# Impact of the protein composition on the structure and viscoelasticity of
polymer-like gluten gels

Laurence Ramos1, Amélie Banc1, Ameur Louhichi1, Justine Pincemaille1,2,
Jacques Jestin3, Zhendong Fu4, Marie-Sousai Appavou4, Paul Menut2,5, Marie-
Hélène Morel2 1Laboratoire Charles Coulomb (L2C), Univ. Montpellier, CNRS,
Montpellier, France  2Ingénierie des Agro-polymères et Technologies Emergentes
(IATE), Univ. Montpellier, CIRAD, INRAE, Montpellier SupAgro, Montpellier,
France  3Laboratoire Léon Brillouin, UMR 12, Université Paris-Saclay,
IRAMIS/CEA Saclay, 91191 Gif-sur-Yvette Cedex, France  4Forschungszentrum
Jülich GmbH JCNS am MLZ Lichtenbergstr. 1, 85748 Garching, Germany
5Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, 91300 Massy,
France<EMAIL_ADDRESS>
###### Abstract

We investigate the structure of gluten polymer-like gels in a binary mixture
of water/ethanol, $50/50$ v/v, a good solvent for gluten proteins. Gluten
comprises two main families of proteins, monomeric gliadins and polymer
glutenins. In the semi-dilute regime, scattering experiments highlight two
classes of behavior, akin to standard polymer solution and polymer gel,
depending on the protein composition. We demonstrate that these two classes
are encoded in the structural features of the proteins in very dilute
solution, and are correlated with the presence of proteins assemblies of
typical size tens of nanometers. The assemblies only exist when the protein
mixture is sufficiently enriched in glutenins. They are found directly
associated to the presence in the gel of domains enriched in non-exchangeable
H-bonds and of size comparable to that of the protein assemblies. The domains
are probed in neutron scattering experiments thanks to their unique contrast.
We show that the sample visco-elasticity is also directly correlated to the
quantity of domains enriched in H-bonds, showing the key role of H-bonds in
ruling the visco-elasticity of polymer gluten gels.

  * January, 18 2021

##  1 Introduction

Polymer materials may exhibit a large variety of unique properties, ranging
from high water content, softness, and flexibility for hydrogels to resilience
and temperature sensitivity for elastomers. The specific properties of polymer
materials entail specific uses in different contexts including biomedical
applications for hydrogels made of natural or biodegradable synthetic polymers
[1], construction materials, and sensors for elastomers [2]. Nature also
abounds in polymer gels and elastomers with unique properties dedicated to
specific functions, as mucus [3], synovial fluid [4], the gelatinous layer of
tension wood [5], seed mucilage hydrogels [6] and natural rubber [7]. In all
these examples, the complex multi-component composition and the many types of
interactions at play are intimately connected to drive the hierarchical
structures of the polymer-like gel materials and control their mechanical
properties.

This complexity and this intricacy also hold for gluten. Gluten, the insoluble
protein of wheat, forms in its water hydrated state a highly cohesive and
viscoelastic mass (comprising typically $2$ g of water per g of protein), akin
to an elastomer [8]. Gluten visco-elasticity is crucial in food science as it
allows wheat flour to be baked into bread and biscuit. Gluten proteins belong
to the broad family of prolamins; they are proteins rich in proline and
glutamine amino-acids, which may confer texture to food materials because of
the formation of protein strands under extensional flow [9]. Gluten is a
complex mixture of several types of protein, which can be divided into two
main classes, monomeric gliadins and polymeric glutenins. In glutenins,
glutenin sub-units are linked together by disulfide bonds yielding polymers
with molar mass up to several millions g/mol [10, 11]. Gluten proteins belong
to the wider class of intrinsically disordered proteins that are currently
extensively investigated because of their crucial role in many biological
processes. Gluten proteins are certainly the most documented elastomeric plant
proteins [12]. However, the study of gluten is very difficult because gluten
proteins are broadly polydisperse and essentially insoluble in water. Hence,
despite several decades of investigation, a full understanding of the
structure of gluten in relation to its viscoelastic properties is still
lacking. The active debate about gluten being regarded preferentially as a
particulate gel or as a polymer gel is not fully closed (see [13] and the
references therein), although we strongly believe that the polymeric nature
plays a major role, as inferred especially from our recent investigations [14,
15, 16, 17, 18]. Previous works have also pointed out the important role of
disulfide bonds and hydrogen bonds in the structuration of gluten [19, 20,
21].

In order to shed light on the structure of gluten and on the relationship
between structure and viscoelasticity, our strategy is to study model systems,
produced by dispersing dedicated protein extracts in a good solvent, a mixture
of water and ethanol, allowing an efficient solubilization of the proteins.
Thanks to this approach, homogeneous samples with a wide range of protein
concentration, spanning several orders of magnitude, can be studied. Note that
this could not be performed with pure water as a solvent: water being a bad
solvent for gluten, homogenous samples can only be produced at very high
protein concentration. In our case, thanks to the choice of water/ethanol good
solvent, unprecedented structural and mechanical data have been obtained. In
particular, we have shown, for a given protein extract comprising equal
amounts of gliadin and gliadin, that most structural and viscoelastic
properties of protein dispersions can be qualitatively and quantitatively
rationalized in the framework of polymer gels [14, 15]. More recently, we have
developed a protocol to obtain from industrial gluten model protein extracts
with controlled and tunable composition in gliadin and glutenin [22]. In this
paper, we leverage on this recent advancement and investigate the structure
and visco-elasticity of suspensions of gluten proteins of various composition.
This study allows us to demonstrate a correlation between the presence of
large proteins assemblies in very dilute regime and the presence of domains
enriched in H-bonds, of size comparable to the assemblies, in the semi-dilute
regime, and a direct link between the hierarchical structures of proteins in
the dilute regime and the sample visco-elasticity.

The paper is organized as follows. We first describe the materials and the
different experimental methods. We then present and discuss the experimental
results regarding the structure and visco-elasticity of the samples as probed
thanks to a combination of complementary scattering and rheology techniques.
We finally conclude by emphasizing the crucial role of protein assemblies in
the materials.

##  2 Materials and methods

###  2.1 Materials and sample preparation

Gluten protein extracts are prepared from an industrial gluten (courtesy of
TEREOS-SYRAL, Aalst, Belgium), following a protocol described elsewhere [17,
22]. In brief, gluten is first dispersed at room temperature in a
water/ethanol $50/50$ v/v solvent. Only the proteins well dispersed in the
solvent are kept (the insoluble part is discarded). The dispersion is then
quenched to a low temperature $T_{q}$, leading to a liquid-liquid phase
separation into a light phase and a heavy phase. In the following, both the
light and heavy phases are used after freeze-drying. The composition of the
freeze-dried protein extracts are characterized by chromatography in a
denaturating solvent, in which weak intra- and intermolecular interactions are
suppressed [17]. Overall, the light phase is enriched in gliadin, the
monomeric proteins, whose molar mass, $M_{\rm{w}}$, lies in the range
$(25-65)\times 10^{3}$ g/mol. Conversely, the heavy phase is enriched in
glutenin, the polymeric proteins, with $M_{\rm{w}}$ from $90\times 10^{3}$
g/mol to several $10^{6}$ g/mol. Interestingly, the exact composition of the
protein extracts is varied by changing $T_{q}$, allowing a fine tuning of the
protein composition. In our experiments, $T_{q}$ ranges between $-0.8$ and
$12.5^{\circ}$C. The composition of the protein extracts is characterized by
its mass fraction of glutenin as
$GLU=\frac{m_{\rm{Glu}}}{m_{\rm{Glu}}+m_{\rm{Gli}}}$ with $m_{\rm{Glu}}$,
respectively $m_{\rm{Gli}}$, the mass of glutenin, respectively gliadin, in
the extract. We obtain protein extracts with $GLU$ in the range $(1-66)$ %.

Samples are prepared by dispersing the freeze-dried protein extracts in the
appropriate volume of solvent, a water/ethanol $50/50$ v/v mixture.
Hydrogenated and deuterated solvents are used. Deuterated solvent comprises OD
ethanol (C2H5OD) and heavy water (D2O). The protein concentration $C$ ranges
between $4$ and $400$ mg/mL.

###  2.2 Methods

####  2.2.1 Small-angle X-Ray scattering

Small-angle X-ray scattering experiments are performed in house and in the
European Radiation Synchrotron Facility, ESRF, (Grenoble, France). The in-
house set-up comprises a high brightness X-ray tube with low power and an
aspheric multilayer optic (GeniX 3D from Xenocs) delivering an ultra low
divergent beam ($0.5$ mrad); a two-dimensional Schneider 2D image plate
detector prototype is used to collect the scattering intensity. The sample-
detector distance is set at $1.9$ m. Synchrotron experiments are conducted at
the ID02 beamline of ESRF [23], using three different sample-detector
distances ($d=1.5$, $7$ and $30$ m) in combination with a wavelength $0.0995$
nm, yielding scattering vectors $q$ in the range $(2\times 10^{-3}-7)$ nm-1.
In all experiments, the samples (prepared with hydrogenated or deuterated
solvents and with protein concentration in the range ($10-400$ mg/mL) are held
in glass capillaries of diameter $1.5$ mm. Standard non-linear fitting
procedures are used to analyze the data.

####  2.2.2 Small-angle and very small-angle neutron scattering

Several facilities are used for small-angle neutron scattering experiments
(SANS) and very small-angle neutron scattering experiments (VSANS). SANS
measurements at Laboratoire Léon Brillouin (Saclay, France) are performed on
instrument PA20 using three configurations with the following wavelength
$\lambda$ and sample-detector distance $d$ ($\lambda=0.6$ nm and $d=1.5$ m;
$\lambda=0.6$ nm and $d=8$ m; $\lambda=1.5$ nm and $d=19$ m) yielding
scattering vector $q$ in the range $(10^{-2}-2)$ nm-1. VSANS and SANS are also
conducted on two instruments operated by JCNS at the Heinz Maier-Leibnitz
Zentrum (MLZ, Garching Germany). SANS experiments are performed on KWS2 [24]
using three configurations with $\lambda=0.7$ nm and $d=2$ m; $\lambda=0.7$ nm
and $d=8$ m; $\lambda=1$ nm and $d=20$ m, yielding scattering vector $q$ in
the range $(2\times 10^{-2}-3)$ nm-1. VSANS experiments are performed at KWS3
[25] with $\lambda=1.28$ nm and $d=10$ m yielding $q$ in the range ($2.2\times
10^{-3}-2\times 10^{-2})$ nm-1 and at KWS2 with $\lambda=0.7$ nm and $d=20$ m
using the focusing mode with MgF2 lenses [26], yielding $q$ in the range
$(3\times 10^{-3}-3\times 10^{-2})$ nm-1. In all cases, samples (prepared with
hydrogenated or deuterated solvents and with protein concentration in the
range ($210-302$ mg/mL)) are held in quartz Hellma cells with thickness of $1$
mm or $2$ mm. Standard data correction and calibration are performed to
analyze the data. Data are corrected for empty cell scattering, solvent
scattering, transmission, and detector sensitivity. Absolute scale
transformation is performed using standard procedures [27]. Standard non-
linear fitting procedures are used to analyze the scattering profiles.

####  2.2.3 Rheological measurements

Linear viscoelastic measurements are performed using a Anton Paar MC302
stress-controlled rheometer. We use cone and plate geometries, with different
diameters ($8$, $25$ or $50$ mm), depending on the sample visco-elasticity.
The whole geometry is immersed in a bath of silicon oil to avoid solvent
evaporation. After loading the sample (with a spatula for gels or simply by
pouring liquid samples), the gap between the cone and the plate is set to its
prescribed value ($100\mu$m, for the $50$ mm diameter plate, $50\mu$m for
smaller plates). We let the samples equilibrate until the normal force acting
on the cone relaxes to zero before starting the measurements. The frequency
dependence storage, $G^{\prime}$, and loss, $G"$, moduli are measured in the
linear regime, with a typical strain amplitude of $1$ %. Measurements are
performed with samples prepared with deuterated solvents with a fixed protein
concentration $C=237$ mg/mL and different $GLU$ content. The temperature is
fixed at $25^{\circ}$C.

##  3 Results and discussion

###  3.1 Structural features in very dilute regime

Figure 1 reports the composition of the species present in very dilute
suspensions ($C=4$ mg/mL) of the different protein extracts as determined
thanks to asymmetrical flow field-flow fractionation [17]. Three classes of
objects, monomeric gliadins, glutenin polymers and protein assemblies, are
identified depending on their average size, $<R>$, and molar mass
$<M_{\rm{w}}>$. For gliadins $<R>=7$ nm, $<M_{\rm{w}}>=8\times 10^{4}$ g/mol,
for glutenin polymers $<R>=20$ nm and $<M_{\rm{w}}>=4\times 10^{5}$ g/mol and
for protein assemblies $<R>=85$ nm, $<M_{\rm{w}}>=3\times 10^{7}$ g/mol
111Note that for gliadins and polymers, $<R>$ refers to a mean hydrodynamic
radius, whereas it refers to a mean radius of gyration for the assemblies.. We
find that the proportion of the different species evolves with the protein
composition. As anticipated, at small $GLU$ ($GLU<$ ca $30$ %), the proportion
of polymers increases with $GLU$. Interestingly, above $30$ %, the proportion
of polymer is roughly constant and one observes the emergence of large protein
assemblies as a third species, whose amount increases with $GLU$.

Figure 1: Mass fraction of monomers, polymers and assemblies (see text) in
dilute suspensions of the different protein extracts. Adapted from [17]. The
relative error, as evaluated from 3 replicated measurement with the extract
with $GLU=47$ % is less than $5$ %.

###  3.2 Structural features in the semi-dilute regime

####  3.2.1 Spatial distribution of the gluten proteins

X-ray scattering is sensitive to the electronic densities of the species.
Hence in small-angle X-ray scattering experiments, the contrast originates
from the difference in the scattering densities between the gluten proteins
and the solvent. Experiments therefore probes the spatial distribution of the
proteins in the solvent. We show (inset Fig. 2) that the scattering profile of
a low concentration sample depleted in glutenin ($C=10$ mg/mL, $GLU=13$ %) is
typical of a solution of polymer coils in the dilute regime. Note that this is
in accordance with the fact that gluten proteins are intrinsically disordered
proteins [28, 29, 30]. At intermediate scattering vectors ($0.5$
nm${}^{-1}<q<3$ nm-1), the scattered intensity, $I$, varies as $q^{-p}$, with
$p=2$. This power law scaling is characteristic of Gaussian chains in a theta-
solvent [31]. At smaller length scale, the transition from the $q^{-2}$
scaling to a $q^{-1}$ scaling, at a scattering vector $q_{c}$, allows the
determination of the persistence length $l_{p}$ of the chains, following
$q_{c}\times l_{p}=1.9$ [32]. We measure $q_{c}$ of the order of $2.8$ nm-1
yielding $l_{p}$ of the order of $0.7$ nm. This small value indicates that the
polypeptide chains of the gluten proteins are very flexible, which is typical
for intrinsically disordered proteins [28, 33]. On the other hand, for length
scales larger than the radius of gyration of the coils, at small $q$, a
plateau of the scattered intensity is observed in a log-log representation. By
modeling the transition from the plateau to the power law decrease with a
Lorentzian function as predicted with a Orstein-Zernicke (OZ) model,
$I(q)=\frac{A}{1+(q\xi)^{2}}$, one can evaluate the correlation length $\xi$
[31]. This length is equal to the radius of gyration of the scattering objects
in dilute regime, and is expected to decrease with concentration in the semi-
dilute regime. The fit with OZ (line in the inset Fig. 2) gives $\xi=3.1$ nm,
a numerical value consistent with the size of gliadin [17, 34]. Note that data
are equally well fitted using a Debye function [35], which is the form factor
for Gaussian chains, yielding a comparable size ($4.6$ nm).

Figure 2: Correlation length, $\xi$, as a function of the protein
concentration for a protein extract with $GLU=13$ %. $\xi$ is extracted from a
fit with a Lorentzian function (see text). The inset shows the scattered
intensity measured at room temperature as a function of the scattering vector
for a sample with $C=10$ mg/mL and $GLU=13$ %. Black symbols are experimental
data points and the green solid line is the best fit. Measurements are
performed at room temperature. Figure 3: Scattering profiles measured by
small-angle X-ray scattering, for samples prepared with a hydrogenated solvent
at various protein concentrations, as indicated in the legends, for protein
extracts with (a) $GLU=13$ %, and (b) $GLU=45$ %. Measurements are performed
at room temperature. Data are acquired at ESRF facility.

Data acquired at different protein concentrations (Fig. 3a) all superpose at
large scattering vectors, when the scattered intensity, $I$, is normalized by
$C$, indicating a unique structure at small length scales, independent of the
protein concentration, as expected. All data also exhibit a plateau at small
$q$ but whose height, normalized by $C$ decreases as $C$ increases. This
indicates a higher compressibility of the samples as $C$ increases, due to the
interpenetration of the polymer coils, which signs the transition from dilute
to semi-dilute regimes, at $C^{*}$. Above the overlap concentration $C^{*}$,
the correlation length $\xi$ decreases due to coil interpenetration. We find
that $\xi$, as obtained from fits of the scattering profiles with the OZ
model, decreases from about $3$ nm down to $1$ nm, as $C$ increases from $10$
to $400$ mg/mL (Fig. 2). The overlap concentration $C^{*}$ is defined as the
concentration from which $\xi$ decreases with increasing $C$. From purely
geometrical arguments, the overlap concentration reads
$C^{*}=\frac{3M_{\rm{w}}}{4\pi R_{\rm{G}}^{3}Na}$, with $R_{\rm{G}}$ the
radius of gyration of the coils, $M_{\rm{w}}$ their molar mass and $Na$ the
Avogadro number. Experimentally, one measures $C^{*}\simeq 100$ mg/mL. Taking
$M_{w}=40000$ g/mol as average molar mass for gliadins, one determines a
characteristic size of the order of $5.4$ nm, a numerical value in agreement
with the values directly measured in the dilute regime. Finally, we note that
the experimental evolution of $\xi$ with $C>C^{*}$ is consistent with the
theoretically expected $C^{-1}$ scaling for a polymer in a theta-solvent [36],
in full agreement with the $p=2$ power law exponent found for the scattered
intensity at intermediate $q$-range.

Overall, glutenin depleted samples exhibit the structural features of polymer
coils in theta-solvent conditions. By contrast, the structural features of the
samples enriched in glutenin polymer are more complex. We show in Figure 3b
the scattering profiles, normalized by the protein concentration, for samples
prepared with a gluten extract with $GLU=45$ %. Data overlap at large $q$ and
display the scattering features expected for polymer chains in theta-solvent,
in agreement with what is measured for glutenin depleted samples (Fig. 3a). At
larger length scale, i.e. at small $q$, on the other hand, the scattered
intensity is found to vary as a power law with the scattering vector: $I\sim
q^{-d_{\rm{f}}}$. This power law indicates large length scale heterogeneities
in the spatial organization of the chains, which are characterized by a
fractal dimension $d_{\rm{f}}$. The fractal dimension is the same at all
concentrations but the amplitude of the power law slightly varies with $C$.
The fractal structure is measured up to the smallest accessible $q$
($q_{\rm{min}}=10^{-2}$ nm-1), hence up to length scales of the order of
$2\pi/q_{\rm{min}}\approx 600$ nm. This length scale is much larger than the
typical size of the protein assemblies ($<R>=85$ nm, see above). Considering
the presence of much larger polymer-like objects for the samples prepared with
$GLU=45$ % than for the samples prepared with $GLU=13$ %, we expect for the
samples with $GLU=45$ % an overlap concentration smaller than the one for the
samples with $GLU=13$ ($C^{*}\simeq 100$ mg/mL). Hence, it is reasonable to
state that data shown in Figure 3b very likely correspond to the semi-dilute
regime.

Similar analysis as the ones described above are performed for samples
prepared with different protein extracts, with $GLU$ ranging from $4$ to $66$
%. The evolutions with the glutenin content of several structural parameters,
the persistence length, the power law exponent at intermediate $q$, and the
fractal dimension at smaller $q$, are plotted in Figure 4. Results show a
polymer-like behavior of all extracts, independently of their composition,
with a same persistence length ($l_{p}=0.74\pm 0.1$ nm) and a same behavior at
small length scale ($p=2.0\pm 0.2$). Interestingly, however, two regimes are
clearly evidenced, by the onset of a power law evolution of the scattered
intensity at small $q$, characterized by a fractal dimension of the order of
$2$. This onset coincides with the threshold for the presence of protein
assemblies detected in very dilute regime ($GLU$ larger than $30$ %
typically).

Figure 4: Persistence length, $l_{p}$ (a), exponent of the scattered intensity
with wave vector $q$ at large $q$, $p$ (b) and fractal dimension measured at
small $q$ (c) as a function of the glutenin fraction of the protein extract.
Open symbols correspond to data obtained with slightly different set-ups and
protocols [14], which may explain the difference, in particular for the
parameter $p$. The dotted lines in (a, b) correspond to the average of $l_{p}$
(a), and $p$ (b).

####  3.2.2 Indirect probing of H-bonds between proteins

Figure 5: Scattering profiles measured by (a) SAXS and VSANS/SANS, for a
sample with $GLU=66$ % prepared in a hydrogenated or deuterated solvent, (b)
SANS and VSANS for samples prepared in deuterated solvent with different
protein extracts as indicated in the legend. The symbols are data points and
the lines are best fits using a Debye-Bueche model (see text). In (a, b) the
protein concentration is $C=237$ mg/mL. In (a), because of different contrast
in SAXS and VSANS/SANS, data are shifted vertically to allow an overlap of the
scattered intensity at large $q$. Measurements are performed at room
temperature. SAXS data are acquired at ESRF facility and VSANS/SANS data are
acquired at MLZ facility.

Figure 5a summarizes the features of the scattering profiles, as measured by
small-angle X-ray scattering (SAXS), and by very small-angle and small-angle
neutron scattering (VSANS and SANS), for samples prepared in hydrogenated and
deuterated solvents. Data are only displayed for fixed protein concentration
($C=237$ mg/mL) and composition ($GLU=66$ %) but similar results are obtained
for other $GLU$ (data not shown). As mentioned above, in SAXS, one probes the
spatial distribution of the protein chains in the solvent. A neutron
scattering experiment by contrast is sensitive to the scattering length
densities of the different species, and the main contrast probed in the
experiment changes depending whether the solvent is hydrogenated or
deuterated. In the case of a hydrogenated solvent, the main contrast is the
one between proteins and solvent. We find that the scattering profiles
measured in SAXS (for both solvents) and in SANS using an hydrogenated solvent
nicely overlap in the whole range of scattering vectors. The perfect overlap
of the SAXS data for hydrogenated and deuterated solvents in the whole
$q$-range demonstrates that replacing H by D in the solvent molecules does not
change the spatial organization of the proteins, for length scales ranging
from $\sim 1$ nm to $\sim 1$ $\mu$m, suggesting identical interactions at
play. In the case of neutron scattering measurements with a deuterated
solvent, the contrast mainly originates from the differences between the
scattering length density of H and D. In that case, we observe that data at
large $q$ ($q>0.2$ nm-1) also overlap with the other scattering profiles,
showing a unique structure of the protein chains at small length scale. In
sharp contrast, however, drastically different scattering profiles are
measured by VSANS and SANS for a deuterated solvent in the low $q$ region:
instead of the $q^{-2}$ scaling, due to large scale fractal organization of
heterogeneities in the proteins spatial organization, a $q^{-4}$ scaling
followed by a pseudo-plateau at very small $q$ is measured. As already
discussed previously for a sample with $GLU=52$ % [37], the striking
difference between the scattering profiles originates from the heterogeneous
exchange between the deuterium atoms comprised in the solvent and the labile
hydrogen atoms of the protein chains. In certain regions of the sample, strong
and/or multiple H-bonds between proteins prevent the standard D/H exchange
between solvent and proteins. In these domains, one expects an enrichment in H
as opposed to other parts of the samples. Neutron scattering experiments are
sensitive to the H/D contrast, hence are probing the H-rich domains, which are
domains enriched in H-bonds between proteins. The $q^{-4}$ scaling indicates
well defined H-rich domains with sharp interfaces. The transition from this
scaling towards a plateau at smaller $q$ allows an evaluation of the
characteristic size of these domains. More quantitatively, the scattering
profiles can be fitted with a Debye Bueche model (DB), conventionally used to
describe micro-phase separated solids with sharp interfaces [38]:
$I=\frac{I_{0}}{[1+(q\Xi)^{2}]^{2}}$. Here $I_{0}$ is the plateau value of the
scattering intensity at low $q$ and $\Xi$ is the characteristic size of the
phase-separated domains. We find that the DB model accounts well for the
experimental data, as exemplified by the fits of some selected data (Fig. 5b).
We find moreover that the characteristic size of the domains enriched in
H-bonds between proteins are constant and independent on the protein
composition, $\Xi=(67\pm 2)$ nm. Interestingly, the size $\Xi$ is comparable
to the size of the protein assemblies measured in very dilute solution
(average size $85$ nm).

Figure 6: Scattered intensity at very low $q$ as measured in VSANS normalized
by the protein concentration as a function of the amount of assemblies in the
samples. Full and empty symbols correspond to measurements performed with
protein extracts as obtained with the protocol described in the text . Gluten
concentration is $C=237$ mg/mL for full symbols (as in Fig. 5b), for the
cross-filled symbol (resp. dot-filled), $C=210$ mg/ml (resp. $302$ mg/ml) and
$GLU=56$ % (resp. $44$ %). Half-filled symbols correspond to measurements
performed with mixtures of two extracts with $GLU=13$ % and $GLU=66$ %.

To check more quantitatively the link between the domains enriched in H-bonds
between proteins, measured in semi-dilute regime, and the presence of protein
assemblies as inferred from measurements in very dilute regime, we plot in
Figure 6 $I_{0}/C$, with $I_{0}$ the value of the low $q$ plateau of the
scattered intensity, as a function of the % of assemblies in the sample. The %
of assemblies is evaluated in dilute samples using asymmetrical flow field-
flow fractionation coupled to a differential refractive index detection. We
measure that $I_{0}/C$ varies roughly linearly with the % of assemblies in the
samples. Note that because the size of the domains is measured to be constant
and independent of the composition of the protein extract, $I_{0}$ is expected
to be directly proportional to the number of domains enriched H-bonds between
proteins per unit volume in the sample (assuming a constant H/D contrast and a
constant composition of the domains). Hence Figure 6 suggests a direct
proportionality constant between the number of protein assemblies and the
number of H-bonds rich domains. In Figure 6, full symbols correspond to
measurements performed with different protein extracts as obtained with the
protocol described above with different quenching temperature $T_{q}$, and
half-filled symbols correspond to VSANS measurements performed with mixtures
of two extracts with $GLU=13$ % and $GLU=66$ %. The reasonable collapse of all
data onto a single curve indicate that a simple dilution law holds. It
moreover suggests that the protein assemblies are stable whatever their
environment (quantity of solvent and presence of gliadins).

To better assess the stability of the regions enriched in H-bonds between
proteins, we investigate the evolution of the scattering profiles with
temperature. Measurements acquired at different temperatures, from $8$ to
$35^{\circ}$C, in SAXS and SANS and for hydrogenated and deuterated solvents,
are shown in Figure 7 for a sample with $C=237$ mg/mL and $GLU=66$ %. As
temperature decreases, the onset of a liquid-liquid phase-separation is
evidenced in SAXS by a transition from a $q^{-2}$ to a $q^{-4}$ power law
dependence at small $q$. As anticipated from previous experiments in a fully
hydrogenated solvent [39], the liquid-liquid phase separation is detected,
both by SAXS and SANS, for samples prepared with a hydrogenated solvent
(H-solvent). We find that a liquid-liquid phase separation also takes place
for a sample prepared in a deuterated solvent (D-solvent). For a sample
prepared in a H-solvent, slightly different temperatures are determined with
the two techniques (which might be explained by the different set-ups used).
Using a same SAXS apparatus, we measure a significantly higher temperature in
a D-solvent (about $18^{\circ}$C) than in a H-solvent (about $14^{\circ}$C),
consistently with previous measurements by differential scanning calorimetry
for other samples [18]. This is in line with differences in the strength of
hydrogen bonds for H and for D and hints as a role of hydrogen-bonding as
driving force for liquid-liquid phase-separation. Interestingly, we do not
find any modification with temperature of the SANS pattern of a sample
prepared in a D-solvent (Fig. 7d). Hence, the number and size of the domains
enriched in H-bonds between proteins are not affected by the liquid-liquid
phase separation. We believe these domains get concentrated in the rich-phase,
which is enriched in glutenins [22], in agreement also with infrared data
showing that the heavy phase in enriched in H (and depleted in D) as compared
to the light phase [39]. In addition, the fact that these domains are not
perturbed when the temperature varies in the one-phase region (in the range
$[18-35]^{\circ}$C) strongly suggests that they are very stable and robust
structures.

Figure 7: Scattering profiles measured at different temperatures, as indicated
in the legend, by small angle X-ray scattering (a, b) and neutron scattering
(c, d), for samples with a fixed protein concentration ($C=237$ mg/mL) and a
fixed glutenin content ($GLU=66$ %) prepared in a hydrogenated solvent (a, c)
or deuterated solvent (b, d). SAXS measurements have been acquired using a in-
house set-up and SANS measurements have been acquired at LLB (c) and MLZ (d)
facilities.

###  3.3 Linear visco-elasticity

We report in Figure 8, the frequency dependence of the storage, $G^{\prime}$,
and loss, $G"$, moduli as a function of frequency, for samples with a fixed
concentration ($C=237$ mg/mL), but different composition of the gluten
extract. Samples depleted in protein assemblies ($GLU=13$ % and $GLU=19$ %)
are purely viscous. The storage modulus is too low to be measured reliably and
the loss modulus is found to be proportional to the frequency:
$G"=\eta\omega$, with a viscosity $\eta\simeq 100$ mPas. This value is in
agreement with measurements performed with gliadin suspensions prepared using
a different protocol [40]. By contrast, the samples prepared with protein
extracts comprising protein assemblies ($GLU>30$ %) display a marked visco-
elastic signature. The two most enriched in glutenin samples ($GLU=56$ and
$66$ %) are essentially elastic: their storage modulus is nearly frequency-
independent, and is larger than their loss modulus, in most or the whole
experimentally investigated frequency range (from $10^{-2}$ to $10^{2}$
rad/s).

Concentration-dependent aging of gels prepared with $GLU=52$ % has been
previously investigated in detail by some of us and we have shown that the
visco-elasticity and the gelation process could be quantitatively rationalized
in the framework of near-critical gels [15, 41, 42, 43]. We show here that the
same features occurs for a whole class of gluten gels. The near-critical gel
features is especially exemplified in the sample with $GLU=44$ %. In the
window of experimentally accessible frequencies, a fresh sample exhibits the
visco-elastic properties of a critical gel, with $G^{\prime}\sim
G"\sim\omega^{0.85}$ and $G">G^{\prime}$. For an aged gel on the other hand,
an elastic plateau ($G^{\prime}>G"$) is measured at low frequency and the
transition to power evolution of the two moduli is measured at higher
frequency. This is the signature of near-critical visco-elasticity above the
gel point.

Visco-elasticity of the gluten gels is governed by H-bonds [15, 44, 18], and
aging, as related to the increase of the elastic modulus with the time elapsed
since sample preparation, is likely due to the reorganization of the H-bonds
in the sample. In accordance, we note that the purely viscous samples do not
exhibit any aging features, as opposed to the visco-elastic samples.
Interestingly, we also find that the sample that is the most enriched in
glutenin ($GLU=66$ %) does not seem to exhibit any aging over the investigated
period (data for a fresh sample and a $35$ day-old sample perfectly overlap),
as opposed to the samples with $GLU=44$ % and $GLU=56$ %, which exhibit
significant increase of their complex modulus with time. Although, this
finding should deserve a deeper investigation, we believe this might be due to
the hindrance of the H-bonds reorganization in a highly elastic material.

Overall we find that the rheological properties of the samples are directly
related to their structure. The emergence of visco-elasticity is directly
correlated to the presence of protein assemblies in the dilute regime, which
is also associated to the presence of H-bonds-rich domains in semi-dilute
regime.

Figure 8: Storage and loss moduli as a function of frequency, for fresh and
$35$ days old samples prepared with a fixed protein concentration $C=237$
mg/mL, and with protein extracts comprising various amount of glutenin, as
indicated in the legend. In (a, b), the lines are power law evolution with an
exponent of $1$ (a) and $0.85$ (b).

##  4 Conclusion

We have investigated the structure of dispersion of gluten proteins, with
tuneable composition, in a good solvent. Gluten proteins are mainly composed
of a blend of monomeric proteins, gliadins, and polymeric proteins, glutenins.
In principle, gluten proteins are by themselves at the cross-road between
polymers and colloids. Despite the complexity and the numerous interactions at
play in gluten, however, our experiments show that the material properties are
dominated by the polymer nature of the constituents. Thanks to a combination
of several techniques that probe the sample properties in different
concentration regimes, we have evidenced the major role played by the protein
assemblies. These assemblies are non compact and very stable structures with a
size of the order of $100$ nm, which form even in very dilute regime, once the
proportion of glutenins in the protein blend is sufficiently high. They can be
assimilated to microgels with polymer chains held together by weak hydrogen
bonds. Thanks to their distinctive contrast in neutron scattering, we have
been able to identify their signature in semi-dilute regime and quantify their
amount as a function of the initial protein composition. The fact that sizes
of the same order of magnitude are measured when the protein concentrations
varies by almost two orders of magnitude is intriguing and would deserve
further investigation. Finally, when varying the protein composition, we find
that the emergence of visco-elasticity coincides with the emergence of the
protein assemblies, demonstrating their crucial function tuning gluten gel
mechanical properties. Gluten being an essential ingredient of dough, and
being the ingredient largely responsible for the unique visco-elastic
properties of wheat dough, characterizing and rationalizing the properties of
gluten gels is obviously crucial in many technological and industrial
applications.

## Acknowledgements

Financial supports from ANR Elastobio (ANR 18 CE06 0012 01) and from Labex
Numev (ANR-10-LAB-20) are acknowledged. This work is also based upon
experiments performed at the KWS-2 and KWS-3 instruments operated by JCNS at
the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany, at PA-20 beamline
operated by Laboratoire Léon Brillouin, Gif-sur-Yvette, France and at ID02
beamline operated at the European Synchrotron Radiation Facility (ESRF),
Grenoble, France. The authors thank Theyencheri Narayanan and Alessandro
Mariani for assistance in using beamline ID02 at ESRF and Philippe Dieudonné
for help in the in-house SAXS measurements.

## Bibliography

## References

  * [1] Li Y 2012 Chemical Society Reviews 41 2193–2221
  * [2] Cankaya N 2017 Elastomers (IntechOpen)
  * [3] Meldrum O W, Yakubov G E, Bonilla M R, Deshmukh O, McGuckin M A and Gidley M J 2018 Scientific Reports 8 5802
  * [4] Jay G D, Torres J R, Warman M L, Laderer M C and Breuer K S 2007 Proceedings of the National Academy of Sciences 104 6194–6199
  * [5] Nishikubo N, Awano T, Banasiak A, Bourquin V, Ibatullin F, Funada R, Brumer H, Teeri T T, Hayashi T, Sundberg B and Mellerowicz E J 2007 Plant and Cell Physiology 48 843–855
  * [6] Yu L, Yakubov G E, Gilbert E P, Sewell K, van de Meene A M and Stokes J R 2019 Carbohydrate Polymers 207 333–342
  * [7] Sabu T, Chin H C, Laly P, Rajisha K R, Jithin J and Maria H 2013 Natural Rubber Materials (RSC Polymer Chemistry Series)
  * [8] Wrigley C, Bekes F and Bushuk W 2006 Gliadin and Glutenin: The Unique Balance of Wheat Quality (American Association of Cereal Chemists, Inc.)
  * [9] Shewry P R and Tatham A S 1990 Biochemical Journal 267 1–12
  * [10] Wrigley C W 1996 Nature 381 738–739
  * [11] Wieser H 2007 Food Microbiology 24 115–119
  * [12] Tatham A S, Hayes L, Shewry P R and Urry D W 2001 Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1548 187–193
  * [13] MacRitchie F 2014 Journal of Cereal Science 60 4–6 ISSN 07335210
  * [14] Dahesh M, Banc A, Duri A, Morel M H and Ramos L 2014 J. Phys. Chem. B 118 11065–11076
  * [15] Dahesh M, Banc A, Duri A, Morel M H and Ramos L 2016 Food Hydrocolloids 52 1–10
  * [16] Banc A, Dahesh M, Wolf M, Morel M H and Ramos L 2017 Journal of Cereal Science 75 175–178
  * [17] Morel M H, Pincemaille J, Chauveau E, Louhichi A, Violleau F, Menut P, Ramos L and Banc A 2020 Food Hydrocolloids 103 105676
  * [18] Costanzo S, Banc A, Louhichi A, Chauveau E, Wu B, Morel M H and Ramos L 2020 Macromolecules 53 9470–9479
  * [19] Belton P S 1999 Journal of Cereal Science 29 103–107
  * [20] Morel M H, Redl A and Guilbert S 2002 Biomacromolecules 3 488–497
  * [21] Ng T S K, McKinley G H and Ewoldt R H 2011 Journal of Rheology 55 627–654
  * [22] Pincemaille J, Lecacheaux L, Banc A, Menut P, Ramos L and Morel M H manuscript in preparation
  * [23] Narayanan T, Sztucki M, Van Vaerenbergh P, Léonardon J, Gorini J, Claustre L, Sever F, Morse J and Boesecke P 2018 Journal of Applied Crystallography 51 1511–1524
  * [24] Radulescu A, Szekely N K, Appavou M S, Pipich V, Kohnke T, Ossovyi V, Staringer S, Schneider G J, Amann M, Zhang-Haagen B, Brandl G, Drochner M, Engels R, Hanslik R and Kemmerling G 2016 Journal of Visualized Experiments 54639
  * [25] Pipich, V and Fu, Z 2015 Journal of Large-Scale Research Facilities 1 A31
  * [26] Frielinghaus H, Pipich V, Radulescu A, Heiderich M, Hanslik R, Dahlhoff K, Iwase H, Koizumi S and Schwahn D 2009 Journal of Applied Crystallography 681–690
  * [27] http://qtisas.com/
  * [28] Hofmann H, Soranno A, Borgia A, Gast K, Nettels D and Schuler B 2012 Proceedings of the National Academy of Sciences 109 16155–16160
  * [29] Kikhney A G and Svergun D I 2015 FEBS Letters 589 2570–2577
  * [30] Balu R, Mata J P, Knott R, Elvin C M, Hill A J, Choudhury N R and Dutta N K 2016 The Journal of Physical Chemistry B 120 6490–6503
  * [31] Rubinstein M and Colby R H 2003 Polymer Physics (Oxford University Press, Oxford)
  * [32] Denkinger P and Burchard W 1991 Journal of Polymer Science Part B: Polymer Physics 29 589–600
  * [33] Ohashi T, Galiacy S D, Briscoe G and Erickson H P 2007 Protein Science 16 1429–1438
  * [34] Thomson N H, Miles M J, Popineau Y, Harries J, Shewry P and Tatham A S 1999 Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology 1430 359–366
  * [35] Pedersen J S and Schurtenberger P 2004 Journal of Polymer Science Part B: Polymer Physics 42 3081–3094 ISSN 0887-6266, 1099-0488 URL http://doi.wiley.com/10.1002/polb.20173
  * [36] Schaefer D W 1984 Polymer 25 387–394
  * [37] Banc A, Charbonneau C, Dahesh M, Appavou M S, Fu Z, Morel M H and Ramos L 2016 Soft Matter 12 5340–5352
  * [38] Debye P and Bueche A M 1949 Journal of Applied Physics 20 518–525
  * [39] Banc A, Pincemaille J, Costanzo S, Chauveau E, Appavou M S, Morel M H, Menut P and Ramos L 2019 Soft Matter 15 6160–6170
  * [40] Boire A, Menut P, Morel M H and Sanchez C 2015 The Journal of Physical Chemistry B 119 5412–5421
  * [41] Winter H H and Chambon F 1987 Journal of Rheology 30 367–382
  * [42] Martin J E, Adolf D and Wilcoxon J P 1988 Physical Review Letters 61 2620
  * [43] Martin J E, Adolf D and Wilcoxon J P 1989 Physical Review A 39 1325–1332
  * [44] Ng T S K and McKinley G H 2008 Journal of Rheology 52 417–449