six
samples ranged from 4.52 to 22.04. B-type starch granules contribute
more to a robust structure of the bread (Roman et al., 2018). In this
regard Xinmai 26, Jimai 229 and Jimai 44, harboring larger number of
B-type starch granules, can be predicted as genotypes of choice for
introducing stability to the dough.
3.5. Water distribution in wheat dough
Water mobility in dough sample can indirectly reflect the in­
teractions between gluten and starch during dough formation (Bosmans
et al., 2012). The peaks T21 and T22 correspond to bond water and bulk
water, respectively in the dough system. The water mobility distribution
in the dough samples of all six genotypes was determined, revealing
similar T2-magnitude profiles, except that no T21 peak was detected in
P13 (Fig. S2). This indicated that the bond water in P13 dough was
negligible. A21 corresponds to the amount of bond water with lower
mobility and A22 corresponds to the amount of bulk water with higher
mobility (Wang et al., 2014). A21 in the dough of Jimai 44 (7.98%) is
significantly higher than that of Xinmai 26, 954072, Jimai 229 and
Jinan 17 (Table 5). Thus, it can be predicted that the dough of Jimai 44
contains more bond water with the closest-fitting interaction between
water and gluten. On the contrary, both P13 and Jinan 17 have rela­
tively higher content of bulk water. This indicated that more water was
distributed in the hydration sites of the gluten proteins and on the starch
surface with high mobility and Jimai 44 had the lowest amount of bulk
water. Given that the bulk water with high mobility can weaken the
dough stability (Bock, 2019; Wang et al., 2014), the above result
confirmed that in Jimai 44 there is a more stable interaction between
water and gluten, indicating better dough stability, consistent with the
results derived from dough mixing process.
3.6. Interactions between starch and gluten network
Wheat flour dough can be empirically regarded as a complex mixture
in which gluten forms a continuous three-dimensional reticular struc­
ture, filled with starch particles. The contrasting differences of the
gluten network of the six wheat lines (Fig. 1), affected by the size dis­
tribution of starch granules and the interaction between gluten protein
and starch granules, resulted in different dough properties. To illustrate
the interaction between gluten network and starch granules, we
observed the dough samples using CLSM. Fig. 2A–C demonstrate the
captures of the same view under different acquisition in the size of 212
� 212 μm. The structure of gluten network was observed under the 559
nm (Fig. 2A), and attention was also given to the starch granules and
their state within the gluten network (Fig. 2B) observed under bright
field. Interestingly, when the illustration of gluten network was over­
lapped with the embedded starch granules (Fig. 2C), the incorporation
status can be seen from the image. To further display the gaps among
Table 5
The spin-spin relaxation times and the amplitudes for the two types of water in
dough made from the six wheat varieties (lines).
Line T21 (ms) T22 (ms) A21 (%) A22 (%)
954072 0.35 � 0.04 a 20.03 � 0.70 a 1.34 � 0.58 b 95.04 � 0.38 a
Jinan 17 0.22 � 0.01 c 21.47 � 0.75 a 0.58 � 0.13 b 97.60 � 0.04 a
Jimai 44 0.37 � 0.05 a 19.34 � 0.00 ab 7.98 � 1.59 a 90.58 � 1.63 b
P13 – 17.44 � 0.61 b – 97.68 � 0.24 a
Jimai 229 0.25 � 0.03 bc 21.47 � 0.75 a 1.12 � 0.53 b 96.81 � 0.60 a
Xinmai 26 0.29 � 0.03 bc 21.47 � 0.75 a 2.95 � 0.86 b 95.27 � 0.76 a
T21: spin-spin relaxation time of the water tightly bound to gluten proteins; T22:
spin-spin relaxation time of the water hydration sites in the gluten and on the
starch surface; A21: amplitudes of the water tightly bound to gluten proteins;
A22: amplitudes of the water hydration sites in the gluten and on the starch
surface.
7
Food Hydrocolloids 106 (2020) 105885
starch granules and gluten network, the images were captured in a
smaller scale (106 � 106 μm). It can be seen from the images that some
of the starch granules are less tightly embedded by the gluten, indicated
with the yellow arrows. The different incorporation status among the six
wheat lines can be visualized in this smaller scale, compared to that in a
large scale in Fig. 1. The dough samples of the six wheat lines exhibit
distinct performances between gluten network and starch granules
(Fig. 2D–I). In accordance with our previous results (Cao et al., 2019),
954072 and P13 have more irregular starch granules and large A-type
starches (Fig. 2A and D). As shown in the images, the irregular starch
granules tend to create gaps between the protein network and the starch
granules. Compared with the large starch granules, the small starch
granules are obviously combined closely with the protein network.
Jimai 44 (Fig. 2C) and Xinmai 26 (Fig. 2F) contain more regular and
B-type starch granules, which are well distributed in the gluten network
and fill the holes perfectly. In addition, it is often observed that some
starch granules are very close to each other (Fig. 2C and E), embedded in
one hole, which is also indicated by the irregular and larger hole in
Fig. 1. This could explain the various distribution of holes in the gluten
network among different lines. Compared with large starch granules, the
distribution of small granules in the same hole seems more flexible,
which is much easier to fit into the gluten network and to form a
closely-packed dough (Martínez & G� omez, 2017; Roman et al., 2018).
The physical connection due to the packing density between starch
granules and gluten networks is termed as interaction level. The better
incorporation of small starch granules makes less gaps between starches
and gluten, resulting in a more compact dough. This phenomenon in­
dicates that there are different interaction levels between starch gran­
ules and gluten network in wheat dough. The previous study has found
that small starch granules have positive effects on the rheological
properties of dough, while the lacunarity of gluten network negatively
affect the dough behavior (Edwards et al., 2002; Gao et al., 2018).
Higher lacunarity represents the gaps are more irregularly distributed in
the network, that is, the gaps exhibit more sizes in the gluten network
with high lacunarity. Large-sized gaps have more possibility to hold
A-type starch granules or have more ability to hold B-type granules. For
wheat dough with long stability time, the micro-structure of dough is
more stable, containing less gaps between gluten and starch, which re­
quires the gluten network and starch granules fully integrated with each
other in their sizes. It can be hypothesized that the interaction level
between starch granules and gluten network may affect the dough
performance. In the present study, this relationship has been observed
intuitively (Fig. 2): for Jimai 44 and Xinmai 26 with good dough quality,
regular starch granules with low average diameters resulted in less
lacunarity of gluten network (Fig. 1), whereas 954072 and P13 with
poor dough quality showed the contrast.
3.7. Indicators proposed to reflect the interaction between gluten and
starch
The study of microscopic parameters of protein network structure
(Gao et al., 2018) and the characteristics of starch granules (Edwards
et al., 2002; Tao et al., 2016) have enabled to perform quantitative
analysis. To explore the physical connection between starch granules
and gluten networks, it was assumed that the interaction level of starch
granules into gluten network can be described by quantitative in­
dicators. Initially, B-type starch content, A-type starch content and ‘B/A
ratio’ in dough were used to represent the number distribution of starch
granule size (Table 4). Lacunarity was used to indicate the void distri­
bution of the gluten network, and to establish the parameters reflecting
the interaction levels of different sized starch particles in gluten network
on micro-structure level: B/lacunarity (B-type starch granule content
divided by lacunarity), A/lacunarity (A-type starch granule content
divided by lacunarity) and B/A/lacunarity (B/A ratio divided by
lacunarity).
X. Gao et al.
3.8. Relationship between dough mixing properties and indicators
reflecting the gluten-starch interaction
The relationships between the selected parameters relative to the
interaction level reflecting the reticular skeleton of gluten integrated
with starch granules and dough quality parameters of the six wheat lines
were analyzed (Fig. 3). Lacunarity decreased with the increased amount
of bond water (Fig. 3A), indicating that the high uniformity of gluten
network embeds more bond water. Notably, there was no obvious
relationship between lacunarity and dough stability based on the results
of the six wheat lines (Fig. 3B), which disagrees with our previous study
reporting that lacunarity was negatively associated with dough mixing
properties (Gao et al., 2018). This could be explained by the different
genotypes in different studies. The wheat genotypes used previously
(Gao et al., 2018) were near isogenic lines with the only variations in
HMW-GS composition, whereas the wheat genotypes in the present
study have different genetic backgrounds. Thus, it is suggested that more
wheat materials, with either consistent or inconsistent genetic back­
ground, need to be further quantitatively analyzed with lacunarity to
indicate wheat dough behavior. As discussed above, the phenomenon
that lacunarity negatively associates with the amount of bond water, but
not always associates with dough stability time, may indicate that the
more bond water with low mobility helps the stability of dough, but is
not the determinant for dough stability time. UPP% and dough stability
time increase with the increase of B-type starch content (Fig. 3C and D).
Given that UPP% positively correlates with dough quality properties
8
Food Hydrocolloids 106 (2020) 105885
Fig. 2. The illustrations of gluten and
starch granules in dough of the six
wheat lines observed by CLSM. Fig. 2A
is the structure of gluten network
observed under the 559 nm; Fig. 2B
image showing the starch granules and
their state within the gluten network
observed under bright field; Fig. 2C is
the illustration of gluten network over­
lapped with the embedded starch gran­
ules, demonstrating the interaction
status between gluten network and
starch granules. Fig. 2D–I are the over­
lapped illustrations of the six dough
samples. The scale bar are as indicated.
The yellow arrows indicate the gaps
between gluten and starch granules.
(For interpretation of the references to
colour in this figure legend, the reader is
referred to the Web version of this
article.)
(Liu et al., 2016), these results indicate that the B-type starch content
affects the dough processing quality of wheat, which is consistent with
the previous study that reported more large starch particles or fewer
small starch particles are both associated with weak dough processing
properties (Zi et al., 2019). Besides, the amount of B-granules has been
reported to impact gluten-free dough rheology, indicating that the size
distribution of starch granules per se can also influence dough properties
even in the absence of gluten (Roman et al., 2018). Moreover, it is of
interest to see that B/Lacunarity is positively related with bond water
and dough stability (Fig. 3E and F), in contrast to A/Lacunarity (Fig. 3F
and G). The results indicate that the selected parameters relative to the
interactions between starch granules and gluten network affect dough
stability. Besides, dough stability and B/A/Lacunarity exhibit similar
trends (Fig. 3H). Higher B/A indicates more small starch granules and
higher lacunarity indicates the heterogeneity of the gaps and holes in
gluten protein network. The ratio of B/A to lacunarity (B/A/Lacunarity),
terminated as filling degree, indicates the interaction level between
various sized starch granules and gluten network. Higher filling degree
is related to the strength of the dough itself, with longer dough devel­
opment and stability time. Given that gluten proteins serve as a network
mainly filled with starch granules, size distribution of starch granules
and lacunarity of protein skeleton have impact on each other and
consequently co-determine dough behavior (Delcour et al., 2012;
Shevkani et al., 2017). Higher B/A/Lacunarity, attributed to greater
filling degree, indicates a harmonious interaction between starch and
gluten protein, finally resulting in the improvement of dough quality.
X. Gao et al.
Fig. 3. The dot plots exhibiting relationships between the selected parameters relative to the interaction reflecting the reticular skeleton of gluten incorporated with
starch granules and dough quality parameters of the six wheat lines.
Thus, A/Lacunarity and B/A/Lacunarity, are suggested to be further
studied with more wheat materials so that they can be applied as novel
indicators to demonstrate the interaction between gluten and starch as
determinants of superior dough quality. Since different flours ground
from different wheat lines were used in this study, there could be also
other factors influencing the end-use quality, which were not analyzed.
It is suggested that future work could be done with more wheat materials
or a model system with controlled gluten and starch components to
validate the correlation between the interaction level of gluten and
starch and dough behaviors.
9
Food Hydrocolloids 106 (2020) 105885
4. Conclusion
Six wheat lines with different genetic background were used to
analyze the size distribution of starch granules, moisture distribution in
dough, gluten secondary structures, UPP%, H/L ratio and gluten micro-
structure. The relationships between starch granules and gluten network
were quantitatively analyzed. A/Lacunarity and B/A/Lacunarity,
reflecting the interaction level of various sized starch granules to gluten
protein networks in dough, can be further applied to illustrate the
interaction between starch and gluten protein and dough quality for
better evaluation of dough properties. Taken together, our results sug­
gest that the quality of gluten network and the size distribution of starch
X. Gao et al.
granules within the gluten network can affect the mixing properties of
the wheat dough, and the interaction level of starch granules and gluten
network can also affect the dough behavior.
Declaration of competing interest
The authors declare no competing financial interest.
CRediT authorship contribution statement
Xin Gao: Investigation, Formal analysis, Writing - original draft,
Writing - review & editing. Jingyang Tong: Methodology, Data cura­
tion, Writing - review & editing. Lei Guo: Methodology, Data curation.
Liwei Yu: Investigation, Data curation. Shaopeng Li: Investigation,
Data curation. Bingpeng Yang: Methodology. Libin Wang: Methodol­
ogy. Yang Liu: Resources. Faji Li: Resources. Jun Guo: Writing - review
& editing. Shengnan Zhai: Writing - review & editing. Cheng Liu:
Writing - review & editing. Ata-ur Rehman: Writing - review & editing.
Asgar Farahnaky: Writing - review & editing. Pei Wang: Writing -
review & editing. Zhonghua Wang: Project administration, Writing -
review & editing. Xinyou Cao: Supervision, Project administration,
Writing - review & editing.
Acknowledgements
The authors thank Mrs Mengyun Ding for her assistance in the
operation of instruments based at the Crop Biology Innovation Center in
the College of Agronomy, NWAFU. This work was supported by ‘Na­
tional Natural Science Foundation of China’ (grant number 31501300),
‘China Postdoctoral Science Foundation’ (grant number
2016M600817), ‘The Key Research and Development Program of
Shaanxi Province’ (grant number 2018NY-030, 056), ‘Science Fund for
the Cultivation of the Excellent Youth Scholars’, NWAFU (grant number
Z109021710)’, ‘Shandong Province Science & Technology Major Inno­
vation Project (2018YFJH0602); ‘The National Key Research and
Development Program of China’ (2016YFD0101802 and
2016YFE108600) and ‘TaiShan Industrial Experts Programme (No.
tscy20190106)’.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodhyd.2020.105885.
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