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. 2026 Mar 4;35:103730. doi: 10.1016/j.fochx.2026.103730

NaCl-mediated gluten protein-wheat starch interaction changes in high-, medium- and low-gluten model doughs during heating stage

Xin Feng a,b,1, Dandan Li b,1, Hankun Zhu b,c, Yong Yu b,c, Shaolin Huang b, Liang Ma b,c,d, Hongjie Dai b,c,d,⁎, Yuhao Zhang a,b,c,d,⁎
PMCID: PMC12972703  PMID: 41816768

Abstract

This study investigated how NaCl affects gluten-starch interactions in model doughs with varying gluten contents during heating. Results showed adding NaCl weakened the hydrogen bonds between proteins in the initial dough mixing (C1) → protein weakening (C2) stage and enhanced hydrogen bond interaction in the C2 → starch gelatinization (C3) stage in model doughs. Hydrophobic force and ordered structure increased continuously during heating, and NaCl addition effectively inhibited the damage of starch crystallinity. The total hydrophobic force increased, and the total hydrogen bond content of the high-gluten group increased. The total hydrogen bond content of medium-gluten group decreased in the C1 → C2 stage and increased in the C2 → C3 stage. The total hydrogen bond content of the low-gluten group increased. CLSM indicated NaCl and protein influenced the formation of dense and continuous networks structure during heating. Therefore, this work can provide a theoretical basis for the development and utilization of low-salt flour products.

Keywords: Gluten, Wheat starch, Model dough, Interaction, Heating stage

Graphical abstract

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Highlights

  • •

    Various high-, medium- and low-gluten model doughs were prepared.

  • •

    Component gluten protein and starch interactions during dough heating were studied.

  • •

    NaCl regulated changes of dough structure and composition during heating stage.

1. Introduction

Wheat dough can be considered as a network of protein matrices with inlaid starch granules. During dough thermo-mechanical processing (mainly continuous mixing and heating), the protein and starch in dough are subjected to a complex series of protein-protein and protein-starch interactions (Wang et al., 2020; Wang et al., 2020; Wang, Guo, Yang, Xing, & Zhu, 2021). Gluten protein and wheat starch, as the main components in dough, inevitably interact to affect the quality, nutritional and sensory properties of the end-products (Wang et al., 2021; Wang, Guo, et al., 2021). Heating causes changes in the physicochemical properties and structure of proteins and starches during dough processing. For example, when the dough temperature increases to approximately 80 °C, heating facilitates the formation of disulfide bond crosslinking between glutenin and gliadin (Wang et al., 2017; Wang et al., 2017). Wang, Appels, et al. (2017) and Wang, Luo, et al. (2017) prepared gluten protein gels treated at different temperatures. The results showed that with the increase of temperature, the content of hydrogen bonds and ionic bonds that maintain the gel structure decreased, and the protein network structure became disordered and destroyed. Ai and Jane (2015) heated starch in the presence of excess water to gelatinize starch, resulting in the destruction of the semi-crystalline structure of starch granules and the dissolution of amylose molecules, but this process would be primarily affected by salt (Chen et al., 2019) and protein (Jing et al., 2021).

Recently, the study about the interaction between starch and protein in doughs has attracted more and more attention. For examples, Jekle, Mühlberger, and Becker (2016) pointed out that there are two main interactions between gluten protein and starch during heating: (I) gluten protein can form a physical barrier on the surface of starch granules, which hinders the water expansion of starch granules; (II) competitive hydration between gluten protein and starch. With regards the protein-starch interactions in dough processing, Chen, Deng, Wu, Tian, and Xie (2010) found that the starch pasting properties were significantly affected by both the quantity and quality of gluten. Rosell, Altamirano Fortoul, Don, and Dubat (2013) proved that the starch gelatinization was inversely related to protein content and directly related to carbohydrate content.

It is worth noting that the research on starch-protein mixtures during heating mainly focused on the thermal behavior, and rheological property characteristics (Wang, Guo, et al., 2021; Wang, Ma, et al., 2021; Yang, Guan, Zhang, Li, & Bian, 2022; Zhang et al., 2021). At the same time, the study of changes in protein and starch during the heating process of grain food mainly aimed at the single properties of protein or starch (Pang et al., 2022; Zhu et al., 2020), while the starch-protein interaction mechanism during various heating stage (30–90 °C) according to Mixolab curves has little information available. As an essential ingredient for dough-made foods such as bread and noodle, salt (NaCl) controls various important properties by effectively enhancing the gluten network, and consequently affecting stability, mixing time, and rheological properties of doughs (Chen et al., 2019). According to our preliminary research (Huang et al., 2023), during the heating stage of thermal mechanics, an appropriate addition of NaCl effectively increased the weakness and decreased the maximum viscosity index of the high-, medium- and low-gluten model dough. However, the mechanism of NaCl in regulating protein-starch interaction in different gluten content models has still not been sufficiently explained.

Therefore, this study prepared the high-, medium- and low-gluten model doughs at different heating stages, and mainly analyzed the influence of NaCl addition on the interaction between gluten protein and wheat starch in model doughs during heating. This study aims to elucidate the molecular mechanism by which NaCl regulates protein-starch interactions in model doughs with different gluten contents, and to provide a theoretical basis for the development and utilization of low-salt flour products.

2. Material and methods

2.1. Material and reagents

The wheat gluten protein (protein content >75%) was obtained from Fengqiu Huafeng Fenye Co, Ltd., China (amylose content: ∼ 30%, protein content: 0.39%, ash content: 0.23%). The wheat starch was supplied by Liangshan County Hong Dafan Industry Co. Ltd. NaCl was supported by Chongqing Chuandong Chemical Industry Co. Ltd. (China). Urea and Rhodamine B were supplied by Chengdu Kelong Chemical Reagent Factory (China). 5,5′-disulfide di-2-nitrobenzoic acid was supplied by BIOSHARP. Fluorescein isothiocyanate (FITC) was supplied by Solibao Biotechnology Co. Ltd. All other chemicals and solvents used in this study were of analytical grade.

2.2. Preparation of model dough using Mixolab

The high-, medium- and low-gluten mixed powders were facilely prepared at specific ratios using gluten protein and wheat starch as raw materials. These ratios were determined in accordance with the respective national product standards, namely GB/T 8607–1988, GB/T 1355–1986, and GB/T 8608–1988. Namely, the proportion of gluten protein and wheat starch added in high-, medium-, and low-gluten mixed flours was 15.4% and 84.6%, 12.9% and 87.1%, 9.4% and 90.6%, respectively.

Then, the high-, medium- and low-gluten mixed flours with various salt addition (0–1.5% NaCl) were studied using the Mixolab device (Chopin Technologies, Paris, France) following the Chopin + protocol (ICC standard method 173). Briefly, the standard protocol shows the following parameters: dough weight 75 g and mixing speed 80 rpm. Adjust the mixing powder and adding water so that the torque reaches the maximum C1 in the dough formation stage, and the target torque C1 of the dough was within (1.1 ± 0.05) N·m. Subsequently, the temperature was kept at 30 °C for 8 min, raised to 90 °C at a rate of 4 °C/min and kept at 90 °C for 7 min, then cool to 50 °C at a rate of 4 °C/min and kept at 50 °C for 5 min. At this heating stage (30–90 °C), the samples during different heating stage were obtained according to Mixolab curves, including initial dough mixing stage (C1), protein weakening during the heating period (C2), starch gelatinization during the heating period (C3) (Kim et al., 2023). The obtained samples were immediately frozen in liquid nitrogen. After further freeze-drying and grinding, the samples were available and store at 25 °C.

According to our previous results of the Mixolab curves and the characteristic parameters of the model dough with various NaCl addition (0–1.5%) during the heating stage (Huang, et al., 2022). When the amount of NaCl added was 1.5% for high-gluten model dough, the peak time of the inverted “V” curve (A) was significantly delayed, and the weakening degree (C1-C2) increased significantly (P < 0.05), the maximum viscosity index (C3-C2) was significantly decreased (P < 0.05). When NaCl addition was 1.0% for medium-gluten model dough, the peak time of inverted “V” curve (A) was obviously delayed, and the weakening degree was significantly increased (P < 0.05), and the maximum viscosity index decreased significantly when NaCl addition was 1.5% (P < 0.05). When NaCl addition was 0.5% for low-gluten model dough, the peak time of inverted “V” curve was significantly delayed, and the weakening degree was significantly increased (P < 0.05), the maximum viscosity index decreased significantly (P < 0.05) at NaCl addition 1.5%. Based on the above analysis, the selection of NaCl addition amounts in high-, medium- and low-gluten model doughs is listed in Table 1.

Table 1.

Various NaCl addition in high-, medium- and low-gluten model doughs during the heating stage.

Model doughs Group NaCl addition/% Heating stage
High-gluten model H-C 0 A, C2, C3
H-1.5% 1.5
Medium-gluten model M-C 0
M-1.0% 1.0
M-1.5% 1.5
Low-gluten model L-C 0
L-0.5% 0.5
L-1.5% 1.5
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2.3. Non-covalent interactions of doughs

Typical non-covalent bonds including ionic bond, hydrogen bond and hydrophobic interaction were determined (Li et al., 2021). Briefly, the freeze-dried dough (1.0 g) was respectively mixed with 9.0 mL of 0.05 M NaCl (Solution A), 0.6 M NaCl (Solution B), 0.6 M NaCl +1.5 M urea (Solution C), 0.6 M NaCl +8.0 M urea (Solution D). After being stirred at room temperature for 2 h and then centrifuged at 12,000 rpm for 10 min, the resulting supernatant (0.1 mL) was taken and mixed with 5.0 mL of Kaumas brilliant blue G-250 solution. Then absorbance of the mixture was recorded at 595 nm and the protein content was determined using bovine serum albumin (BSA) as a standard (Sigma, Darmstadt, Germany). Ionic bond was expressed as the difference between soluble wheat gluten protein content in Solution B and Solution A. Hydrogen bond was determined as the difference between soluble protein content in Solution C and Solution B. Hydrophobic interactions were expressed as the difference between soluble protein content in Solution D and Solution C.

2.4. Free sulfhydryl content of doughs

The content of free sulfhydryl in doughs was measured according to the method of Han, Ma, Li, Zheng, and Wang (2019) with appropriate modifications. Briefly, the freeze-dried dough (50 mg) was suspended in 10 mL of Tris-Gly-8 M urea buffer (pH 8.0). After being thoroughly mixed and reacted for 60 min at room temperature, 0.5 mL of 4 mg/mL Ellman's reagent was added and mixed quickly, and then stored at 25 °C for 30 min in the dark (shake and mix once every 10 min). After centrifugation, the absorbance of the supernatant was measured at 412 nm (Yang et al., 2022; Yang, Guan, et al., 2022).

2.5. Raman spectroscopy

Raman spectrum of doughs was recorded using a Raman spectrometer (DXR2, Thermo Fisher Scientific, USA), equipped with a 785 nm argon ion laser source. The parameters were set as follows: light slit 50 μm, spectrum recording range 400–3375 cm−1, laser power 11 mW, exposure time 2 s, exposure 45 times. The data transformation and deconvolution were conducted by OMNIC V8.2 software. After being normalized against the phenvlalanine band at 1003 cm−1, Peakfit Version software was applied to fit and analyze the content of secondary structure components of protein in each doughs (Pu et al., 2021).

2.6. X-ray diffraction (XRD)

The XRD patterns of doughs was recorded using a X'pert Pro X-ray diffractometer (Netherlands), equipped with Ni-filtered Cu-Kα (wavelength 1.5418 Å) under 40 kV and 40 mA operation conditions. The diffraction intensity was collected in the range of 10–50° (2θ) at a scanning speed of 2°/min (Yang et al., 2021).

2.7. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra was obtained using a PerkinElmer spectrum 100 FTIR spectrometer (Akron, USA). The spectra data were collected over the range of 4000–600 cm−1 with a resolution of 4 cm−1.

2.8. Confocal laser scanning microscopy (CLSM)

CLSM images of doughs was recorded according to the method described by Huang et al. (Ze-Hua et al., 2017) with appropriate modifications. The dough samples (10 μm) were sliced by freezing microtome (CM1850 UV, Leica, Germany) and then stained with a mixed fluorescent dye (1:1), namely Rhodamine B (0.0025%, w/v, preferentially staining protein) and fluorescein 5-isothiocyanate (FITC, 0.025%, w/v, preferentially staining starch). Then, the stained samples were captured using a CLSM (LSM880, Zeiss, Germany) with excitation/emission wavelengths at 568/625 nm for Rhodamine B and 488/518 nm for FITC.

2.9. Statistical analysis

All measurements were repeated at least in triplicate and reported as mean values ± standard deviation (SD). Data analysis was performed by SPSS Statistics 19.0, and the figures were drawn with origin 2018. Differences between mean values were analyzed using ANOVA and Duncan's multiple range tests at a significance level of 0.05.

3. Results and discussion

3.1. Changes in the properties of gluten protein during the heating stage

3.1.1. Non-covalent interaction analysis

The non-covalent bond contents of high-, medium- and low-gluten model doughs with various NaCl addition (0–1.5%) during heating stages are shown in Fig. 1. As can be observed from these figures, the non-covalent forces for maintaining the structure of all model doughs were mainly hydrogen bonds and hydrophobic forces. The content of ionic bonds was relatively low with a slight change for all doughs, and some studies have also confirmed that the contribution of ionic bond is negligible due to its weak contribution (Yang, Guan, et al., 2022; Yang, Wang, et al., 2022). For all model doughs at C1 stage, hydrogen bonds and hydrophobic forces between proteins were enhanced with the increase of gluten protein content from low gluten to high gluten model, indicating that more gluten proteins were involved in the formation of hydrogen bonds and hydrophobic forces (Li et al., 2021).

Fig. 1.

Fig. 1

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Non-covalent contents of high (A, B), medium (C, D, E) and low (F, G, H) gluten model doughs during different heating stage. Note: a–c represents a significant difference of ionic bond content (P < 0.05), e–h represents a significant difference of hydrogen bond content (P < 0.05), i–l represents the significant difference of hydrophobic force (P < 0.05).

With the increase of NaCl addition, these interactions in high-, medium- and low-gluten model doughs showed an increased trend. In particular, H-1.5% group (NaCl addition 1.5%) showed the most increase in hydrogen bond and hydrophobic force, thus resulting in more stable dough structure. With the extension of heating process (C1 → C3), the hydrophobic force and hydrogen bond in high-, medium- and low-gluten model doughs decreased significantly (P < 0.05). After adding NaCl, in the low-temperature heating stage (A → C2), the hydrogen bond was lower than the control group, while the hydrophobic force was higher than the control group, indicating that the reduction of hydrogen bonds between proteins at this stage dominated the weakening of the dough. In the high-temperature heating stage (C2 → C3), these interactions in high-, medium- and low-gluten model doughs were all higher than the control group, indicating that the enhanced hydrogen bonds and hydrophobic forces between proteins could promote the aggregation of protein molecular chains, which may be related to the increase of the interaction between gluten protein molecular chains and wheat starch (Han, Ma, Li, & Sun, 2020).

3.1.2. Free sulfhydryl content

The change of free sulfhydryl content is considered as a convincing indicator of disulfide bond change, which is directly related to the structure of gluten protein network (Guang et al., 2017). Free sulfhydryl content of high-, medium- and low-gluten model doughs during different heating stage is shown in Fig. 2. From low- to high-gluten model doughs, the content of free sulfhydryl in the control group decreased with increasing gluten protein content, indicating that more disulfide bonds were formed in high-gluten control group (H-C group). With the extension of heating processing (C1 → C3), the content of free sulfhydryl in high-, medium- and low-gluten control groups (L-C, M-C and H-C groups) decreased significantly (P < 0.05), indicating that more free sulfhydryl was exposed and oxidized to disulfide bonds during the heating stage (Wang, Peng, Appels, Tian, & Zou, 2022).

Fig. 2.

Fig. 2

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Free sulfhydryl content of high-, medium- and low-gluten model doughs during different heating stage. Note: Capital letters (A–C) represent a significant difference of free sulfhydryl content affected by NaCl addition (P < 0.05), and the lowercase letters represent a significant difference of free sulfhydryl groups affected by heating (P < 0.05).

With the increase of NaCl addition, the content of free sulfhydryl in high-, medium- and low-gluten model doughs decreased, indicating that NaCl promoted the formation of disulfide bonds. For high-gluten dough model, no matter in the raw dough or during the heating stage, the effect of NaCl addition on the formation of disulfide bonds was not obvious (Chen et al., 2019). For medium- and low-gluten dough models, the formation of disulfide bonds was promoted. With the progress of heating (C1 → C3), the addition of NaCl still promoted the formation of disulfide bonds. When the addition of NaCl was 1.5% (H-1.5%, M-1.5%, and L-1.5%), the formation of disulfide bonds in the high-temperature heating period (C2 → C3) was more obvious in the model dough with medium- and low-gluten protein (M-1.5% and L-1.5%).

3.1.3. Raman spectrometry

Both NaCl and heating treatment can change the interaction between gluten protein and wheat starch, thus affecting the secondary structure of proteins in the model dough. The secondary structure content of high-, medium- and low-gluten model doughs during different heating stage is shown in Fig. 3. The amide I band (1600–1700 cm−1) was used to analyze the protein structure quantitatively. The peaks at 1647–1660, 1670–1680, 1680–1690 and 1660–1670 cm−1 were respectively attributed to α-helical, β-fold, β-turn, and random coils (Wang, Appels, et al., 2020; Wang, Li, et al., 2020). As shown in Fig. 3, α-helix is the main secondary structure in high-, medium- and low-gluten model doughs (C1). For the control groups (L-C, M-C and H-C), with the increase of gluten protein content, the content of ordered structure (α-helix and β-fold) of doughs increased. This may be related to the high protein content in the system provides greater probability of interaction between protein molecules, thus forming a tighter protein network structure (Kanakis et al., 2011). After introducing NaCl, the α-helix content in high-, medium- and low-gluten model doughs is higher than that of the control doughs (L-C, M-C and H-C groups) without NaCl addition, which was consistent with the trends of increasing molecular forces in model doughs.

Fig. 3.

Fig. 3

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Secondary structure content of high-, medium- and low-gluten model doughs during different heating stage.

During the heating process (C1 → C3), the content of ordered structure of high-, medium- and low-gluten control models decreased, indicating that the doughs tended to be loose and disordered. With the addition of NaCl, at the stage of C1 → C2, compared with the control models, the disorder degree of doughs in high-, medium- and low-gluten model doughs decreased. For example, the disorder degree of doughs in H-1.5% group was relatively lower (26.11% → 31.67%) than that of H-C group (28.87% → 43.60%) at the stage of C1 → C2. This phenomenon was inconsistent with the result that the dough weakening degree was significantly increased by adding NaCl, indicating that the dough weakening in the initial heating process (C1 → C2) was not caused by the damage of protein structure. This may be related to the structure of wheat starch and the interaction between gluten protein and wheat starch (Wang et al., 2022). At the stage of C2 → C3, compared with the control models, the disorder degree of dough in the high-, medium- and low-gluten model doughs declined, which was consistent with the result that the maximum viscosity index of the dough was significantly reduced after adding NaCl. Under this condition, NaCl can promote the aggregation of protein chains by increasing the hydrogen bonds and hydrophobic forces between proteins, which may be related to the increased interaction between protein chains and wheat starch (Obadi, Zhang, & Xu, 2022).

3.2. Changes in the properties of wheat starch during the heating stage

3.2.1. XRD analysis

XRD patterns of high-, medium- and low-gluten model doughs during different heating stage are shown in Fig. 4, and the relative crystallinity is listed in Table 2. All raw model doughs (C1) showed obvious double-shoulder characteristic diffraction peaks at 2θ of 17° and 18°, and the single diffraction peak around 15° and 23°, showing a typical A-type XRD patterns (Niu, Wu, & Xiao, 2017). With the increase of gluten protein and NaCl addition, the shape and location of diffraction peaks in high-, medium- and low-gluten doughs (C1) did not change, but the relative crystallinity (Table 2) of starch was improved, suggesting the formation of a more orderly structure (Yang et al., 2019).

Fig. 4.

Fig. 4

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XRD patterns of high-, medium-, and low-gluten model doughs during different heating stage.

Table 2.

Relative crystallinity of high-, medium- and low-gluten model doughs during different heating stages.

Group C1 A C2 C3
H-C 33.51% 26.48% 16.63% 4.67%
H-1.5% 35.02% 29.79% 25.25% 11.51%
M-C 30.91% 23.39% 14.97% 2.61%
M-1.0% 31.91% 26.55% 15.72% 7.34%
M-1.5% 31.61% 28.24% 22.28% 10.73%
L-C 29.87% 23.07% 11.80% 2.37%
L-0.5% 30.57% 24.64% 19.21% 7.52%
L-1.5% 31.43% 26.34% 23.03% 9.63%
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As can be seen from Fig. 4 and Table 2, during the heating processing stage (C1 → C3), the shape and location of the starch diffraction peak in high-, medium- and low-gluten control model doughs (H-C, M-C and L-C) did not change, but the intensity of the diffraction peak gradually weakened, and the peak width and relative crystallinity decreased. As depicted in Fig. 4A, C, F and Table 2, when the highest temperature (C3) was reached, the diffraction peak intensity and relative crystallinity of the control model doughs sharply decreased due to the gelatinization of starch molecule. Compared with the control model doughs, adding NaCl significantly inhibited the destruction of starch crystallization. Thus, at the stage of C1 → C2, the increase of weakening degree of model doughs after adding NaCl was due to the destruction of starch crystallization and the interaction changes between gluten protein and wheat starch. At the stage of C2 → C3, the maximum viscosity index of model doughs decreased after adding NaCl. NaCl addition increased the hydrogen bond and hydrophobic force between proteins, promoted the aggregation of protein molecular chains, inhibited the destruction of starch crystallization by heating, and increased the interaction between protein molecular chains and wheat starch.

3.3. Interaction between gluten protein and wheat starch during the heating stage

3.3.1. FTIR analysis

The -OH stretching vibration and -CH3 symmetric deformation vibration can be used to analyze hydrogen bond and hydrophobic interactions respectively, whose peaks and intensities are summarized in Fig. S1-S4 and Table 3. After adding NaCl and heating treatment, the characteristic absorption peaks of high-, medium- and low-gluten model doughs still maintained without new peak appeared, indicating the presences of non-covalent interaction between gluten protein and wheat starch. The absorption peak at 3200–3600 cm−1 is attributed to -OH stretching vibration (Aurea, Gerardo, Norma, Luis, & Leopoldo, 2017). It can be seen that for the control model (C1), with the increase of gluten protein content, the stretching vibration peak of -OH appeared a red-shift, and the hydrogen bond content in the high-, medium- and low-gluten control model showed little difference. During the heating stage (C1 → C3), the stretching vibration peak of –OH in the high-gluten, medium-gluten and low-gluten control model doughs gradually moved to high wavenumbers, indicating that the hydrogen bonds in the dough were gradually destroyed (Sha, Sushil, Song, Na, & Gang, 2021), which was consistent with the changes in the structure of dough protein network and the crystallinity of starch. After adding NaCl, the stretching vibration peak of -OH in the high-, medium- and low-gluten doughs was red-shifted, indicating the enhancement of hydrogen bond interaction (Diao et al., 2017), which also promoted the increase of protein ordered structure and the crystallinity of starch. Notably, NaCl addition did not change the overall trend of hydrogen bond content during the heating stage. At the heating stage of C1 → C2, NaCl addition destroyed the hydrogen bonds between proteins and inhibited the destruction of starch crystallinity, so as to increase the content of hydrogen bonds between starches. At the stage of C2 → C3, NaCl addition increased hydrogen bonds between proteins, inhibited the destruction of starch crystallinity with increased hydrogen bonds between starches.

Table 3.

Wavelengths and peak intensities of characteristic absorption peaks in high-, medium- and low-gluten model doughs during different heating stages.

Samples C1 A C2 C3
—OH stretching vibration peak (cm−1)
 H-C 3278.02 3283.25 3283.66 3284.81
 H-1.5% 3273.80 3281.50 3282.12 3283.74
 M-C 3278.14 3282.92 3288.51 3290.29
 M-1.0% 3276.48 3285.11 3285.16 3285.23
 M-1.5% 3277.92 3285.03 3285.21 3285.64
 L-C 3278.19 3282.11 3289.90 3292.92
 L-0.5% 3273.23 3285.19 3290.04 3294.35
 L-1.5% 3273.04 3284.56 3285.01 3293.15



—CH3 symmetric deformation vibration (peak intensity)
 H-C 53.7 51 51.08 54.67
 H-1.5% 51.44 55.6 55.8 55.08
 M-C 55.2 52.8 53.46 57.42
 M-1.0% 52.82 53.05 54.24 62.43
 M-1.5% 52.94 54.3 54.17 54.41
 L-C 55.89 55.91 55.75 56.14
 L-0.5% 58.65 57.77 56.59 56.13
 L-1.5% 58.78 58.32 60.99 56.81
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Compared with the control model, the influence of NaCl addition on hydrogen bond content changes in high-, medium- and low-gluten dough models was different. For the high-gluten model dough, the addition of NaCl promoted the hydrogen bond interaction between protein and starch. During heating stage and NaCl condition, the protein was more likely to form a continuous network. The gelatinized and expanded starch increased the degree of cross-linking with protein, thus forming a strong hydrogen bond interaction between protein and starch (Wang, Guo, et al., 2021; Wang, Ma, et al., 2021; Zhang et al., 2023). Therefore, the hydrogen bond interaction in the high-gluten model dough with NaCl addition was stronger than that in the group without adding salt. For the medium-gluten model dough, due to the decrease of the ratio of protein and starch in the dough, the continuity of protein network structure was relatively low during heating. At the early stage of heating (A), the model dough with NaCl addition could appear a situation in which starch and protein networks compete for space during heating (Yu et al., 2021). At the same time, NaCl alleviated the damage of starch crystallization and the degree of protein disorder caused by heating, thus decreasing the hydrogen bond interaction between starch and protein. At the heating stage of C2, starch began to gelatinize. In the presence of NaCl, some expanded starches continued to compete for space with the protein network, which greatly promoted the hydrogen bond interaction between starch and protein. At the end of heating stage (C3), the increase of starch gelatinization degree resulted in the formation of starch gel network, in which the protein network may interspersed among them that further strengthens the hydrogen bond interaction between starch and protein. Therefore, the hydrogen bond interaction in the medium-gluten model dough after adding NaCl was also stronger than that in the group without adding salt (Peng et al., 2021). For the low-gluten model dough, the process was similar to that for the medium-gluten model dough. However, at the end of heating (C3), the gelatinization degree of starch increased and a continuous starch network structure was formed, and the protein was gradually encapsulated in it due to its low content. This decreased the hydrogen bond interaction between starch and protein, so that the hydrogen bond in the low-gluten model dough with NaCl was weaker than that in the group without NaCl (Zhang et al., 2022).

The peak around 1400 cm−1 was attributed to the -CH3 symmetric deformation vibration. The increase of vibration peak intensity of -CH3 symmetric deformation indicated the enhanced hydrophobic interaction (Chen et al., 2020). With the increase of gluten content, the hydrophobic force in the control model dough decreased. Previously, it has been proved from the perspective of protein interaction that the force of hydrophobic interaction in dough increased with the increase of gluten protein content, which indicates that the hydrophobic interaction between gluten protein and starch decreased with the increase of gluten protein content. With the progress of heating (C1 → C3), at the low temperature heating stage (A → C2), all control model doughs showed a decreased peak intensity of -CH3 symmetrical. However, at the high temperature heating stage (C2 → C3), the peak intensity increased, indicating the increase of hydrophobic force between starch and protein with heating (Fig. S1-S4). After adding NaCl, the peak intensity of the -CH3 symmetrical deformation vibration in the low-gluten model dough increased, implying the enhanced hydrophobic force (Yang, Guan, et al., 2022; Yang, Wang, et al., 2022). However, the high- and medium-gluten model doughs showed a decrease of hydrophobic force due to the decreased peak intensity. This may be related to the higher protein content in the high- and medium-gluten groups resulted in an easier connection between gluten proteins through hydrogen bonding. With the progress of heating (C1 → C3), the model doughs with NaCl addition showed a higher peak intensity than the control group model dough, indicating NaCl promoted the hydrophobic interaction between protein and starch during the whole heating process.

3.3.2. CLSM

The microstructure of high-, medium- and low-gluten model dough at different heating stages is shown in Fig. 5. FITC can non-covalently bind with starch with a green color, Rhodamine B can non-covalently bind with protein with a red color, and the complex structure of protein and starch shows a yellow color (Chen et al., 2020; Jia et al., 2019). It can be seen from the figure that starch granules maintained their shape in all models and adhered to the protein matrix. For the control model dough (C1), with the increase of gluten protein content, the protein network structure was gradually enhanced, the proportion of yellow area gradually increased, and more round or oval starch granules were embedded in the gluten network structure. Therefore, the high-gluten control group formed a relatively strong network structure. After adding NaCl, due to changes in interactions (i.e., hydrogen bonds, hydrophobic interactions, or electrostatic shielding effects), the starch granules exposed outside the network were aggregated and arranged in a more regular manner, which increased the degree of cross-linking between gluten protein and wheat starch and formed a more stable and compact structure.

Fig. 5.

Fig. 5

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CLSM images of high-, medium- and low-gluten model doughs during different heating stage.

For the high-gluten control model dough, the stronger protein network still retained with a good continuity during the whole heating process. At the initial heating stage (A), a small amount of starch granules was exposed in the control group (H-C-A), which reduced the degree of cross-linking with the protein network and formed an inhomogeneous network structure. As the heating progressed (H-C-C2), a small amount of starch granules exposed outside the network expanded in volume, further reducing the degree of cross-linking with the protein network. At the end of heating (C3), as more starch granules expanded, the crosslinking with proteins was significantly reduced. However, after adding NaCl (H-1.5% group), the gluten protein network remained well throughout the heating process. With the progress of heating (C1 → C2), the starch granules swelled slightly and still maintained their shape, and a small amount of swollen starch granules increased the degree of crosslinking with protein. As the heating progressed (H-1.5%-C2), it can be found that the starch distribution in the protein network is slightly dispersed, but it is still surrounded by the protein network and forms a uniform network structure. At the end of heating (H-1.5%-C3), the starch granules further expanded and embedded in the protein network structure, thus increased the crosslinking strength between protein and starch.

For the medium-gluten control model dough, the yellow area was relatively decreased due to the reduced ratio of protein and starch. At the initial stage of heating (A), the protein network structure of the control group (M-C-A) appeared hollow with poor continuity. Some starch granules appeared swelling that could strengthen protein cross-linking networks. With the continuous of heating (M-C-C2), the void area of the protein network increased, and the starch granules exposed outside the network expanded further that caused the crosslinking to deteriorate. At the end of heating (M-C-C3), the number of voids in the protein network decreased due to that most of the starch granules swelled, but the voids appeared on the starch granules thus formed a network structure interspersed with proteins unevenly. After adding NaCl, in the early stage of heating (A), the protein network structure appeared hollow, and the continuity became poor. The heating treatment made the starch granules exposed outside the network disperse, which destroyed the hydrogen bond interaction and cross-linking between starch and protein. At the heating stage of C2, the content of swollen starch granules increased, further competing for space with the protein network. At the same time, starch gelatinization and protein formed a network structure interspersed with each other unevenly, thus forming an enhanced cross-linked network structure. At the end of heating (C3), the further expanded starch granules generated a uniform network structure interspersed by swollen starch granules and protein, which increased the crosslinking between protein and starch.

For the low-gluten control model doughs, the ratio of protein to starch was lower, showing an increase in the green area in CLSM. At the initial stage of heating (L-C-A), the starch granules exposed outside the protein network were dispersed, and a small part of the starch granules expanded and gradually blurred the edges, which adhered to the protein network structure and strengthened the cross-linking with protein. With the heating progress (L-C-C2), the protein network structure appeared hollow and the starch granules with blurred edges increased greatly, and the brightness of the center of the starch granules decreased (from green to light gray), and the cross-linking between protein and starch also became worse. At the end of heating (L-C-C3), the area of the FITC-labeled “cloudy” area increased, the color was weakened, and empty pockets appeared in the center of starch granules, showing a uniform network structure with fully expanded starch particles wrapped around the protein. After adding NaCl, the starch granules dispersed better at the initial stage of heating (L-C-A and L-1.5%-A), and the reduced yellow area in CLSM implied the weaker interaction between starch and protein. At heating stage of C2, the starch granules expanded further and formed a relatively uniform network structure, increasing the crosslinking degree. At the end of heating (C3), a small amount of starch granules still swelled, and most of the starch granules ruptured, forming an inhomogeneous structure of protein wrapped by starch viscous network, resulting in a decrease in the cross-linking degree.

3.4. Molecular mechanism

Based on the above results, the mechanism diagram was proposed for better understanding the NaCl mediated interaction between gluten protein and wheat starch in high-, medium- and low-gluten model doughs and shown in Fig. 6. In the formation stage of the model dough (C1), the interaction between gluten protein and wheat starch in high- and medium-gluten model doughs is mainly hydrogen bonds, while the interaction in low-gluten model doughs is mainly hydrophobic interactions. With the increase of NaCl addition, the hydrogen bonds in all model doughs are further enhanced, and the hydrophobic interactions in the low-gluten model doughs are also strengthened, which may improve the structural stability of the model dough. At the heating stage of C1 → C2, the hydrogen bonds, hydrophobic interactions and relative crystallinity in the control model dough were all reduced, while NaCl can enhance the hydrophobic interactions in all treatment groups. Therefore, heating mainly destroyed the structure of the model dough by destroying the hydrogen bonds between gluten protein and wheat starch. Furthermore, with the increase of gluten protein content (L-H), the enhanced hydrogen bonds and relative crystallinity inhibited the destruction of the model dough structure to a certain extent, while with the increase of NaCl addition, the hydrogen bonds of the model dough in all treatment groups were gradually destroyed, resulting in further destruction of the model dough. In the gelatinization stage (C2 → C3), the hydrogen bonds and crystallinity of all treatment groups further decreased. The increased gluten protein content can reduce the damage of heating to the dough by enhancing hydrogen bonds, and the increase of NaCl addition promoted the hydrogen bonds and hydrophobic interactions between gluten protein and wheat starch, inhibiting the gelatinization and destruction of starch granules.

Fig. 6.

Fig. 6

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Molecular mechanism of the interaction between gluten protein and wheat starch in high-, medium- and low-gluten model doughs mediated by NaCl.

4. Conclusion

Various NaCl addition in high-, medium- and low-gluten model doughs can affect the interaction between gluten protein and wheat starch during heating process. With the increase of heating temperature (C1 → C3), the contents of molecular force and ordered structure (α-helical and β-fold) in all model doughs decreased. The content of molecular force and ordered structure in the control model dough increased as gluten protein content increased. With the increase of NaCl addition, the hydrogen bond content of high-, medium-, and low-gluten model doughs decreased in a low temperature heating region (A → C2) and increased in a high temperature heating region (C2 → C3). Meanwhile, the hydrophobic force and the ordered structure content increased during the whole heating stage. The relative crystallinity of starch in all model doughs decreased with increasing temperature (C1 → C3), while increasing gluten protein or NaCl content could inhibit the damage of starch by heating with an increased relative crystallinity. Furthermore, NaCl addition could destroy the hydrogen bond interaction and enhance hydrophobic interaction between gluten protein and wheat starch in model doughs. Therefore, this work can elucidate the molecular mechanism of NaCl mediated interaction between gluten protein and wheat starch, and provide a theoretical basis for the development and utilization of low-salt flour products.

CRediT authorship contribution statement

Xin Feng: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Dandan Li: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Hankun Zhu: Visualization, Supervision. Yong Yu: Visualization, Supervision. Shaolin Huang: Methodology, Formal analysis, Data curation. Liang Ma: Visualization, Supervision, Resources, Conceptualization. Hongjie Dai: Visualization, Supervision, Formal analysis, Data curation. Yuhao Zhang: Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by National Natural Science Foundation of China (32372353), and Fundamental Research Funds for the Central Universities (Nos. SWU-KT22046 and SWU-XJPY202310).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2026.103730.

Contributor Information

Hongjie Dai, Email: daihjdemo@163.com.

Yuhao Zhang, Email: zhy1203@163.com.

Appendix A. Supplementary data

Supplementary material
mmc1.docx (1.6MB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
mmc1.docx (1.6MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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