LWT - Food Science and Technology 148 (2021) 111624

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LWT
journal homepage: www.elsevier.com/locate/lwt

Interaction between gliadin/glutenin and starch granules in dough

during mixing
Mingfei Li, Qinghua Yue, Chong Liu *, Xueling Zheng **, Jing Hong, Nannan Wang, Ke Bian
College of Food Science and Engineering, Henan University of Technology, Zhengzhou, 450001, PR China

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

Keywords: In order to explore the interaction between gliadin/glutenin (gli/glu) and starch granules, rheological, micro­
Gliadin scopic and molecular structural properties of gluten-starch dough with varied gli/glu ratios during mixing was
Glutenin investigated. The effect of gli/glu ratio on dough elasticity (G′ ) and strength (extension resistance) depended on
Starch
the mixing state. The increase in gliadin ratio could weaken the dough strength at optimum-mixing stage while
Rheology
Molecular interaction
enhance it at under and over-mixing stages. At optimum stage, increasing gliadin could embed in and reduce the
intermolecular attraction of glutenins, leading to lower stability (higher z values) of dough. At under and over-
mixing stages, increasing gliadin could promote the formation of a temporary gli/glu-starch physical network
structure, leading to higher stability (lower z values). Polymerization or depolymerization of glutenin macro­
polymer (GMP) depended on both the gli/glu ratio and mixing time, which could be correlated to the changes in
rheological behaviors. The cohesiveness of glutenin caused large clumps in dough with high glutenin content,
while the increase in gliadin ratio was favor for the dispersion of glutenin, promoting the formation of gluten
network. Covalent and hydrogen bonds were the main interactions for high glutenin content dough, while hy­
drophobic and hydrogen interaction became predominate with increasing gliadin ratio.

1. Introduction extension properties (Veraverbeke & Delcour, 2002).

Glutenin and gliadin are two major storage proteins of wheat. Glu­
Dough is a viscoelastic mixture of flour and water. Rheological tenins are polymerized proteins in which individual subunits cross-
properties play an important role in processing of dough-based products linked via interchain S–S bonds. Gliadins in their native state are
and end-product quality. (Dobraszczyk & Morgenstern, 2003). The monomeric proteins involved with intrachain S–S bonds (Carceller &
higher the elasticity of dough is, the higher the deformation resistance Aussenac, 2001). Gluten composition and gli/glu ratio are the key fac­
and gas retention of the dough are (Xu & Zhou, 2018). tors influencing dough mixing, strength and final product characteristics
The mixing is one of the most important steps of dough processing. (Clarke & Bekes, 2003). The effect of gli/glu ratio on the functional
During mixing, hydrated gluten proteins form a three-dimension properties of dough has been studied by adding glutenin or gliadin to the
network which is stabilized by S–S and non-covalent bonds (Bohlin & original flour (Singh & Singh, 2013; Uthayakumaran & Bekes, 1999).
Carlson, 1980). Insight into these interactions may be derived from GMP Fido et al. (1997) found that the dough extension resistance was
and secondary protein structures (Weegels & Schofield, 1996). Weegels decreased and the extensibility was increased with increasing gliadin
et al. (1997) reported that the glutenin depolymerization led to a content. From measurements on gluten reconstituted at various gli/glu
decrease in GMP content during mixing. Robertson et al. (2006) found ratios, Janssen et al. (1996) found that at a constant protein content the
that viscoelasticity of bread dough attributed to the interactions be­ main factor determining the rheological behavior of hydrated gluten
tween aligned β-sheets formed in the repetitive domains through a was the gli/glu ratio. Veraverbeke and Delcour (2002) showed that the
network of hydrogen bonds between hydrophilic side chains. The loaf volume was significantly negative correlated with gli/glu ratio.
interaction between proteins in the gluten network plays a key role in Previously, the effect of gli/glu ratio on the physicochemical and
the rheological properties of dough, especially the strength and structural properties of dough was mainly studied by selecting flour with

* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (C. Liu), [email protected] (X. Zheng).

https://doi.org/10.1016/j.lwt.2021.111624
Received 28 December 2020; Received in revised form 28 March 2021; Accepted 28 April 2021
Available online 30 April 2021
0023-6438/© 2021 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Li et al. LWT 148 (2021) 111624

different gli/glu ratios or adding gli/glu into the original flour to make (National Manufacturing Division, TMCO, Lincoln, NE). The dough at 1
dough. However, the influence of non-starch polysaccharides couldn’t min (under-developed), 4 min (optimum-developed) and 10 min (over-
be excluded. The mechanism of interaction between gluten components, developed) during mixing was taken as the samples. Three parallel
and between starch and protein during dough formation is unclear. The samples were performed at each time.
model dough system involving starch and gli/glu could be used as it
decreased the complexity of the real system. In addition, interactions 2.6. Rheological properties
between specific components can be also investigated.
In the current study, the effect of the gli/glu ratio on the rheological 2.6.1. Frequency sweep
properties, microstructure and molecular interaction of gli/glu-starch Rheological behavior of dough was measured by a Haake RS6000
dough during mixing are studied. The purpose of this study is to rheometer (Thermo Fisher Haake, Germany). The method referred to
clarify the interaction mechanism of gli/glu, gli-starch and glu-starch in our previous studies (Li, Yue, Liu, Zheng, & Bian, 2020a, 2020b).
the dough.
2.7. Uniaxial extension
2. Materials and methods
The uniaxial extension properties of dough was measured with a TA-
2.1. Materials XT Plus Instrument (SMS,UK) using an A/KIE rig according to
Dobraszczyk and Morgenstern (2003). The pre-test, test and post-test
The AK58 wheat was provided by Henan Academy of Agricultural speeds were 2.0 mm/s, 3.3 mm/s and 10.0 mm/s, respectively. The
Sciences, China. The moisture, protein, ash and starch contents of AK58 trigger force was 5 g and the distance was 50.0 mm. The prepared dough
wheat flour were 13.14%, 12.6%, 0.44% and 74.22%, respectively. In was placed in the environment of 90% humidity, 30 ◦ C for 20 min before
total, the ratio of starch to protein in the wheat flour was 84:16. analysis. Each at least 6 measurements per dough were performed.

2.2. Wheat starch and gluten preparations 2.8. Confocal laser scanning microscopy (CLSM)

1.0 kg wheat flour was mixed with 0.5 L water for 5 min. The The CLSM images of dough with different gli/glu ratio were observed
resulting dough was rested for 20 min at room temperature. After at room temperature with a Leica TCS SP5 Confocal Laser Scanning
resting, 1.0 L water was added, and then stirred for a while. The gluten Microscope (Leica Microsystems Inc.,Heidelberg, Germany), equipped
was collected, whereas the starches were filtrated. The starch suspension with an inverted microscope (Model Leica DMI6000). The methods
was centrifugated at 4000 g for 15 min. The sediment consisted of a referred to our previous studies (Li et al., 2020a, 2020b).
yellow-brown layer of sludge fraction with a starch layer underneath.
The top layer was scraped off and the white bottom layer consisted of 2.9. GMP content
wheat starches were washed in anhydrous ethanol, starch and gluten
were dried in the freeze dryer for 24 h. The dried samples were ground, The GMP contents of the samples were determined following a
passed through an 80-mesh sieve, and stored at 4 ◦ C. The starch, protein, method reported by Liu & Wei (2015) with some modifications. The
and moisture content of the isolated starch fraction were 90.02%, sample (1.0 g) was suspended in 40 mL of 1.5% SDS solution by shaking
2.60%, and 5.34% and that of the gluten fraction were 4.83%, 78.2% for 60 min at room temperature and centrifuged at 15,500×g for 15 min.
and 8.60%, respectively. The supernatant was discarded and the sediment was re-suspended in
SDS solution, then shook and centrifuged as above. The sediment was
2.3. Gliadin and glutenin extraction collected, the nitrogen content was measured by Kjeldahl method and
identified as GMP content.
The gluten (100 g) was dispersed in 3 L of 70% ethanol. The sus­
pension was stirred for 3 h at 25 ◦ C, followed by centrifugation for 20 2.10. Fourier transform infrared spectroscopy (FTIR)
min at 4000 g and 4 ◦ C. The supernatant was collected, the lower pre­
cipitation layer was again extracted with 70% ethanol, and the above The freeze-dried samples (2 mg) were mixed with KBr (200 mg) and
steps were repeated 3 times. The glutenin precipitation was then ground into powder in an agate mortar incubated with infrared light,
collected. The supernatants were pooled and diluted at 1:2 (distilled which was then pressed into a slice. Fourier transform infrared spectra
water). The supernatant pH was adjusted to 6.2, and the gliadin was was recorded using a Nicolet 380 FTIR spectrometer (Thermo Nicolet
obtained. The gliadin and glutenin were freeze-dried for 24 h. The dried Corporation, USA) from 400 to 4000 cm− 1 wavenumbers with a reso­
samples were ground, passed through an 80-mesh sieve, and stored at lution of 2 cm− 1 for 128 scans. The overlapping amide I band
4 ◦ C. The protein, lipid content of isolated gliadin were 94.23%, 0.27%, (1600–1700 cm− 1) components were further interpreted by deconvo­
respectively, and the protein, lipid content of the isolated glutenin were lution using Peak-Fit v4.12 software (Peak-Fit v4.12, Systat Software,
88.57%, 0.34%, respectively. Inc. Chicago, USA).
The secondary structure was calculated as the proportion of area of
2.4. Gli/glu-starch blend preparation the corresponding secondary structure in the total amide I band area. A
baseline adjustment was performed prior to the second-derivative
The gluten with different gli/glu ratios was mixed with starch in a analysis to accurately measure the band areas of the second derivative
bag by fixing the ratio of starch, e.g., 100 g starch-gli or glu mixtures spectra in the amide I region. Then, the bands were resolved with a
were prepared, based on five different gli/glu ratios (0:10, 3:7, 5:5, 7:3, Gaussian curve fit, followed by a second-derivative analysis. The band
and 10:0) to give the composition of starch-gli or glu mixtures of 84/0/ assignment was conducted based on a previous study (Wang et al.,
16, 84/4.8/11.2, 84/8/8, 84/11.2/4.8, and 84/16/0, respectively. 2014). The secondary structural contents were curve fitted using a
parabola pattern (Y = a + bX + cX2) to show a clearer trend.
2.5. Dough preparation
2.11. Chemical interactions
The dough was prepared by mixing the blend sample (10 g) with the
deionised water (25 ◦ C) determined from the optimum water absorption Chemical interactions were determined using a modified version of
(AACCI approval method 54–70), using a 10 g dough mixograph the procedure described by Cao et al. (2017). Selective buffers (prepared

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in 0.05 M phosphate buffer, pH 7.0) were used to creak certain types of 3. Results and discussion
bonds as following: (1) 0.05 M NaCl (PA); (2) 0.6 M NaCl (PB); (3) 0.6 M
NaCl + 1.5 M urea (PC); (4) 0.6 M NaCl + 8 M urea (PD); (5) 0.6 M 3.1. Rheological properties
NaCl+8 M urea+50 mM DTT (PE). Briefly, freeze-dried dough powder
(0.2–2 g) was homogenized in 5 mL of reagent, stirred at room tem­ 3.1.1. Frequency sweep
perature for 120 min and then centrifuged at 10,000 g for 20 min. 0.1 mL Fig. 1a shows the sweep curves of under-developed dough. The
supernatant was put into the test tube, and then 5 mL Coomassie bril­ elastic modulus (G′ ) and viscous modulus (G′′ ) were decreased as the gli/
liant blue G-250 was added, and the suspension was vortexed and glu ratio increased from 0:10 to 3:7, and then increased as gli/glu ratio
mixed. The absorbance of the supernatant was measured at 412 nm further increased from 3:7 to 10:0. And the G′ and G′′ of model dough
using an UV762 ultraviolet spectrophotometer (Inesa Analytical In­ with a gli/glu of 3:7 were closed to that of native dough. The G′ and G′′ of
strument Co., Ltd., Shanghai, China). The standard curve was prepared gluten-starch dough with a gli/glu of 7:3 and 10:0 were higher than that
by using BSA as a reference. Ionic bonds were expressed as the difference of others dough. This indicated that the effect of gliadin on the elasticity
between soluble dough powder in PB and PA, hydrogen bonds were of under-developed dough was greater than that of glutenin. Possibly,
expressed as the difference between PC and PB. Hydrophobic in­ for the insufficient energy input, the degree of dispersion and hydration
teractions were expressed as the difference between PD and PC, and is greater for monomeric gliadin than polymeric glutenin to form
covalent bonds were calculated as the difference between PE and PD. viscoelastic network structure. Because gliadin is spherical while glu­
tenin is fibrous proteins and the molecular weight and surface hydro­
2.12. Statistical analysis phobicity of gliadin was lower than that of glutenin (Wang et al., 2015;
Markgren et al., 2020).
Data was analyzed by one-way analysis of variance (ANOVA) and Fig. 1b shows the sweep curves of optimum-dough. With increasing
SPSS 16.0 Statistical Software Program (SPSS Incorporated, Chicago, gli/glu ratio, the G′ and G′′ showed a trend of decrease. The sufficient
USA). hydration of glutenin facilitated a full expansion of the peptide chain
and the formation of an ordered network structure, resulting in a higher
G’ (Bohlin & Carlson, 1980). Previous studies (Khatkar & Schofield,
2002) have shown that glutenin content had a significant positive

Fig. 1. The frequency sweep curves of doughs with different gli-glu ratios during mixing. a: mixing at 1min; b: mixing at 4min; c: mixing at 10min.

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correlation with dough strength. Belton et al. (1995) suggested that 1999). Labat et al. (2002) showed that water soluble pentosan can
hydrated glutenin in dough contained a considerable amount of inter­ enhance the cross-linking between gluten proteins and promote the
molecular β-sheet, and reported that the elasticity could be resulted from formation of viscoelastic network. For the same mixing time, a decrease
intermolecular interactions involving these sheet structures. This also in the z and an increase in the K values with increasing gli/glu ratio were
explains the above behavior. found in both the under and over-developed states. This indicated that
Fig. 1c shows the sweep curves of over-developed dough. Results high ratio gliadin (10:0) could enhance dough strength and stabilize
showed that the G′ and G′′ of model dough with a gli/glu ratio of 0:10 network of under and over-developed doughs. With increasing gli/glu
were lower than those of others. As the mixing time increase, the GMP ratio, the z and K values of the optimum dough were gradually
was depolymerized into smaller molecules, resulting in the decrease of increased. The continuous network structure formed by glutenin could
G’. The over-developed dough with high gliadin ratio showed higher G′ be interrupted by high content of gliadin resulting in a network structure
and G". Possibly, gliadin can embed in the polymers and reduce their having low elasticity and high rigidity.
intermolecular attraction, thus promote the formation of three-
dimensional network structure and increase the elasticity (Kim & Cor­ 3.1.2. Uniaxial extension
nillon, 2001). The uniaxial extension results are shown in Table 1. The extension
Fig. 1 shows that the G′ of model doughs were higher than that of resistance was sharply decreased and then increased as the gli/glu ratios
native dough. Besides that the content of starch and gluten protein in the increased, reaching respectively the minimum and maximum at a gli/
model dough is higher than that in the original flour, it may be also due glu ratio of 3:7 and 10:0, regardless of mixing time. In the absence of
to the lack of water-soluble fraction in the model dough. Miller and gliadin, the hydrated glutenin peptide chains are stretched by mechan­
Hoseney (1999) found that water-soluble fraction had a weakening ac­ ical force and form a polymer through disulfide bonds (Lagrain et al.,
tion on the dough strength. In the whole range of frequency, G′ was 2008). These large polymers are entangled by strong secondary bonds
higher than G′′ for all doughs, which is typical of highly structured and thus difficult to slide, resulting in relatively high extension resis­
materials. The breaking and making of bonds between the components tance. Similar to starch, the embedded spherical gliadin molecular at a
may lead to structural changes affecting the rheological properties. Both gli/glu ratio of 3:7 would increase the intermolecular distance of the
moduli increased with frequency, following an exponential equation. glutenin network, resulting in a loose network structure, and thus a
The results of the fitting are reported in Table 1. The z values show the decrease in the extension resistance. However, at higher gli/glu ratio
degree of dependence of G′ on frequency, which can reflect the type of (from 5:5 to 10:0) the extension resistance was unexpectedly increased.
molecular interaction (Zhang et al., 2017). The z = 0 suggests a covalent We attributed this abnormal phenomenon to the formation of a compact
linkage with a stable network structure, whereas z > 0 suggests a temporary physical network structure between starch granular and
physical linkage with a less stable network structure. The K values show gliadin. As the mixing time increased, the extension resistance was
the dough rigidity, with a higher K value indicating higher dough ri­ slightly decreased for the model dough with a gli/glu ratio of 0:10,
gidity (W & Yoo, 2008). Table 1 showed that the z values of the under increased and then decreased for the gli/glu ratios of 3:7 and 5:5, while
and over-developed model dough were lower than that of the wheat slightly increased for the gli/glu ratios of 7:3 and 10:0. This implied that
dough, the trend of K value was opposite, indicating more stable the interaction between gliadin and glutenin in the model dough with
network structure of the model doughs than the wheat dough at under gli/glu ratios of 3:7 and 5:5 was stronger than that of others. Results
and over-developed states. This may be due to the effect of water-soluble showed that, at the appropriate gli/glu ratio, with increasing energy
fraction removal during model doughs formation. However, at the input, glutenin and gliadin would greatly promote the association of
optimum-mixing stage, most of the z values of the model doughs were non-covalent bonds such as hydrogen bonds, leading to the mutual as­
higher than that of the wheat dough, and the K values were lower than sociation of free chemical bonds (Lazarev et al., 2010). At the same time,
the wheat dough except for a gli/glu ratio of 10:0. This implied that at the gluten network structure would change from disorder to laminar
the appropriate input energy, the water-soluble fraction could promote order and then to disorder state, resulting in the regular change of dough
the formation of more elastic network structure (Miller & Hoseney, extension resistance (Ktenioudaki et al., 2010). However, the absence of

Table 1
The parameters obtained from Frequency sweep, stress-relaxation and uniaxial extension test of model dough with different gli-glu ratios during mixing.
Mixing Time Sample Frequency sweep Uniaxial extension

z K × 105 R2 Extension resistance/g Displacement/mm

bcC cA bA
1 min Gli:Glu0:10 0.113 ± 0.001 1.453 ± 0.002 0.999 ± 0.000 25.10 ± 0.05 8.34 ± 0.55cA
Gli:Glu3:7 0.144 ± 0.003bB 1.202 ± 0.004dA 0.999 ± 0.000 8.88 ± 0.05dB 12.90 ± 0.74bA
Gli:Glu5:5 0.098 ± 0.002cB 1.363 ± 0.023cdA 0.999 ± 0.000 15.48 ± 0.16cB 7.74 ± 0.14cA
Gli:Glu7:3 0.097 ± 0.004cC 2.875 ± 0.022bA 0.999 ± 0.000 22.24 ± 0.71bB 5.29 ± 0.14dB
Gli:Glu10:0 0.093 ± 0.004cB 4.732 ± 0.026aA 0.999 ± 0.000 34.36 ± 3.06aB 6.26 ± 0.29cdA
AK58F 0.255 ± 0.003aC 0.409 ± 0.013eB 0.999 ± 0.000 27.80 ± 0.66abA 32.08 ± 2.23aA
4 min Gli:Glu0:10 0.205 ± 0.005dA 0.193 ± 0.001dC 0.999 ± 0.000 22.27 ± 0.33bB 8.52 ± 0.16bA
Gli:Glu3:7 0.219 ± 0.008cdA 0.150 ± 0.008dB 0.999 ± 0.000 10.31 ± 0.05eA 8.19 ± 0.02bB
Gli:Glu5:5 0.253 ± 0.001cA 0.287 ± 0.005cC 0.999 ± 0.000 18.87 ± 0.05cA 8.70 ± 0.69bA
Gli:Glu7:3 0.270 ± 0.003bA 0.480 ± 0.005bB 0.999 ± 0.000 23.42 ± 0.11bAB 7.51 ± 0.35bAB
Gli:Glu10:0 0.313 ± 0.006aA 0.996 ± 0.002aB 0.999 ± 0.000 34.80 ± 0.11aAB 7.26 ± 0.00bA
AK58F 0.221 ± 0.002cdB 0.448 ± 0.002bA 0.999 ± 0.000 14.74 ± 0.33dB 19.64 ± 0.19aB
10 min Gli:Glu0:10 0.143 ± 0.001bcB 0.520 ± 0.003bB 0.999 ± 0.000 21.34 ± 0.38cB 8.40 ± 0.28bA
Gli:Glu3:7 0.189 ± 0.003bAB 0.145 ± 0.004dB 0.998 ± 0.000 8.84 ± 0.16eB 6.02 ± 0.00cC
Gli:Glu5:5 0.111 ± 0.002dB 0.935 ± 0.023aB 0.999 ± 0.000 9.65 ± 0.00eC 8.11 ± 0.82bA
Gli:Glu7:3 0.149 ± 0.005bcB 0.269 ± 0.031cC 0.997 ± 0.000 24.85 ± 0.44bA 8.57 ± 0.28bA
Gli:Glu10:0 0.091 ± 0.004eB 0.973 ± 0.021aB 0.999 ± 0.000 35.98 ± 0.22aA 7.03 ± 0.36bA
AK58F 0.342 ± 0.003aA 0.103 ± 0.005eC 0.999 ± 0.000 12.63 ± 0.27dC 12.79 ± 1.06aC

The z = 0 suggests a covalent linkage with a stable network structure, whereas z > 0 suggests a physical linkage with a less stable network structure. The K values show
the dough rigidity, with a higher K value indicating higher dough rigidity. Different lowercase letters on the same line indicated significant difference (P < 0.05)
between different samples at the same mixing time, and different capital letters on the same line indicated significant difference (P < 0.05) between different mixing
time at the same sample.

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gliadin would cause a lack of lubrication and thus a more rapid disin­ known that glutenins are elastic proteins capable of forming inter and
tegration of glutenin during mixing. intrachain disulfide bonds, which can form a highly network structure
and thus act as a backbone of the gluten network (Barak et al., 2013).
Under the action of external forces during mixing, the cross-linking
3.2. CSLM between glutenin molecules was easier to broken. These breaks were
favorable for the sliding between adjacent glutenin molecules, so that
The microstructures were measured by CSLM and shown in Fig. 2. the dough network structure has the maximum homogeneity. It was also
Dough was comprised of a composite material with starch granules found that the number of gas holes (black block) first decreased from 1
(green) and gluten proteins (red). The under-developed dough with a min to 4 min, and then gradually increased from 4 min to 10 min,
gli/glu ratio of 0:10 showed a discontinuous protein agglomerates regardless of gli/glu ratios. This may be due to that during the initial
(yellow). There was a strong inherent cohesion between the glutenin mixing state, large gas holes is formed by the air incorporated into the
molecules, while the energy input was not sufficient to break the glu­ uneven dough. As the mixing time increased, large gas holes were
tenin macropolymers. As the mixing time increased, the size of the gradually broken into small gas holes and more evenly distributed in the
glutenin agglomerates was gradually reduced but still dispersed un­ dough network.
evenly in the model dough. It was also observed that the partially
depolymerized glutenin was adsorbed on the surface of starch granules,
implying the presence of some interaction between glutenin and starch. 3.3. GMP contents
As the gli/glu ratios increased, the red block content was gradually
decreased, and the more open gluten network structure was evenly Fig. 3 shows the GMP content for the test doughs. The GMP content
distributed in the dough, which was especially obvious for the doughs for the model dough with a gli/glu ratio of 0:10 was only slightly
with gli/glu ratios of 7:3 and 10:0. This may be due to the partial sub­ decreased as the mixing time increased from 1 to 4 min, and there was
stitution of the intermolecular secondary forces of glutenin by the sec­ no further change as mixing proceeded. Probably, the energy input
ondary forces between glutenin and gliadin, when gliadin was under current conditions was insufficient to break down the very
embedded in the network structure of glutenin. As the mixing time cohesive glutenin chains in the model dough (Jazaeri & Seetharaman,
increased, the rough network structure of starch-gli/glu gradually 2015). A more obvious decrease in the GMP content during mixing was
formed a homogeneous structure and then was broken again. It was well found for the dough with a gli/glu ratio of 3:7. This indicated that

Fig. 2. CLSM of dough with different gli-glu ratios during mixing.

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M. Li et al. LWT 148 (2021) 111624

Fig. 2. (continued).

adding gliadin to the glutenin-starch dough would promote the S–S structural feature, followed by β-turns and α-helix structures (Georget &
bonds break and thus the depolymerization of GMP into SDS soluble Belton, 2006). The β-sheet, random, α-helix and β-turn structure con­
fraction by the mechanical force (Weegels & Schofield, 1996). The GMP tents of gluten in the samples are displayed in Table 2. The β-sheet
content was increased from 1 to 4 min and then decreased from 4 to 10 structure contents of model dough with a gli/glu ratio of 0:10 were
min for the dough with a gli/glu ratio of 5:5. This implied that at this gradually decreased during mixing. This may be attributed to the
gli/glu ratio the glutenin undergone an polymerization process before depolymerization of GMP, which led to a reduction of parallel alignment
depolymerization as the mixing time increased, similar results have been of peptide chains. The β-sheet structure contents of the model dough
observed by other authors (Melnyk & Seetharaman, 2012). Probably, as with a gli/glu ratio of 3:7 were increased and then decreased, which was
the mixing time increased from 1 to 4 min, the influence of glutenin consistent with the change of extension resistance. It can be inferred that
folding and aggregation through hydrophobic interaction between the large deformation properties for the dough with higher glutenin
gliadin and glutenin exceeded its dissociation, however, with further content was mainly caused by the covalent bond and hydrogen bond
mixing, the trend reversed (Weegels & Schofield, 1996). There were no resulted from the conversion of β-turns to β-sheet structure during
significant changes in the GMP content at the gli/glu ratios of 7:3 and mixing. The β-sheet content of the model dough with a gli/glu ratio of
10:0 during mixing. In generally, the breakdown of GMP can be 5:5 were decreased and then increased during mixing. Interestingly, the
attributed to a chemical depolymerization mechanism which involved β-turn structure contents of the model dough with a gli/glu ratio of 5:5
the fracture of disulfide bond and secondary bond (Tanaka & Bushuk, were increased and then decreased during mixing, which is consistent
1973). The much greater amounts of gliadin than glutenin would with the change of G’. This implied that the large deformation changes
significantly reduce the stretch effect of mechanical force on glutenin of dough with a gli/glu ratio of 5:5 was mainly caused by covalent bond
molecules and thus the fracture of covalent and non-covalent bonds, and hydrogen bond which is caused by the conversion of β-sheet to
because of the embedded and barrier effect of gliadin (Melnyk et al., β-turns structure. This suggested that β-turns in the repetitive domains of
2012; Steertegem et al., 2013). At the same mixing time, the changing the glutenin subunits may transform to β-sheets, which can be stabilized
trend of the GMP content for different gli/glu ratios was consistent with by new covalent bonds and by hydrogen bonds between adjacent glu­
that of the content of glutenin. tenin subunits (Robertson et al., 2006). However, at the high gliadin
ratio (7:3, 10:0), the β-turns contents were decreased and then increased
3.4. Secondary structure during mixing, which was consistent with our frequency sweep results. It
can be inferred that in the high gliadin ratio model dough, the change of
In wheat dough, the β-sheets are the predominant secondary β-turns during mixing mainly affected the small deformation behavior.

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Fig. 3. GMP content (A) and Contributions of intermolecular forces (B) in the gluten-starch dough with different gli-glu ratios during mixing. (1) Different lowercase
letters on the same line indicated significant difference (P < 0.05) between different samples at the same mixing time, and different capital letters on the same line
indicated significant difference (P < 0.05) between different mixing time at the same sample.

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Table 2 others bonds. This implied that the hydrophobic interactions were
The secondary structure content of gluten-starch dough with different gli-glu nonnegligible for maintaining the conformation of gluten protein mol­
ratios during mixing. ecules. This may be because gluten contains more non-polar amino acids
Mixing Sample β-sheet/% Random α-helix/% β-turn/% (Veraverbeke & Delcour, 2002). After hydration, more non-polar amino
Time Coil acid side chains interacted with each other leading to more hydrophobic
1610-1640 1640-1652 1652- 1660- interactions.
cm− 1 cm− 1 1660 cm− 1
1685 cm− 1
Fig. 3B(a) shows the ionic bonds contribution. There are many
1min Gli: 40.96 ± 14.81 ± 13.47 ± 30.82 ± dissociated side-chain groups in the amino acid residues that make up
Glu0:10 0.44aA 0.31abB 0.92bA 0.59bcB proteins. In solutions, more amino and carboxyl groups become charged
Gli: 39.08 ± 14.78 ± 14.43 ± 31.71 ± groups, leading to the formation of ionic bonds. Much smaller amount of
Glu3:7 0.32aAB 0.21abB 0.10aA 0.43cC ionic bond compared to hydrophobic bond was observed during mixing.
Gli: 39.12 ± 14.88 ± 13.35 ± 31.58 ±
Glu5:5 0.59aA 0.10aB 1.53bA 1.10cC
At under-mixing stage, as gli/glu ratio increased, the ionic bond was
Gli: 36.43 ± 14.28 ± 13.89 ± 35.39 ± increased and then decreased, reaching the maximum value at the ratio
Glu7:3 0.86bA 0.24bB 0.15bA 1.26bB of 5:5. At optimum mixing stage, the ionic bond showed an upward
Gli: 34.82 ± 14.56 ± 14.31 ± 36.82 ± trend with increasing gli/glu ratio, while had no significant change was
Glu10:0 0.59cB 0.59abB 0.59aA 0.59bB
observed at over mixing stage. The ionic bond reached the maximum
AK58F 34.14 ± 13.62 ± 14.12 ± 38.10 ±
0.39cB 0.29bB 0.45aA 1.26aA value at 4 min during mixing, regardless of gli/glu ratio. Results also
4min Gli: 40.79 ± 17.90 ± 12.70 ± 34.47 ± showed that the ionic bonds contribution of starch-gli/glu dough was
Glu0:10 3.01aA 1.26aA 1.55aB 1.96abA lower than that of wheat dough. This may be due to higher amount of
Gli: 40.42 ± 15.24 ± 12.66 ± 35.48 ± mineral existed in wheat flour, while the separation of gluten and starch
Glu3:7 0.78aA 0.22bcA 0.68aC 0.59aA
Gli: 37.78 ± 16.16 ± 12.62 ± 35.42 ±
led to the decrease of ionic bond.
Glu5:5 1.67bcC 0.39bA 0.69aB 0.59aA Fig. 3B(b) shows the contribution of hydrogen bonds. In general,
Gli: 36.70 ± 16.59 ± 12.88 ± 32.82 ± hydrogen bonds are the main forces to maintain stability of the sec­
Glu7:3 0.47cA 0.32bA 0.59aB 0.88bC ondary structure in proteins (Georget & Belton, 2006). Hydrogen bonds
Gli: 36.87 ± 16.82 ± 12.90 ± 33.52 ±
were mainly formed between water molecules, between bound water
Glu10:0 0.12cA 0.19bA 0.69aB 0.59bC
AK58F 39.88 ± 14.96 ± 12.89 ± 34.26 ± and some groups on the surface of proteins. For the dough with a high
0.07bA 0.08cA 0.08aB 0.06abB glutenin ratio, hydrogen bonding force remained stable until 4 min
10min Gli: 36.50 ± 14.02 ± 12.47 ± 34.02 ± during mixing, while it was significantly reduced at over-mixing stage.
Glu0:10 1.07aB 0.66aB 1.60bB 3.33bA Probably, hydrogen bonds between glutenin may be partially replaced
Gli: 37.95 ± 14.61 ± 13.28 ± 33.16 ±
Glu3:7 0.30aB 0.74aB 0.63bB 1.08bB
by water-glutenin molecules due to the depolymerization of GMP when
Gli: 38.28 ± 14.53 ± 13.79 ± 33.40 ± excessive stirring, resulting in reduced hydrogen bonds. For other ratios
Glu5:5 0.62aB 0.33aB 0.74bA 0.44bB dough, hydrogen bond was increased and then decreased during mixing.
Gli: 35.49 ± 13.56 ± 14.10 ± 36.86 ± Possibly, as the mixing time increases, gluten were hydrated leading to
Glu7:3 0.92bB 1.37bB 1.04aA 3.33abA
the formation of unstable hydrogen bond forces between hydrophilic
Gli: 34.04 ± 13.85 ± 14.04 ± 38.07 ±
Glu10:0 0.11bB 0.45bB 0.37aA 0.71aA amino acids on the surface of gliadin and glutenin, or hydroxyl groups
AK58F 33.48 ± 13.60 ± 14.35 ± 38.52 ± free from starch granules. Over-mixing, however, caused the unstable
0.14cB 1.34bB 0.27aA 0.51aA hydrogen bonds to break.
Different lowercase letters on the same line indicated significant difference (P < The contributions of hydrophobic interactions did not change
0.05) between different samples at the same mixing time, and different capital significantly for the lowest gli/glu ratio, while they were increased and
letters on the same line indicated significant difference (P < 0.05) between then decreased for other ratios during mixing (Fig. 3B(c)). Possibly, at
different mixing time at the same sample. the optimum stage, mixing led to the exposure of hydrophobic groups as
the unfolding of protein molecules (Lagrain et al., 2008), resulting in
Georget and Belton (2006) found that in gliadin, the content of β-turns more hydrophobic interactions to stabilize the network structure.
was decreased and then increased with increasing hydration level, Over-mixing destroyed the hydrophobic core domain and interrupted
which was in agreement with our finding. This demonstrated the asso­ the hydrophobic interaction of the stable network structure, thus
ciation of β-sheet structures with β-turns as originally described by resulting in structural damage. At the same mixing time, the contribu­
Belton et al. (1995), who suggested that sheet structures formed at the tions of hydrophobic interaction were gradually decreased with
expense of turn structures (loops) led to more orderly gluten network. increasing gli/glu ratio. In general, glutenin is fibrous structure with
In Table 2, it was also found that the α-helix structure was slightly many repetitive hydrophobic regions existed on the surface of the
decreased and then increased during mixing. Interestingly, the random molecule, and gliadin is a spherical structure with many hydrophobic
coils exhibited the trend opposite to the α-helix structure. In general, the amino acid side chains being embedded in the spherical molecule, and
α-helix is regarded as a fairly stable conformation, as all peptide bonds in hydrophilic amino acid side chains being mainly exposed on the surface
the backbone was involved in hydrogen. Therefore, the slight decrease of the molecule (Marquié, 2001; Markgren et al., 2020), leading to the
in α-helix structure from 1 min to 4 min suggested the free sulfhydryl decrease in the contributions of hydrophobic interactions with
groups buried in the “cage” structure were exposed by the mechanical increasing gli/glu ratio.
force and conversed to random coils (Seabourn et al., 2008). The contributions of covalent bond are shown in Fig. 3B(d). The
contributions of covalent bond of the model dough with low gli/glu ratio
(0:10, 3:7) were gradually decreased during mixing. The fracture of
3.5. The molecular interactions disulfide bonds between glutenin molecules may be the main reason for
above trend, which is consistent with the depolymerization of GMP
The chemical interactions involved in dough formation are mainly (section 3.5). The contributions of covalent bonds of others model dough
ionic bonds, hydrogen bonds, and disulfide bonds, as well as hydro­ were increased and then decreased during mixing. This indicated that
phobic interactions (Cao et al., 2017). These chemical interactions can gliadin could promote the covalent cross-linking of gluten network at
be disrupted using different buffers, and the chemical interactions in test these levels during mixing. For the under and over-developed dough, the
dough are shown in Fig. 3B. As shown in Fig. 3B, the contribution of covalent bond was sharply decreased with increasing gli/glu ratio. For
hydrophobic interactions to gluten network was more significant than the optimum-developed dough, the covalent bond was slightly

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M. Li et al. LWT 148 (2021) 111624

decreased for 3:7 and 5:5 while dramatically decreased for 7:3 and 10:0 complex depolymerized, and non-covalent and covalent bonds were
with increasing gli/glu ratio. It was also found that the contributions of broken, leading to a decrease in dough strength. 3: A model dough with
covalent bond to the model dough were lower than to wheat dough. a gli/glu ratio of 10:0 was shown. At 1 min, gliadin competed with
Possibly, the presence of non-starch polysaccharides in wheat dough, starch to absorb water, forming phase separation or adsorption of
facilitated the catalysis of thiol/disufide exchange (Auvergne & Robin, gliadin on the surface of starch. As the mixing progressed, the hydration
2008), thereby resulting in higher contribution of covalent bond to the of gliadin led to an increase in hydrophobic force and hydrogen bond,
former. thereby increasing the strength of the model dough.

4. Conclusion
3.6. Simplified model
The current study showed the interaction between gliadin and glu­
A simple model was proposed to describe the mixing process of tenin and the effect on viscoelasticity of gli/glu-starch model dough was
model dough with different gli/glu ratios and clarify the interactions strongly dependent on both the gli/glu ratio and mixing state. At
between starch and wheat gluten components (Fig. 4). 1: A model dough optimum-mixing stage increasing gliadin ratio could weaken while at
with a gli/glu ratio of 0:10 was shown. At 1 min, the dough showed under and over-mixing stages could enhance the stability of gluten
polymerized glutenin and discontinuous protein domain, with the main network. These were due to the reduction of intermolecular attraction of
force being hydrophobic forces, S–S bond, and hydrogen bonds; as the glutenins by gliadin at optimum-mixing stage, while higher dispens­
mixing progressed, the exchange of sulfhydryl and disulfide bonds oc­ ability of gliadin than glutenin at under-mixing stages or the formation
curs inter- and intra-molecular of glutenin subunits, and the hydroxyl of of a temporary gli/glu-starch physical network structure at over-mixing
starch formed intra and inter-molecular hydrogen bonds with the free stages. These changes can be negatively or positively correlated to the
SH group of protein chain, and the hydrophobic side chain of protein dough strength or elasticity, respectively. Adding gliadin to the glutenin-
was exposed to form hydrophobic force. At 4 min, the glutenin subunits starch dough would promote the S–S bonds break and thus the depo­
formed an ordered fibrous macromolecular polymer through disulfide lymerization of GMP at a certain proportion (gli/glu ratio of 3:7).
bonds, and starch granules were embedded in the ordered glutenin However, at other gli/glu ratio, the glutenin could undergone an poly­
network in the form of non-covalent bonds. At 10 min, the unstable non- merization process before depolymerization as the mixing time
covalent bonds were broken, and the covalent bonds of the glutenin increased, indicating the gliadin could promote the covalent cross-
macropolymers were also partially broken, forming protein fragments. linking of gluten before optimum-mixing. The contributions of hydro­
2: A model dough with a gli/glu ratio of 5:5 was shown. Gliadins are phobic interactions did not change significantly for the lowest gli/glu
monomeric proteins containing only intra-molecular disulfide bonds, ratio, while they were increased and then decreased for other ratios as
and a large number of hydrophobic amino acid residues wrapped in the the mixing time increased. Results showed that the large deformation
spherical interior, leading to a lower S–S bond and hydrophobic force at properties for the dough with higher glutenin content was mainly caused
under-mixing stage. As the mixing progressed, the gliadin and starch by the covalent bond and hydrogen bond resulted from the conversion of
granules embedded in the fibrous network structure of glutenin polymer β-turns to β-sheet structure during mixing. While for the dough with
may form a loops structure due to the formation of intra-chain covalent higher gliadin ratio, the change of β-turns structure during mixing led to
and non-covalent bonds. At over-mixing stages, the large molecular

Fig. 4. Proposed simplified model for the interactions between starch and gliadins and/or glutenins in the dough with different gli-glu ratios during mixing. (1)
Mixing of gluten-starch dough with a gli-glu ratio of 0:10; (2) Mixing of gluten-starch dough with a gli-glu ratio of 5:5; (3) Mixing of dough with a gli-glu ratio of 10:0.

9
M. Li et al. LWT 148 (2021) 111624

the hydrophobic and hydrogen interaction became predominate, which Labat, E., Rouau, X., & Morel, M. H. (2002). Effect of flour water-extractable pentosans
on molecular associations in gluten during mixing. Lebensmittel-Wissenschaft und
mainly affected the small deformation behavior.
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Lagrain, B., Brijs, K., & Delcour, J. A. (2008). Reaction kinetics of gliadin-glutenin cross-
Author statement linking in model systems and in bread making. Journal of Agricultural and Food
Chemistry, 56(22), 10660–10666.
Lazarev, Y. A., Grishkovsky, B. A., & Khromova, T. B. (2010). Amide I band of IR
Mingfei Li: Writing Original Draft, Formal analysis, Investigation. spectrum and structure of collagen and related polypeptides. Biopolymers, 24(8),
Qinghua Yue: Formal analysis. Chong Liu: Conceptualization, Method­ 1449–1478.
ology, Review & Editing. Limin Li: Investigation. Jing Hong: Investiga­ Liu, R., Xing, Y., Zhang, Y., Zhang, B., Jiang, X., & Wei, Y. (2015). Effect of mixing time
on the structural characteristics of noodle dough under vacuum. Food Chemistry, 188
tion. Xueling Zheng: Supervision. Ke Bian: Resources. Nannan Wang: (1), 328–336.
Language revising. Li, M., Yue, Q., Liu, C., Zheng, X., & Bian, K. (2020a). Effect of gliadin/glutenin ratio on
pasting, thermal, and structural properties of wheat starch. Journal of Cereal Science,
93, 102973.
Declaration of competing interest Li, M., Yue, Q., Liu, C., Zheng, X., & Bian, K. (2020b). Comparative study of rheology and
steamed bread quality of wheat dough and gluten - starch doughs. Journal of Food
The manuscript has not been published or submitted to other jour­ Processing and Preservation, 15160.
Markgren, J., Hedenqvist, M., Rasheed, F., Skepö, Marie, & Johansson, Eva (2020).
nals for publication. Each author has read the manuscript and approved Glutenin and gliadin, a piece in the puzzle of their structural properties in the cell
to submit it to this journal for possible publication. described through Monte Carlo simulations. Biomolecules, 10, 1095.
Marquié, C. (2001). Chemical reactions in cottonseed protein cross-linking by
formaldehyde, glutaraldehyde, and glyoxal for the formation of protein films with
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