RESEARCH ARTICLE

OPEN ACCESS

# Water Molecular Dynamics During Dough Heating by Fast Field Cycling Nuclear Magnetic Resonance

Corinne Rondeau-Mouro¹ ID | Anne-Laure Rollet² ID

¹OPAALE, INRAE, Rennes, France | ²Laboratoire Physico-Chimie des Électrolytes et Nano-Systèmes Interfaciaux (PHENIX), Sorbonne Université/CNRS, Paris, France

**Correspondence** Corinne Rondeau-Mouro (corinne.rondeau-mouro@inrae.fr) | Anne-Laure Rollet (anne-laure rollet@sorbonne-universite.fr)

**Received:** 29 April 2025 | **Revised:** 21 July 2025 | **Accepted:** 30 July 2025

**Funding:** The authors received no specific funding for this work.

**Keywords:** ¹H NMR | ²D NMR | dough | fast field cycling NMR | gluten | starch

## ABSTRACT

Measurements of the water ¹H nuclear magnetic resonance (NMR) relaxation rate $R_1$ in hydrated starch (SW), gluten (GW) and dough (DW) were performed at different frequencies using fast field cycling, at 20, 40, 60, and 80°C and again at 20°C after cooling. The originality of this study lies in the measurements performed during the heat-induced dough transformation to investigate the impact of temperature on water distribution and interaction with other dough constituents. Rather than measuring NMRD on off-line heated samples, real-time heating and cooling was applied directly in the spectrometer in such a way that sample temperatures were fully controlled when measuring $T_1$ at various frequencies. Measurements before heating enabled the detection and interpretation of potential differences in the dynamics of each sample's NMR dispersion (NMRD) profile. At low temperatures, water dynamics in DW and interactions with other dough constituents were dominated by the interaction of the water with starch granules. The NMRD profile for GW differed significantly from the others, suggesting that gluten retains more water when it is mixed with starch. By heating each sample, the hindering effect of gluten on hydrothermal changes in starch (swelling and gelatinization) was confirmed. Last, the competitive interactions between hydroxylated groups of each macromolecule and the hydrogen in water molecules were shown to be continuously driven by chemical exchanges that influenced $R_1$ values in all samples.

## 1 | Introduction

Understanding the role of water in cereal products is essential for several reasons. In raw materials such as wheat or corn, water influences the grains' growth and development (cell size, nutrient flow, growth cessation). Their use and processing, too, is also highly dependent on the water both initially present and subsequently accessible to constituents for their transformation into a more digestible product. Bread typifies cereal products for which the presence of water is a prerequisite for the flour to transform upon heating. The approximate composition of wheat flours used in bakery products is 71% starch, 12% proteins and 14% water (calculated in g for 100 g of flour) [1, 2], with minor

components (insoluble and soluble pentosans and lipids) making up less than 3% of the flour. Bread dough preparation requires the addition of water and minimal quantities of ingredients such as yeast and salt, before mixing all constituents together to homogenize the mixture. Last, the final hydration level of bread dough varies between 40% and 46% (wet basis), depending on the choice of flour and the end product desired [3].

Starch in its native form, is composed of granules with diameters between 3 and 100 µm, depending on the botanical source. In the presence of water, starch strongly hydrates due to the hydrophilic nature of the macromolecules that make up the granules, amylose and amylopectin. Moreover, macropores can be

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© 2025 The Author(s). *Magnetic Resonance in Chemistry* published by John Wiley & Sons Ltd.
observed on the surface of starch granules, suggesting a capillary internal structure that facilitates their hydration [4].

The addition of water to starchy products is of particular interest because the starch-to-water ratio determines the extent of starch hydrothermal transformation. The latter occurs through gelatinization, not as a single event, but as a group of combined events that are challenging to analyze in full [5]. Indeed, it has been established that starch gelatinization involves some combination of starch granule hydration and swelling, crystallite melting or loss of crystallinity, leaching of soluble carbohydrates, and irreversible loss of granular structure.

Furthermore, gluten, which is the main protein fraction of wheat flour, is organized in a sheet structure where the network is formed by cross-linked protein sheets layered one over the other [6–8]. In this structure, confined unfreezable water, which interacts strongly with the gluten matrix, is thought to be found within nanopores [7, 9, 10]. The heating of gluten results in the unfolding and aggregation of glutenin and gliadin, the two protein constituents of gluten. The effects are due to the disruption of non-covalent hydrogen interactions and the formation of S-S bridges between exposed free sulfhydryl (SH) groups of glutenin and gliadin, the two proteins contained in gluten [8, 11]. These events result in the loss of viscoelasticity, water retention capacity and solubility [12–15]. In the case of both starch and gluten, the role played by water in the structural changes induced during heating is generally poorly understood.

Of the methods used to investigate the dynamics and interaction of water with biopolymers, time domain NMR (TD-NMR) has been shown to have major benefits due to its non-destructive and non-invasive nature, speed, cheapness and ease of use. This method is based on the measurement of the physical process known as relaxation [16, 17]. Relaxation is commonly analyzed in terms of two separate time constants: spin-lattice relaxation time ($T_1$) associated with the transfer of energy between the spin system and the environment, and spin-spin relaxation time ($T_2$), corresponding to an entropic process that does not affect the energy of the system. As these relaxation phenomena are governed by the dynamics of the spins within their environment, to be efficient, the characteristic times of the spin dynamics must be proportional to the frequency at which the measurements are performed. Consequently, by changing the measurement frequency, it is possible to change the time window at which the dynamical process is studied. In short, high frequencies shed light on rapid motions such as molecular rotations, while low frequencies highlight slow motions such as translational diffusion, especially in confined media [18–20].

TD-NMR at a fixed constant field, has proved to be a powerful tool for the real-time monitoring of the transformation of starch, gluten or model doughs, which varies according to flour origin and sample water content [10, 21-24]. This analytical technique is able to provide quantitative information on simple or complex food systems by probing the state, quantity and distribution of water, along with the mobility of other molecules for all sample states, whether solid, soft, or liquid [24]. For cereal products, $T_2$ measurements have been widely used because of their sensitivity to water compartmentation, chemical and diffusion exchanges with starch, and structural changes in gluten occurring

during hydrothermal transitions. Although less frequently used, $T_1$ have also been helpful for the monitoring of starch gelatinization [23, 25].

When used as a complementary method to studies at fixed fields, the Fast Field Cycling (FFC) can be very useful to the study of water dynamics in a complex system of this type ( cereal products), and has already been used successfully for other complex systems, including protein-based [26–30] and inorganic [31–34] dispersions, hydrogels [35], paints [36, 37], cement [38–40] and a wide variety of porous media [41] (porous silica [42–44], synthetic membrane [45], shale [46, 47], etc. [18–20]). Indeed, this technique makes possible the measurement of relaxation times from 0.01 MHz to tens of MHz (typically 42 MHz for commercially-available relaxometers).

FFC has been recently applied to many foodstuffs to complement knowledge on sample composition, but it has also been used to distinguish inter- and intra-molecular water interactions modulated by molecular dynamics across a wide dynamic range [48]. It may help to decipher the different dynamical processes that are expected to occur in dough (Figure 1). Already in 2011, Curti et al. [49] demonstrated the effectiveness of this method of analysis in the 0.01 to 10 MHz range when investigating water distribution during bread staling at 25°C. In the present study, we focus on FFC

<figure><img src="image_1.png" /><figcaption>**FIGURE 1** | Diagram of the possible different dynamical processes observable in the NMR relaxation experiments. The ¹H relaxation being ruled by dipolar interaction, the low field relaxation mechanisms imply the relative motion of pairs of ¹H, each of them being carried by another water molecule or by one of the macromolecules (amylose, amylopectin, glutenin, and gliadin).</figcaption></figure>
measurements performed in real-time during the heating of dough in a step sequence. First, the $T_1$ relaxation profiles of the main wheat dough constituents, starch and gluten, were analyzed. Measurements on $^2$D were also carried out in order to identify the intramolecular contributions. Analysis was then performed on wheat dough samples with the same water content as the starch (45% wet basis, wb), this being that generally studied in the literature.

## 2 | Experimental Details

### 2.1 | Sample Preparation

Samples were prepared using wheat starch (Sigma-Aldrich, Saint-Quentin-Fallavier, France), wheat gluten (Eurogerm SAS, France) and distilled water. Wheat flour (12.52 ± 0.30% moisture) was provided by Grands Moulins de Strasbourg (GMS Meunerie, “Mirabelle” France). Distilled water was added to flour to achieve a final water content of 45% (wb). The dough was then mixed by a kneading machine (Alvéolab, Chopin) following the protocol described in [50].

Starch was hydrated at 45% and gluten at 60% as described in Rondeau-Mouro et al. [22] and Rakhsi et al. [10]. The hydrated samples were mixed in a beaker with a spatula to obtain an homogeneous mixture. In the case of gluten, the hydrated sample was kept at room temperature for around 16 h before measurements were performed, allowing the gluten to swell.

For hydration in heavy water, the powders have been first re-hydrated with $D_2O$ (99.9% purity, Eurisotop, France) before being freeze dried over one day. The following rehydration for the NMR analyses have been performed using $D_2O$ as described above for light water.

Samples are referred to as follows:

- SW for wheat starch hydrated with distilled light water at 45% (wb),

- GW for wheat gluten hydrated with distilled light water at 60% (wb),

- SD for wheat starch hydrated with heavy water at 45% (wb),

- GD for wheat gluten hydrated with heavy water at 60% (wb),

- DW for wheat dough made with traditional wheat flour hydrated with distilled light water at 45% (wb).

The mixtures were prepared the day before the experiment and left to rest over the night.

### 2.2 | NMR Relaxation Measurements

The $^1H$ NMR relaxation rates $R_1(\omega_0) = 1/T_1(\omega_0)$ were measured on two different NMR relaxometers: the Stelar Spinmaster ($\nu_0$=10 kHz to 30 MHz) and the Stelar Smatracer ($\nu_0$=10 kHz-10 MHz). For the Spinmaster, $T_1$ was measured using a pre-polarized (PP) and a non-polarized (NP) sequence from 10 kHz

to 10 MHz and from 10 MHz to 30 MHz, respectively [51]. For the Smatracer, a PP sequence was used from 10 kHz to 6 MHz while the NP sequence was performed from 6 to 10 MHz. The $^2$D NMR relaxation rates have been measured only using the Spinmaster in the frequency range.

The samples were each placed in a glass tube (external diameter 10mm, length 38 mm) sealed with a silicon cap. The NMR tubes were filled almost completely to prevent evaporation during the experiments [52].

The $^1H$ nuclear magnetic resonance dispersion (NMRD) profile, that is, $R_1(\omega_0)$ versus $\omega_0$, were performed on the hydrated systems (SW, GW, and DW) at 20, 40, 60, and 80°C during heating, and finally for a second time at 20°C after cooling (20°C-cooled). Temperature control is provided by a flow of cold air heated by a thermal resistance in the probe. The temperature ramp is 10°C/min. Measurements are taken 15 min after reaching the targeted temperature to ensure that the sample is homogeneous in temperature (see Supporting Information for the sample stabilization control).

Prior to the main study, reproducibility was tested for the most simple and complex systems, that is, SW and DW. The $^1H$ Nuclear Magnetic Resonance Dispersion (NMRD) profiles were measured for four different DW preparations and three SW mixtures (with the same composition and hydration level). All curves plotted for the same system were then superimposed (see Figure S1).

Each magnetization curve was satisfactorily fitted monoexponentially within the margins of experimental error. However, fitting with a bi-exponential function, tested using the EMILIO-FID software produced unsatisfactory results (large standard errors) [53].

## 3 | Results

In order to investigate water distribution in the wheat dough (DW) and its interaction with the different constituents, we first performed FFC measurements for each separate constituent hydrated with light water (i.e., the starch-water (SW) and gluten-water (GW) mixtures). The results were then compared to $T_1$ and $T_2$ relaxation times measured at fixed field (20 MHz) and published elsewhere [10, 21-25, 54].

### 3.1 | Interaction of Water With Dough Constituents at 20°C

While the hydration was 45% (wb) for DW and SW, it was 60% for GW as below this hydration level, the gluten-water mixture lacks homogeneity, containing non-hydrated assemblies that require several hours of hydration equilibrium [54]. The $R_1$ values measured were assigned to water rather than macromolecules, as the latter are characterized by very long $R_1$ [23].

The NMRD profiles of the three preparations in light water—DW, SW, and GW—are plotted in Figure 2. Each mixture has its own NMRD profile, although those of DW and SW are similar.
<figure><img src="image_2.png" /><figcaption>FIGURE 2 | NMRD profiles at T=20°C for DW (black circles), SW (red squares), GW (blue triangles), and the calculated contribution of gluten in the dough R₁_gw-calc (open blue triangles). Green and purple dotted lines indicate crossovers and the frequency at which the profiles for DW and SW diverge, respectively.</figcaption></figure>

Especially at low fields, from 10 kHz up to approximately 3 MHz, they are almost parallel, with R₁_gw being slightly higher than R₁sw. This indicates that starch is the constituent that mainly influences the state and distribution of water in DW. In other words, the dynamics of water in DW and its interaction with the other dough constituents are dominated by the interaction of water with starch granules.

In the fast exchange regime, R₁(ω₀) can be expressed as the sum of all the R₁'(ω₀) resulting from the various dynamical processes i, where ω₀ is the resonance angular frequency:

$$R_1(\omega_0) = \sum R_1^i(\omega_0) \quad (1)$$

Furthermore, in the absence of paramagnetic species, each R₁' often exhibits a unique feature, as for example, a Lorentzian function for a rotational motion (see Figure S8 in supplementary information). Despite the absence of features as striking as NMR spectra peaks, examination of a slope can yield useful information. Likewise, crossovers, that is, the frequencies at which a slope changes and decreases, are often related to the characteristic durations of the dynamical processes at play [19, 55-57]. Hence, closer examination of Figure 2 reveals three different dynamical processes at work in the DW and SW samples. From 10 to 40 kHz, the NMRD profiles follow a smooth curve corresponding to a very slow process. From 40 kHz to 0.5 MHz, R₁ varies as ω₀⁻¹/⁴ and as ω₀⁻⁰.⁶ above. Assuming that the crossovers would correspond to the characteristic durations of these two dynamical processes, this would give 4 µm for the first slower process and 0.3 µm for the second (τ_c ~ (2πν₀)⁻¹). The slower process may relate to the release of water from the granule's swollen surface. Indeed, the strength of the water-starch interaction causes this phenomenon to take a long time. Above 5 MHz approximately,

the plots for DW and SW diverge. Whereas R₁sw continues to decrease as ω₀⁻⁰.⁶, the evolution of R₁_gw changes around 3 MHz, with a less pronounced decrease than ω₀. Such changes at high field can often suggest the contribution of bulk water. Indeed, the dynamical correlation time for bulk water being shorter than the times window explored here, R₁bulk(ω₀) could be expected to reach a plateau of about 0.5 s⁻¹ [19, 58]. However, in this instance, 0.5 s⁻¹ is far too small a value to affect the experimental curves. The differences between the R₁' of DW and SW observed at fields above 5 MHz can therefore be attributed to the presence of gluten in DW.

The NMRD profile for GW was significantly different from those for DW and SW, especially above 0.1 MHz. To evaluate the gluten contribution for DW, we compared its NMRD profile to that calculated from Equation 2, where the possible gluten contribution (R₁_gw-calc) is calculated by subtracting the starch contribution (R₁sw at 89% in dry mass) to the DW profile R₁_gw (Figure 2).

$$R_{1\text{GW-calc}} = R_{1\text{DW}} - 0.89R_{1\text{sw}} \quad (2)$$

Indeed, the R₁ measurement for the particles contained in the mixture, without any specific interaction between the two, is the simple sum of the R₁ for each particle weighted by its concentration in the mixture, and increases with the quantity of relaxation sources (interfaces, paramagnetic species, proteins, etc.) [59].

The striking point, here, is that the calculated profile for gluten differs from the measured profile for GW hydrated at 60% at frequencies above 0.3 MHz. This result highlights the interaction of the gluten with starch granules, and its impact on the dynamical properties of water. Furthermore, from 10 kHz to 0.3 MHz, the calculated profile of gluten for a hydration level of 45% fits surprisingly well when superimposed on the measured profile for GW hydrated at 60%. This result suggests that the hydration level of the gluten in DW hydrated at 45% was higher than would have been expected for a gluten-only mixture hydrated at 45%. In other words, it would appear that gluten retains a greater quantity of water when it is in a mixture with starch. This interpretation agrees with observations made when preparing samples. The GW sample, hydrated at 60%, even when stabilized at room temperature for 12 hours, appeared to seep, producing water droplets when pressed. By contrast, SW and DW were relatively homogeneous with no seepage of water. This conforms with the inhibitory effect of gluten on starch swelling, and therefore on starch-water interactions in dough, that has recently been demonstrated in Rakhshi et al. [10] using fixed-field TD-NMR, a topic surrounded by some disagreement in the literature.

Between 0.3 MHz and 3 MHz, R₁_gw-calc follows the same R₁ evolution as the SW and DW samples, though with lower R₁ values, but its profile differs strongly from R₁_gw. In the frequency range of 20 kHz to 3 MHz, R₁_gw-calc varies as ω₀⁻⁰.². This surprising result shows that gluten is also strongly impacted by starch granules. Furthermore, it is in the high frequency domain, that is, fast dynamics, that this difference occurs. It would appear that, rather than a change occurring in the characteristic times of a fast dynamical contribution, the latter is instead extinguished
(Figure S8 and further explanation in SI). A tentative explanation for this would be that the water diffusing within the gluten network and in rapid exchange with its surface in GW is highly reduced in DW due to the strong interaction between the gluten and the starch granules. The rapid exchange of water then occurs between the gluten and starch granules, the latter having a high sorption capacity for released water. This would confirm the competition for water between gluten and starch granules [22, 60].

Above 3 MHz, the $R_{1_{GW-calc}}$ profile shows a lesser dependence on $\omega_0$ with a lower $R_1$ at low frequency than $R_{1_{DW}}$ and $R_{1_{SW}}$, indicating that the characteristic time of the fastest dynamical process lies beyond our accessible frequency range.

## 3.2 | Temperature Dependence of $R_1$

In this section, we investigate the influence of temperature on the dynamics of water in the different samples. The temperature range explored here spans from 20°C up to 80°C and back to 20°C. The sets of NMRD profiles for each mixture are shown in Figure 3.

How the profiles vary with temperature is notably different between the three samples. For clarity, we first describe the global evolution of each preparation before discussing the activation energies that can be extracted in some frequency domains. The Arrhenius plots are provided in SI (Figures S5, S6 and S7).

### 3.2.1 | Gluten GW

As the temperature increases from 20 to 80°C, relaxation rates $R_{1_{GW}}$ fall progressively, indicating that the processes involved, such as diffusion, are activated by temperature. Although the general shape of the profiles appears little affected by temperature, closer examination reveals that the average slope of the profile increases with temperature. This is more pronounced at very low fields (below 0.6 MHz). To add detail to these differences, the slopes in log-log scale, that is, the $\alpha$ values in Equation 3 have been determined and are reported in SI Table 1.

$$R_1^{slow} \propto \omega_0^{\alpha} \quad (3)$$

Individually, these slopes do not provide much information for our purposes, but their comparison does. Indeed, the fact that the evolution in temperature in the 0.01-0.05 MHz range has a steeper slope than in the 0.05-4 MHz, reveals that the NMRD profiles of GW include at least two dynamical processes, in addition to that observed at high frequency (above 10 MHz).

The first ($R_1^{slow}$) is expressed at very low field and can be assigned to very slow dynamics that are probably linked to a strong interaction between the water molecules and gluten hydroxyl groups. In addition, the relationship between the slope of the NMRD profile and $\omega_0$ changes significantly with temperature, giving $R_1^{slow} \propto \omega_0^{-0.15}$ at 20°C, as opposed to $R_1^{slow} \propto \omega_0^{-0.24}$ at 80°C (see Table S1 for details).

Because the response to temperature of the NMRD profile for GW at very low fields was a shift to lower values that left the shape

<figure><img src="image_3.png" /><figcaption>FIGURE 3 | NMRD profiles for each preparation at different temperatures. From top to bottom: gluten, starch, and wheat dough. The purple arrow indicates the position of the quadrupole peaks.</figcaption></figure>

practically unchanged, it was possible to extract the activation energy $E_A$ using to an Arrhenius plot (Figure S5 in SI). $E_A$ was found to be decreasing from 15.7 ± 0.1 kJ above 10 MHz to 12.0 ± 0.1 kJ
below 20 kHz, but nevertheless close to that obtained for the dif-
fusion of bulk water (19 kJ/mol [58]). The relationship of R₁₋ₖW to
ω₀ echoes the intermittent Brownian dynamics over a rigid strand
observed elsewhere [61]. It can therefore be attributed to the dy-
namics of water molecules intermittently interacting with the ac-
cessible hydroxyl groups of gluten amino acids.

The second process ($R_1^{\text{medium}}$) is expressed in the intermediate frequency range. Here, the slope of the profile is much less sensitive to temperature, with a measurement of $R_1^{\text{medium}} \propto \omega_0^{-0.17}$ for all temperatures, approximately, except for the cooled sample at 20°C, where $R_1^{\text{medium}} \propto \omega_0^{-0.22}$.

The third process, at frequencies above 20 MHz, had an activation energy of 16 kJ/mol and could therefore be attributed to “bulk” water, some distance from the gluten accessible surface [58].

It is also worth noting the presence of two peaks at approxi-
mately 2 and 2.8 MHz, signaling the presence of N-H bonds with
a very low mobility (Figure 4). These peaks have been reported
on numerous occasions for protein samples and are due to the
crossing of the energy gap by ¹H and ¹⁴N [62–64]. Energy gaps
are governed by the Zeeman interaction for ¹H and by both the
Zeeman and quadrupolar interactions for ¹⁴N. It is most com-
mon for three quadrupolar peaks to be observed per ¹H-¹⁴N
couple whose their position and number (2 or 3) depend on the
asymmetry parameter η of the ¹⁴N quadrupolar interaction.
Nevertheless, the peak at the lowest fields often harder to detect
due to other dynamical contributions that mask it [65]. Figure 4
shows the two most visible peaks in the GW cooled to 20°C after
heating. It should be also noted that the quadrupolar peaks re-
main visible even at 80°C and after heating (figure S4).

As the temperature increases, R₁ values decrease. The heating of
gluten above 40°C is known to result in irreversible conforma-
tional changes of proteins caused by the disruption of hydrogen
bonds, in which exposed free sulfhydryl groups of glutenin and
gliadin form S-S bridges (crosslinks and aggregates) and hydropho-
bic interactions [15, 66]. It is also known from FT-IR studies that

intermolecular beta sheets form with the loss of alpha structures
[11]. Above 60°C, the unfolding of proteins allows the exposure
of embedded intra-molecular disulfide bonds and the formation
of new inter-chain disulfide bonds, which increase hydrophobic
interactions between gluten proteins. An increase in temperature
thus causes gluten to expel water, which is expressed by the de-
creasing of R₁. This interpretation is in accordance with TD-NMR
experiments carried out at 20 MHz [54], one surprising result was
that the shapes of the NMRD profiles remained identical for all
temperatures other than 20°C after heating (20°C-cooled). The ¹H
NMRD profiles of the sample before and after heat treatment could
not be superimposed, indicating that the gluten had been modified
during the heat treatment and that the process was not reversible,
as would be expected for temperature higher than 40°C [15, 54, 66].
Furthermore, R₁<sub>GW</sub> for the initial sample was significantly higher
than R₁<sub>GW</sub> after heating.

<figure><img src="image_4.png" /><figcaption>**FIGURE 4** | Close-up of the quadrupolar peaks section of the GW NMRD profile at 20°C after heating.</figcaption></figure>

3.2.2 | Starch SW

The variations brought by temperature in NMRD profile of the starch samples differ greatly from those observed in the gluten samples (Figure 3). For starch, from 20°C to 80°C, the profiles remain superimposed at frequencies above 3 MHz. Below 3 MHz, R₁sw increases with temperature, which is the inverse of what is to be expected in a temperature-activated process. This feature is even more pronounced below 0.05 MHz. This change is already significant at 40°C. This shows that the modification of starch begins at relatively low temperatures. At 80°C, in the region of the profile representing the lowest fields (0.01 - 0.1 MHz) R₁ continues to increase, while from 0.1 MHz to 3 MHz, R₁ decreases similarly to what occurs for wheat dough (see next section). Again, the fact that it is possible to distinguish several regions in R₁ variations with different temperature dependence, helps us in identifying the existence of at least two dynamical processes.

The first ($R_1^{\text{slow}}$) is expressed at very low fields, and is probably linked to water embedded within the starch granules. The increase of $R_1^{\text{slow}}$ with temperature appears to be fully reversible as $R_1^{\text{slow}}$ at 20°C-cooled has an identical frequency dependence to that of the sample in its initial state. The small shift in the NMRD profile to higher values can be attributed to a minor loss of water.

The second dynamical regime ($R_1^{\text{medium}}$) is expressed in the in-
termediate field range. Contrary to the equivalent case for glu-
ten, it is not activated by temperature and therefore cannot be
attributed to an intermittent diffusion process. It may be similar
in nature to $R_1^{\text{slow}}$.

The extra-granular component cannot be identified in the starch profiles (high field zone). It may be related to the strong swelling properties of the starch granules that are underhydrated at 45% (wb) (no extra-granular water is present at this level of hydration).

At this point, it is very interesting to look at the evolution of the
slopes produced by plotting ln(T₁) against 1/RT, with R being the
gas constant and T, the temperature (SI, Figure S6). Contrary to
GW where ln(T₁) decreases as expected with 1/RT, ln(T₁) for SW
is almost constant versus 1/RT at high field and progressively in-
creases versus 1/RT when the field is decreased. This surprising
effect has been evidenced and finely characterized by Korb et al. [67]. In controlled pore chromatographic glasses, they recorded NMRD profiles at several temperatures of protic and aprotic liquids [19, 67, 68]. When weak interaction with the surface occurs, the dependence of ln(T₁) versus 1/RT was a decrease as expected according to the Arrhenius law for a diffusion process (T₁ ∝ e^(-(Eₐ-E<sub>m</sub>)/RT)) [68]. When strong interaction with the surface occurs, the contrary dependence was observed because both the diffusion and the interaction with the surface have to be taken into account leading to T₁ ∝ e^(-(Eₐ-E<sub>m</sub>)/RT) [67]. The difference between Eₐ and E<sub>m</sub> leads to this counter-intuitive dependence of ln(T₁) versus 1/RT. Hence the R₁<sub>sw</sub> dependence on temperature testifies the strong interaction between water and the hydroxyl groups of starch at all temperatures.

### 3.2.3 | Wheat Dough DW

The DW sample looks like an intermediate case between the gluten and the starch samples. Indeed, it is possible to recognize features in common with either gluten or starch (Figure 3). From 20°C to 40°C, the low-field region of the profile remains unchanged, while at high fields, R₁ decreases. The high-field R₁ can be again associated with bulk water weakly interacting with polysaccharide chains and its decrease, therefore, with the activation by temperature of the diffusion/rotation of water molecules. At low fields, by contrast, the shape of R₁ can be attributed to water molecules strongly interacting within the matrix. The lack of marked changes in this region of the profile between 20°C and 40°C indicates that little variation occurs in the interactions between water and polysaccharide chains occurs in DW, regardless of temperature, and no structural change in the matrix. From 60°C, this is no longer true. The low field part of the profile is significantly modified with an increase of R₁. In the high-field region of the profile, R₁ continues to decrease indicating the presence of bulk water. From 60°C to 80°C, the modification of the profile shape pursuits with an increase in the overall slope of R₁, although R₁ generally continues to decrease. An interesting difference with SW can be noted, clearly visible in Figure S3, which shows that, the modification of the NMRD profile of DW upon heating is notably weaker than in

the case of SW, especially at low fields. This fact demonstrates the inhibitory effect of gluten on the swelling/gelatinization of starch granules at high temperature.

Notably, the slope for ln(T₁) plotted against 1/RT (Figure S7 in SI) goes from 7.8 ± 0.6 kJ at high frequency to -1.4 ± 0.7 kJ at low field. Similarly to SW, these negative slopes confirm that water intermittently interacts with the matrix as already shown by Rondeau-Mouro et al. [50]. These interactions between the hydroxylated groups of macromolecules and the hydrogen in water molecules are continuously driven by chemical exchanges that explain the reduction in the activation energy compared to that of free water. The link between apparently low activation energies and strong interactions of water molecules with the surface has long been established [19, 67] and has been observed in different systems [19, 59, 67].

Last, for 20°C-cooled, the NMRD profile cannot be superimposed on the initial 20°C NMRD profile (Figure 3), at least at low fields. The increase seen in R₁ at low fields after heating can be linked to the formation of a more rigid structure analogous to crumb, where the interactions between water and the matrix are stronger.

It can be noted that the quadrupolar peaks visible in GW are not visible in DW, probably due to the low content of GW in the mixture.

## 3.3 | Deuterated Mixtures GD and SD

Concerning the ²D nucleus, its relaxation being induced by quadrupolar interaction, and consequently governed by the fluctuation of the electric field gradient, the revealed dynamics is the one of one molecule, and not potentially of a pair molecules as for ¹H. In addition, cross-relaxation and spin diffusion are negligible [69].

Figure 5 shows the ²D and ¹H NMRD profiles for gluten and starch at 20°C and 80°C. They strongly differ from each other for both gluten and starch. This results confirms that, for all kinds

<figure><img src="image_5.png" /><figcaption>**FIGURE 5** | ¹H and ²D NMRD profiles of gluten and starch mixtures with H₂O (open symbol) and D₂O (full symbols) at 20°C and 80°C. The residual ¹H in D₂O (¹H in D₂O) NMRD profile at 20°C is represented by full triangle.</figcaption></figure>
of mixture, ¹H NMRD profiles are not governed by intramolecular relaxation.

In the case of GD, a plateau is reached at low frequency, below 0.03 MHz (roughly corresponding to 5 µs). It could be associated to an escape time of water from the gluten surface. By increasing the temperature, $R_1$ decreases as it was observed for the gluten mixtures with $H_2O$. However, the evolution in temperature is much pronounced at high field and indicates that two dynamical processes are expressed in these ²D profiles. As detailed in the previous section, the weak temperature dependence at low field reveals the exchange of proton between water and ex-changeable protons of gluten. However, to a lesser extent than in the case of starch. The residual ¹H in $D_2O$ (¹H 20°C in $D_2O$) could be associated either to non-exchangeable ¹H of the proteins or to the ¹H of HDO interacting with non-exchangeable ¹H of the proteins. Further characterizations are necessary to conclude on this attribution.

In the case of SD, a plateau is also reached at low field, below 0.02 MHz (roughly corresponding to 8 µs). $R_1$ increases with temperature in the whole range of frequency. It confirms the observation made on ¹H NMRD profiles and the strong interaction of water molecules with the hydroxyl groups of starch. Furthermore, the significant increase in $R_1$ at 80°C shows that the starch gelatinization yields a very dense gel with a weak amount of free water and where the water diffusion is driven by exchanges with hydroxyl groups of starch. The residual ¹H NMRD profile almost superimposes to the one of ¹H for GW. It indicates a non sufficient replacement of ¹H by ²D, confirming the difficulty of removing all the water from starch granules, particularly for those that are not gelatinized, still containing water embedded in strong interaction with starch polymers, like structural water of amylopectin crystals.

## 4 | Conclusions

The ¹H FFC NMR technique was employed to investigate the transformation of dough constituents in real time during heating and subsequent cooling. By exploring the dynamics of water across more than three orders of magnitude in timescale, FFC NMR provided insights into the interactions between starch and gluten mediated by water molecules. The measurement of NMRD profiles for the dough and for its individual constituents (gluten and starch) mixed with water allowed us to decipher the various dynamical processes at work and their evolution with temperature. The ¹H NMRD profiles provide evidence that in dough hydrated at 45% and at low temperatures (20°C and 40°C), gluten hinders the starch water uptake, and consequently limits starch swelling in dough, thus confirming hypotheses already proposed in literature on its role in this respect. The ¹H NMRD profiles also suggested that this hindering effect is a dynamical process. This competition for water is less in evidence at higher temperatures due to irreversible protein denaturation and the high water sorption capacity of starch.

The Arrhenius plots for DW, SW and GW shows the strong interaction of water with starch granules as compared with gluten. Moreover, the ²D NMRD profiles for both gluten and starch have confirmed the intermolecular nature of the ¹H NMRD profiles.

Complex composition combined with organizational heterogeneity make it highly challenging to model the experimental NMRD profiles. Despite this difficulty, FFC NMR technique enlarges the observation window of the different dynamical processes of water, providing additional clues to better understand the role of water in the transformations of dough and its constituents.

## Author Contributions

**Corinne Rondeau-Mouro:** conceptualization, validation, formal analysis, investigation, data curation, resources, writing – original draft, writing – review and editing, visualization, project administration, funding. **Anne-Laure Rollet:** conceptualization, validation, formal analysis, investigation, data curation, resources, writing – original draft, writing – review and editing, visualization.

## Acknowledgments

The authors acknowledge the Fédération de Chimie et Matériaux de Paris-Centre (FCMat) for the access of the NMR relaxometers (RELAXOME facility) that were funded by Sorbonne Université, CNRS and Région Ile de France.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/mrc.70024.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section. MRC_Pain_FFC_SI_revised.pdf