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. 2025 Jul 1;29:102717. doi: 10.1016/j.fochx.2025.102717

The aggregation characteristics of gluten proteins in whole wheat steamed buns at different thermal treatment temperature: An insight from dietary fiber

Siyu Zhang 1, Huining Li 1, Yuchang Nie 1, Lina Yang 1,⁎, Danshi Zhu 1,⁎
PMCID: PMC12272913  PMID: 40686894

Abstract

The consumption of whole wheat steamed buns is increasing year by year. However, the taste quality of whole wheat steamed buns is difficult to be controlled during heat processing because it is rich in dietary fiber (DF) and other elements. Compound agents have attracted widespread attention for their effective improvement of the quality of baked products, but their application in steamed flour products is relatively limited. Therefore, this paper explored the effect of DF combined with compound starter on the aggregation characteristics of wheat gluten protein in whole wheat dough during heat processing, in order to find ways to improve the quality characteristics of whole wheat steamed buns. Research has found that chemical leavening (60–80 °C) increases the unfolding degree of gluten protein and improves the elasticity of dough. The addition of DF delayed the stretching of the gluten network caused by chemical leavening. The hardness and pH value of the dough reached their lowest values at 40 °C, which were 796.90 N and 4.95. At 100 °C, the hardness of the dough gradually increases to 4123.45 N and is elastic. The hydrogen bonding of gluten protein gradually increases during the heating process, and it increases with the increase of whole wheat flour (WWF). WWF promoted the thermal aggregation of gluten protein, improved its thermal stability, and stabilized the electrostatic interactions within the system. Correlation analysis demonstrated that the texture characteristics of the dough were significantly correlated with the gluten protein. These results provide a reference for the processing technology of whole wheat steamed buns.

Keywords: Thermal treatment, Whole wheat steamed buns, Gluten, Aggregation, Chemical leavening

Highlights

  • •

    Chemical leavening promoted the stretching of gluten protein during heating process.

  • •

    Chemical leavening improved the elasticity of dough.

  • •

    Whole wheat flour increased hydrogen bonding between gluten proteins.

  • •

    Appropriate whole wheat flour promoted thermal aggregation of the gluten proteins.

1. Introduction

Steamed buns, a traditional Chinese staple, have a rich history, with 40 % of China's wheat consumed in this form (Zhang et al., 2024). In recent years, with the rise in health consciousness, whole wheat steamed buns have become increasingly popular in flour product consumption (Li et al., 2024). These steamed buns are competitive in the market due to their strong wheat flavor and high nutritional value, and they effectively prevent chronic diseases such as obesity, diabetes, and cardiovascular disease (Sun et al., 2023). However, their coarse texture, high hardness, and processing challenges have long challenged producers and consumers (Li et al., 2024). Research has shown that whole wheat flour (WWF) can reduce the quality of bread and other flour produces, as the wheat bran negatively affects the dough's rheology and interferes with gluten aggregation (Cappelli et al., 2025; Hemdane et al., 2016). The addition of wheat bran decreases the specific volume of whole wheat steamed buns, weakens the gluten structure, creates overly dense pores, increases hardness and chewiness, and lowers sensory ratings. Nonetheless, some studies have reported that WWF can enhance the freeze-thaw resistance of frozen dough and steamed buns and that moderate amounts of WWF can improve the quality of steamed buns (Comino et al., 2016; Nawrocka, Szymańska-Chargot, Miś, Kowalski, & Gruszecki, 2016). The gluten protein in wheat flour accounts for about 80 % of the total wheat protein. The formation of gluten network makes the dough have a stable structure, which is one of the main reasons for the elasticity of steamed buns. Hemdane et al. (2016) stated that arabinoxylan interacts with gluten proteins, resulting in poor edibility of whole wheat foods. Si et al. (2021) found that during thermal processing, water-insoluble arabinoxylan disrupts the covalent cross-linking between gluten proteins by affecting the disulfide bonds between them, and dominates the folding/unfolding process of gluten proteins by competing for water with gluten, resulting in poor quality of whole wheat food (Wang et al., 2018). In whole wheat products, dietary fiber (DF) from wheat bran is the main factor affecting their quality. There is currently widespread research on the effect of DF from wheat bran on dough properties during thermal treatment. The impact of DF on gluten protein network is not only caused by diluting gluten network and disrupting gluten stability, but also related to the interaction between DF and gluten protein (Nawrocka, Szymańska-Chargot, Miś, Wilczewska, & Markiewicz, 2016; Ozyurt & Ötles, 2016; Wang et al., 2016; Yu et al., 2024).

Th proofing process is one of the difficult processes to control in the processing of whole wheat steamed buns, which directly affects the sensory quality of whole wheat steamed buns. At present, the common yeast fermentation and old dough fermentation have the problems of long fermentation time, difficult to control the fermentation process, and it is difficult to adapt to the rapid production of industrialization (Hu et al., 2016; Zhang, Yang, et al., 2025). In recent years, chemical leavening has been widely used in baking fermented flour products to improve the quality of flour products (Gélinas, 2021). Chemical leavening is faster than yeast fermentation and does not require long periods of activation and strict environmental conditions (temperature, humidity, pressure, etc.) to escape gas (Gélinas, 2022).

Thermal treatment is the most crucial step in the processing of flour products. During steaming, the dough undergoes a series of physicochemical reactions including water evaporation, starch gelatinization, and protein denaturation, which contribute to the desirable appearance and elasticity of the steamed buns (Bao et al., 2023; Cui et al., 2022; Wang et al., 2022). Heat induction is the primary factor driving gluten aggregation and the formation of the product's structural network, which determines the final volume and texture. Heating causes gluten proteins to bind together, forming large protein aggregates (Mann et al., 2013; Nawrocka et al., 2015). As the temperature increases, a structured gluten network gradually forms, with disordered polypeptide chains aggregating into an ordered network through chemical bonds (Wang et al., 2014). Recent studies also indicate that DF affects the thermal behavior of gluten proteins at different thermal treatment temperatures, thereby altering the conformation of gluten molecules (Bao et al., 2024; Gu et al., 2023; Wang et al., 2023). Through the preliminary experiments, we found that the mixed fermentation of GDL, NaCO3 and yeast was conducive to the integrity of gluten network formation during thermal treatment. However, the chemical reaction between GDL and NaCO3 must be around 60 °C to violently react to produce CO2, thereby increasing the volume of dough (Zhang, Nie, et al., 2025). Based on the existing research, the mechanism by which the compound starter affects the changes in the aggregation characteristics of dietary fiber and gluten protein in dough during the heating process remains unclear. Therefore, this study can provide reference for shortening the proofing time of whole wheat steamed buns and improving the application of chemical leavening in cooking flour products.

2. Materials and methods

2.1. Materials

Golden dragon fish high gluten wheat flour, Golden dragon fish whole wheat flour (Yihai Kerry Golden Dragon Fish Grain and Oil Food Co., Ltd., China); high activity dry yeast, GDL, sodium bicarbonate (Angel Yeast Co., Ltd., China); sodium gluconate, periodate, ammonium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, ethylenediaminetetraacetic acid and other analytical pure reagents were domestically produced.

2.2. Preparation of whole wheat steamed buns

Wheat flour and WWF were mixed for 500 g at the ratio of 1: 0 (0WWF), 1: 1 (50WWF), 0: 1 (100WWF), and then mixed with 0.8 g of yeast, 0.4 g of NaHCO3, 1.8 g of GDL and 55 g of water for 8 min to form the dough (Zhang, Nie, et al., 2025). Next, the dough was placed into a controlled proofing box (SP-16S, SUN-MATE Co., Ltd., Jiangsu, China) for 10 min at 38 °C and 85 % relative humidity. Take half of the dough in flowing water to wash out the gluten. Subsequently, place the dough and gluten in constant temperature and humidity (85 %) incubators at 20, 40, 60, 80, and 100 °C for 20 min and cooling to room temperature. Freeze-dried and ground for later use.

2.3. Preparation of gluten solution

According to Wang, Ma, et al. (2021); Wang, Yang, et al. (2021), add 1.0 g of gluten freeze-dried powder to 50 mL of 0.5 mol/L acetic acid solution and stir evenly. Centrifuge (10,000 ×g, 20 min), collect the supernatant and store it at 4 °C.

2.4. Determination of gluten content

Dry the wet gluten obtained in Section 2.2 at 80 °C for 12 h to obtain dry gluten. The wet gluten content, dry gluten content, and gluten index of the dough were calculated as follows:

Wetgluten content/%=Wetgluten weight/gFlour usage/g×100
Drygluten content/%=Drygluten weight/gFlour usage/g×100
Gluten Index/%=Wetgluten weight/g−Drygluten weight/gFlour usage/g×100

2.5. Determination of dietary fiber content

Refer to the Zhou et al. (2021) to determine the content of total dietary fiber (TDF), soluble dietary fiber (SDF), and insoluble dietary fiber (IDF) in flour (Chinese Standard, 2023). The main operation is as follows: the sample was homogenized and subjected to enzymatic hydrolysis to remove starch and protein, resulting in an indigestible enzymatic solution. The enzymatic hydrolysate was precipitated with 78 % ethanol, and the precipitate was collected and washed, dried, and weighed to determine the mass of DF, including IDF and precipitable soluble dietary fiber (SDFP), in the residue. Collected the filtrate, desalinated and concentrated it, and used liquid chromatography (LC1260 infinity II, Agilent Technologies China Ltd., China) (internal standard method) to determine the non-precipitable soluble dietary fiber (SDFS). The sum of the two is TDF. Filtered the enzymatic hydrolysate directly and washed it with hot water. Collected the filter residue, washed, dry, and weighed it to determine the mass of IDF residue. Collected the filtrate and precipitated it with 78 % ethanol. After drying and weighing the precipitate, determined the mass of SDFP residue that can be precipitated. For the filtrate, measured SDFS, and the sum of SDFS and SDFP is SDF. The DF content of TDF, IDF, and SDFP residues can be obtained by deducting the residual protein, ash content, and reagent blank mass.

2.6. Texture profile analysis

The texture profile analysis of the whole wheat steamed buns was evaluated using a TA-XT2i analyzer (TA-XT Plus, Lotun Science Co.,Ltd., China) according to Martín-Esparza's (Martín-Esparza et al., 2018) method with some modifications. A 2 × 2 × 2 cm sample was selected from the whole wheat steamed buns core for texture determination. A P50-mm-diameter cylindrical probe was used to compress the steamed bun to 60 % deformation. The pre-test speed was set to 2 mm/s, and the test and post-test speed were set to 1 mm/s. There was a 3-s interval between the first and second compression (Zang et al., 2025).

2.7. Fourier transform infrared spectroscopy (FT-IR)

The gluten protein samples were mixed with KBr at a ratio of 1:100 (w/w), ground into a fine powder, and pressed into a slice. The Fourier Transform Infrared (FT-IR) spectrometer (Agilent Technologies, Santa Clara, USA) had a scan range of 400–4000 cm−1 and a resolution of 4 cm−1 for 64 scans. The spectra were analyzed using Peak Fit and OMNIC software.

2.8. Determination of re-sulfhydryl (-SH) content

Gluten proteins (80 mg) were mixed with 5 mL of 8 mol/L Tris-glycine buffer (pH 8.0), and then add 50 μL Ellman reagent containing 4 mg/mL DTNB, stand at 25 °C for 60 min, centrifuged (5000 ×g, 15 min), took the supernatant, and measured the absorbance at 412 nm. Tris-gly buffer containing Ellman reagent was used as the blank control. The sulfhydryl content was calculated as:

−SHμmol/g=73.53×A412×DC

where 73.53 is the molar extinction coefficient of Ellman's reagent, D is the dilution factor, and C is the concentration of gluten protein(mg/mL).

2.9. Determination of pH

The gluten protein sample was mixed with water at a 1:3 ratio, and stired with a magnetic force for 0.5 h. Then measure the pH of the gluten proteins.

2.10. Particle size distribution

Take 2.0 mL of gluten solution into a U-shaped conductive colorimetric dish. The particle size of all sample solutions was measured using a particle size analyzer (NanoBrook 90Plus, Brookhaven, USA) at 25 °C. The samples were run 14 times per measurement.

2.11. Fluorescence spectroscopy measurement

The fluorescence spectroscopy of gluten solution was determined using an F-7000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). The excitation wavelength was set at 280 nm with a slit width of 5 nm, and the endogenous fluorescence spectra were scanned in the emission wavelength range of 300–400 nm.

2.12. Zeta potential detection

Take 1 mL gluten solution into the cuvette and then equilibrated in the instrument for 2 min. The surface charge of all sample solutions was determined with a Zeta potential analyzer (NanoBrook 90Plus, Brookhaven, USA) at 25 °C. Run The samples were run five times per measurement.

2.13. Determination of surface hydrophobicity

Mix 100 μL of gluten solution with 1 mL of 20 mmol/L phosphate buffer (pH 7.0), add 200 μL of 1 mg/mL bromophenol blue indicator, dilute 10 times, let it stand for 10 min, and measure its absorbance at 595 nm. The binding amount of bromophenol blue is the surface hydrophobicity (Bao et al., 2024). The calculation of the binding amount of bromophenol blue is as follows:

Bromophenol blue binding amountμg=200×Ablank−Asample/Ablank

where, A blank is the absorbance without sample added, and A sample is the absorbance of the sample.

2.14. Thermogravimetric analysis (TGA)

Gluten proteins (5 mg) were placed into a crucible and heat it from 30 °C to 600 °C using a thermogravimetric analyzer (STA 8000, Perkins Elmer, USA) at 10 °C/min. Calculate the degradation temperature (Td) and weight loss rate at 600 °C using Origin 2018.

2.15. Scanning electron microscopy (SEM)

The freeze-dried samples were glued onto a double-sided conductive metal plate and coated with gold. The surface morphology was observed using an SEM (S-4800, Hitachi Limited, Japan) with a magnification of 1000× in normal mode.

2.16. Statistical analysis

Each experiment was repeated three times, and the results were expressed as the mean ± standard deviation. Data were analyzed by a one-way analysis of variance using SPSS 25.0 software. Significance analysis (P < 0.05) was performed using Duncan's test. Plots were generated using Origin Pro 2021 software.

3. Results and discussion

3.1. Gluten and dietary fiber content in flour

The gluten and DF content in flours with varying levels of WWF are presented in Table 1. As the WWF content increased, the wet gluten content gradually decreased, with a significant decrease observed in the 100WWF group. The dry gluten content peaked at 13.52 % in the 50WWF group, with no significant difference in gluten index compared to the control group. The gluten index, which measures gluten strength and significantly affects flour quality, showed no significant difference between the 0WWF and 50WWF groups but significantly decreased at 100WWF. The higher gluten index of the 50WWF group compared to the 100WWF group suggested a higher water holding capacity, which helped maintain water levels within the gluten matrix. However, excessive DF led to the rupture of the gluten network, causing a 21 % decrease in gluten content (Jiang et al., 2019). A combined analysis with DF content revealed a positive correlation between the gluten index and DF content, aligning with previous research that indicated DF content significantly influences gluten protein properties (Si et al., 2018).

Table 1.

Flour gluten and dietary fiber content with different WWF contents.

0WWF 50WWF 100WWF
Wet gluten content/% 39.48 ± 2.42c 35.60 ± 3.31b 29.21 ± 1.13a
Dry gluten content/% 13.49 ± 1.22b 13.52 ± 0.63b 12.47 ± 0.91a
Gluten Index/% 65.83 ± 2.13b 62.01 ± 1.60b 57.31 ± 3.12a
Insoluble dietary fiber content/% 1.35 ± 0.22a 5.54 ± 0.19b 9.04 ± 0.12c
Soluble dietary fiber content/% 2.50 ± 0.25a 2.70 ± 0.04a 2.86 ± 0.21a
Dietary fiber content /% 3.84 ± 0.36a 8.25 ± 0.16b 11.90 ± 1.32c
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Note: Data is presented as mean ± standard deviation. Different letters indicate significant differences (P < 0.05). Each experiment was repeated three times.

3.2. Texture profile analysis (TPA)

The TPA of different doughs during the thermal treatment process is presented in Table 2. The TPA of the three groups first decreased and then increased with the increase in steaming temperature, reaching the highest value at 100 °C. The dough hardness reached its lowest value at 40 °C, while elasticity, cohesiveness, and resilience reached their lowest values at 80 °C. This different is mainly because the yeast in the dough remained active during the initial stage of steaming (20–40 °C). During this time, the dough underwent secondary fermentation, producing excessive acidic substances that caused changes in its network structure, resulting in lower hardness and elasticity. When the temperature continued to rise to around 60 °C, starch granules continuously absorbed water, expanded, beginning to gelatinize; At this point, GDL reacted violently with sodium bicarbonate to release a large amount of CO2, causing further expansion of the dough volume. At 80 °C, starch gelatinization is complete (Jekle et al., 2016; Wang, Ma, et al., 2021;Wang, Yang, et al., 2021); and the chemical leavening agent also reacts completely, ending gas production and generating a large amount of gluconic acid, which lowers the pH of the dough. The gluten network structure gradually forms, but still has low elasticity. Once the temperature rises to 100 °C, an irreversible network structure will be formed inside the dough, tightly wrapped by protein starch, forming an elastic network system (Mir et al., 2016). With the increased of WWF, the DF content gradually increased, leading to increased hardness, adhesiveness, and chewiness, while elasticity, cohesiveness, and resilience gradually decreased. In the later stage of heating, there was no significant difference between the 50WWF and 0WWF group, indicated that the mixed fermentation agent can effectively improve the texture characteristics of the 50WWF group. But for 100WWF group, the spatial hindrance formed by excessive DF hinders the expansion of dough volume during chemical gasification, thereby damaging the gluten network structure.

Table 2.

The TPA of different doughs during the thermal treatment process.

Type Steaming temperature/°C Hardness/N Elasticity Adhesiveness/N·s Resilience Cohesiveness Chewiness/N
0WWF 20 817.76 ± 75.81Ab 0.21 ± 0.03Ab 0.35 ± 0.02Ab 0.04 ± 0.00Aa 288.23 ± 29.92Ab 60.55 ± 12.46Ad
40 796.90 ± 68.14Aa 0.13 ± 0.00Aa 0.26 ± 0.02Aa 0.03 ± 0.00Aa 205.36 ± 33.02Aa 26.88 ± 5.01Aa
60 1128.67 ± 95.42Ac 0.14 ± 0.01Aa 0.25 ± 0.01Aa 0.05 ± 0.00Aa 282.41 ± 30.30Ab 39.87 ± 8.03Ab
80 1735.54 ± 346.78Ad 0.13 ± 0.01Aa 0.20 ± 0.01Aa 0.05 ± 0.00Aa 344.79 ± 85.72Ac 46.02 ± 9.48Ac
100 4123.45 ± 251.75Ae 0.93 ± 0.01Ac 0.78 ± 0.00Ac 0.37 ± 0.01Ab 3223.39 ± 194.94Ad 3001.85 ± 211.78Ae
50WWF 20 1390.67 ± 126.55Ba 0.21 ± 0.00Aa 0.37 ± 0.00Aa 0.06 ± 0.00ABa 508.08 ± 45.58Bb 106.80 ± 8.71Bb
40 1362.81 ± 156.61Ba 0.16 ± 0.00Aa 0.37 ± 0.02Ba 0.07 ± 0.00Ba 500.31 ± 50.73Bb 80.44 ± 11.26Bab
60 1542.81 ± 206.57Bb 0.13 ± 0.01Aa 0.27 ± 0.02Aa 0.06 ± 0.00Aa 419.09 ± 18.83Ba 55.01 ± 1.48Ba
80 1862.43 ± 293.00Ac 0.12 ± 0.01Aa 0.21 ± 0.01Aa 0.06 ± 0.00Aa 390.00 ± 81.76Aa 46.87 ± 7.96Aa
100 4458.88 ± 423.16Ad 0.92 ± 0.04Ab 0.77 ± 0.00Ab 0.37 ± 0.01Ab 3449.53 ± 318.34Ac 3201.17 ± 409.62Ac
100WWF 20 3262.12 ± 92.64Cb 0.30 ± 0.08Ba 0.40 ± 0.03Aa 0.08 ± 0.00Ba 1131.12 ± 111.54Cb 406.96 ± 141.32Cc
40 1524.56 ± 73.97Ba 0.19 ± 0.01Aa 0.38 ± 0.01Ba 0.07 ± 0.00Ba 585.25 ± 33.72Ba 109.33 ± 11.94Cb
60 2082.19 ± 107.58Ca 0.16 ± 0.00Aa 0.34 ± 0.02Ba 0.06 ± 0.00Aa 707.35 ± 3.66Ca 113.11 ± 5.08Cb
80 2455.41 ± 348.27Bab 0.12 ± 0.01Aa 0.24 ± 0.00Aa 0.06 ± 0.00Aa 591.26 ± 92.00Ba 74.15 ± 16.43Ba
100 10,568.61 ± 291.61Bc 0.87 ± 0.05Ab 0.70 ± 0.01Ab 0.30 ± 0.00Ab 7443.96 ± 266.81Bc 6483.19 ± 587.59Bd
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Note: Lowercase letters represent inter-group differences, while uppercase letters represent intra-group differences. Each experiment was repeated 3 times.

3.3. Changes in structural properties of gluten proteins

As shown in Fig. 1a, no new functional groups or chemical bonds appeared in the molecular structure of the three dough groups during thermal treatment at different temperatures. The peak area between 3100 and 3700 cm−1 was used to characterize the hydrogen bond strength. The hydrogen bond strength gradually increased during the heating process, with WWF contributing to this improvement. After baseline correction, Gaussian smoothing, and deconvolution treatment, the protein secondary structure content in the amide I band (1600–1700 cm−1) was presented in Fig. 1b. The content of α-helices and β-turns decreased with the increase in heating temperature, while the content of β-sheet gradually increased, and the random structure content remained unchanged. With the addition of WWF, the amount of β-sheets gradually increased (Nawrocka et al., 2016a). Heating caused the β-turn structure to transition to the β-sheet structure, possibly due to the hydrogen bonding of gluten protein gradually increasing with the increase of heating temperature, resulting in an increase in gluten strength; At the same time, the use of WWF promotes protein folding to a certain extent, and the tight structure formed by proteins increases. The higher content of β-sheet in the gluten protein of 100WWF may be due to the fact that DF in the later stage of thermal treatment tightly wraps and envelops the protein to maintain structural stability (Lagrain et al., 2008). These results are similar to the conclusion of Wang et al. (2021), indicating that the composite fermentation agent did not affect the strength of hydrogen bonding between gluten proteins during thermal treatment.

Fig. 1.

Fig. 1

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Effect of different temperature thermal treatments on the gluten proteins structure of whole wheat dough. (a-c) FTIR, (d) protein secondary structure content, (e) free SH content, (f) pH.

As seen in Fig. 1e, the -SH content of the three doughs first increased and then decreased with increasing temperatures. The highest content was observed at 40 °C, followed by a significant decrease at 80 °C in the 0WWF dough. The -SH content in the 50WWF and 100WWF doughs significantly decreased at 100 °C (P < 0.05). Combining the TPA results in Table 1, it can be explained that between 20 °C ∼ 40 °C, the dough continued to rise, causing the gluten proteins to become fully stretched and relaxed, which led to an increase in thiol content. Between 40 °C and 100 °C, gluten proteins progressively formed a gel network structure and aggregated. As the WWF content increased, the -SH content also increased, duo to the dilution of gluten proteins by DF and the inhibition disulfide bond formation with thiol groups (Hu et al., 2017). Non-covalent interactions between DF and gluten proteins disrupt the covalent interactions within gluten proteins and inhibit the formation of disulfide bonds (Bao et al., 2023). Additionally, the pH results (Fig. 1f) showed a significant decrease at 40 °C, indicating that yeast underwent secondary fermentation, which lowered the system's pH and contributed to the increase in -SH content. The decrease in pH at 80 °C is due to the complete reaction between GDL and NaCO3, which generates gluconic acid and lowers the pH of the system. This also confirms the phenomenon of decreased elasticity and chewiness of the dough at 80 °C. However, when the temperature exceeds 80 °C or 90 °C, the -SH content significantly decreases (Hu et al., 2017; Rahaman et al., 2016). The compound fermentation agent may change the content of -SH by altering the pH of the system during the heating process, thereby affecting the structure and properties of gluten proteins.

3.4. Analysis of gluten protein aggregation characteristics

The changes in protein particle size during thermal treatment are shown in Fig. 2a. As the temperature increased, the particle size of gluten protein in the 0WWF group first decreased and then increased, reaching its maximum value at 100 °C. At this point, the gluten system forms an irreversible network structure, making the dough elastic. The particle size of the 0WWF group was lowest at 60 °C, while the lowest value for 50WWF and 100WWF appeared at 80 °C. These results indicated that during the initial heating stage, the dough produces gas, which gradually increased the gluten network. In the subsequent heating process, starch gelatinization fills the expanded gluten pores, never allowing them to form an elastic gluten network. However, the secondary fermentation of yeast did not have a significant impact on the particle size changes of gluten protein, but the CO2 released by the reaction between GDL and sodium bicarbonate at 60 °C rapidly expanded the gluten protein network, resulting in the minimum particle size of 0WWF at 60 °C. For 50WWF and 100WWF with added WWF, chemical gasification did not immediately expand the gluten network, which may be related to DF content, as a large amount of DF entering the dough system diluted gluten, and the spatial hindrance of DF also affected gluten protein aggregation (Si et al., 2021). The particle size of gluten protein significantly increased with the addition of WWF at 100 °C, which may be due to the accelerated DF encapsulation of gluten protein by thermal treatment, leading to an increase in the degree of gluten protein aggregation. Except for the 50WWF group, the other two groups showed larger particle size at 20 °C, and the gluten particles may not have undergone thermal aggregation at 20 °C. This may be due to the phenomenon of cold shrinkage when the gluten system enters a lower temperature after proofing, that is, the gluten protein shrinks and aggregates abnormally when it is cooled (Zhang et al., 2024). During the subsequent heating process, hot air re-enters the system, causing further cross-linking of the gluten protein. For 50WWF group, there are reported that moderate WWF helps stabilize the dough, while moderate DF may hinder abnormal aggregation of gluten proteins, resulting in smaller particle size. It can also be found that chemical leavening cannot change the damage of gluten network caused by DF during thermal treatment.

Fig. 2.

Fig. 2

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Effect of thermal treatment on the aggregation characteristics of gluten proteins. (a) Changes in particle size, (b) Fluorescence spectra, (c) Changes in Zeta, (d) Bromophenol blue binding amount. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

As shown in Fig. 2b, the 0WWF and 50WWF groups exhibited similar changes in fluorescence intensity and wavelength during the thermal treatment process. The increase of fluorescence intensity indicates an increase in the solubility of gluten protein. At 40 °C, the fluorescence intensity decreased, but the wavelength shifted significantly to the red. Compared to 40 °C, the fluorescence intensity at 60 °C increased significantly, and the lowest fluorescence intensity appeared at 100 °C. These results demonstrated that the tryptophan groups of gluten proteins were more exposed at 40 °C, leading to a decreased in the degree of cross-linking between the proteins. The decrease in fluorescence intensity may have been due to abnormal aggregation of gluten proteins caused by secondary fermentation of the dough, which also confirmed the changes in dough texture characteristics. The unfolding of gluten protein at 60 °C may have been due to the CO2 generated by the reaction between GDL and sodium bicarbonate, which expanded the gluten network and promoted the unfolding of gluten proteins. However, there was no significant difference in fluorescence intensity between 60 °C and 80 °C in the three groups of dough, indicating that the gluconic acid produced by chemical fermentation did not affect the solubility of gluten protein. When the temperature reached 100 °C, the gluten proteins formed an irreversible network structure that was dense and compact, imparting hardness and elasticity to the dough. With the increase in WWF content, the fluorescence intensity of gluten proteins increased. In the 100WWF group, the aggregation degree of gluten proteins increased with the increase in temperature. This influence may have been due to excessive WWF diluting the content of gluten proteins, thereby reducing the damage to the gluten network caused by yeast secondary metabolism. Gluten molecules tended to aggregate at higher heating temperatures, leading to a decrease in fluorescence intensity. With the addition of WWF, the maximum fluorescence intensity of gluten proteins also increased, indicating that WWF promoted the unfolding of the hydrophobic core of gluten proteins, exposing the hydrophobic tryptophan inside, thereby causing an increase in fluorescence intensity. Therefore, it can be inferred that WWF induced the unfolding of gluten protein molecules at lower processing temperatures.

As shown in Fig. 2c, there is a significant change in zeta values. In contrast, the zeta potential of the 100WWF group initially increased and then decreased, reaching its maximum at 80 °C. The gradual decrease in zeta potential with increasing WWF content was may due to the heating process and the incorporation of WWF, which weakened the electrostatic repulsion between protein molecules and disrupted the system's stability. However, from 80 °C to 100 °C, the WWF effectively delayed significant changes in electrostatic interactions. This influence may be attributed to the DF in WWF, which is incorporated into the protein network, forming a more stable system. The surface hydrophobicity measurements, shown in Fig. 2d, revealed that during thermal treatment, the surface hydrophobicity of the 0WWF gradually decreased, whereas that of the 100WWF group gradually increased. Wang et al. (2021) reported that at temperatures reaching 95 °C, gluten proteins undergo polymerization through disulfide bonds and hydrophobic interactions, leading a sharp decrease in surface hydrophobicity. This observation suggests that, compared to refined wheat flour, WWF promotes polymerization during thermal treatment by enchancing disulfide bonds and hydrophobic interactions, thereby increasing the surface hydrophobicity of the system. Combined with the above results, it can be found that the main factors affecting the aggregation degree of gluten protein in the thermal treatment process are chemical leavening gas production and yeast secondary proofing. It has great influence on the binding and expansion degree of gluten protein network.

3.5. Thermal decomposition characteristics of gluten proteins

The change in the thermal stability of gluten proteins during thermal treatment are shown in Fig. 3. Figs. 3a-c depict the differential thermogravimetry analysis (DTG) curves of gluten proteins. As the heating temperature increased, the weight loss of the sample, shown in Fig. 3d, initially increased and then decreased. The presence of WWF was associated with a reduction in weight loss of gluten proteins, with moderated amounts of WWF decreasing weight loss during thermal treatment. Consistent with previous observations, the highest weight loss occurred at 40 °C, indicating that the gluten network was most fragile at this temperature. The unfolding of the gluten network at 60 °C was attribute to chemical leavening (Si et al., 2021). As the temperature increased to rise to 100 °C, the gluten network gradually stabilized. The degradation temperature of gluten proteins during thermal treatment, as shown in Fig. 3e, initially decreased and then increased with increasing temperature, exhibiting poor thermal stability at 40 °C and 60 °C. WWF enhanced the thermal stability of gluten proteins at 100 °C, with the most significant effect observed in the 50WWF group. These results indicated that an appropriate amount of DF could encapsulate gluten proteins at high temperatures, thereby stabilizing them (Qian et al., 2021). However, the use of complex starter culture may cause the decrease of thermal stability of gluten protein. WWF can improve the decrease in thermal stability caused by compound fermentation agents.

Fig. 3.

Fig. 3

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Effect of thermal treatment on the thermal decomposition characteristics of gluten proteins. (a-c) DTG curve, (d) weight loss, (e) Td value.

3.6. Whole wheat dough microstructure analysis

The changes in the microstructure of whole wheat dough during thermal treatment are shown in Fig. 4. As the heating temperature increased, the gluten network gradually formed, exposing more starch particles at 40 °C than 20 °C. The gluten proteins began to aggregate, initiating gluten network formation. At 60 °C, the cross-linking between gluten proteins increased, enveloping starch granules. When the temperature reached 100 °C, starch gelatinization was complete, and the swelling starch particles wrapped by gluten protein, formed a continuous dough structure, contributing significantly to the elasticity of steamed buns (Qian et al., 2021). Compared to the 0WWF group, the surface microstructure of the dough in the 50WWF group was smoother at 100 °C. When the temperature reached 60 °C, the gluten network completely enveloped the starch granules. Compared with 0WWF group, 50WWF and 100WWF group more starch particles were exposed at 80 °C, showing increased volumes. Combined with the analysis of texture and particle size characteristics, it can be seen that at this time, the gas production of chemical starter ends, and DF in dough hinders the expansion of gluten network, thus forming the phenomenon of starch particles exposure. These results indicate that moderate WWF can enhance the formation rate of gluten networks and maintain their continuity. However, excessive WWF can cause discontinuity in the gluten network. At 100 °C, more broken network structures were present, exposing starch granules. The compound starter can significantly affect the properties of dough.

Fig. 4.

Fig. 4

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SEM of thermal treatment whole wheat dough.

3.7. Correlation analysis

Pearson bivariate correlation analysis was used to explore the relationship between dough texture characteristics, gluten protein aggregation, and thermal stability during thermal treatment, and the results are shown in Fig. 5. During the heating process, the texture characteristics, particle size, and β-sheet content of the dough showed a significant positive correlation, with correlations coefficient ranging from 0.55 to 0.99. Conversely, there was a negative correlation with zeta potential, -SH content, β-turn, and α-helix, with correlation coefficients ranging from −0.51 to −0.99, indicating a strong correlation between protein aggregation characteristics and dough texture properties. The zeta potential negatively correlated with the thermal decomposition temperature, β-turn, and α-helix. This finding aligns with previous research. Wang et al. (2024) suggested that during the steaming process, IDF promotes gluten aggregation by reducing electrostatic repulsion, which may be the main reason for the deterioration of steamed buns. The aggregation of gluten proteins significantly impacted the hardness of frozen dough. Preheating led to a decrease in -SH content, gluten polymerization, and an increase in dough rigidity (Ma et al., 2022; Wang et al., 2023). The results indicated that changes in zeta potential, particle size, β-sheet content, and -SH content during the heating process can reflect changes in dough quality.

Fig. 5.

Fig. 5

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Correlation analysis between dough texture characteristics, gluten protein aggregation characteristics, and thermal stability.

4. Conclusion

The results showed that the hardness and pH value of the dough reach their lowest levels at 40 °C, with values of 796.90 N and 4.95. At 100 °C, the hardness of the dough increased to 4123.45 N, and the dough became elastic. The dough from the 50WWF group had texture characteristics similar to the control. During the thermal treatment process, the hydrogen bonding of gluten proteins gradually increased, further increased by the addition of WWF. WWF promoted the thermal aggregation of gluten proteins, improved their stability, and stabilized electrostatic interactions within the system. However, the use of composite starter reduced the thermal stability of gluten protein at 40–60 °C. SEM results showed that with increasing WWF content, the protein network began to break, leading to the gradual exposure of starch granules. The main factors influencing the aggregation of gluten protein during thermal treatment were chemical fermentation gas production (60–80 °C) and yeast secondary proofing (40 °C). Gluten protein undergoes two expansions during the heating process, enriching the connections of the gluten network and gradually making the dough more elastic. Compound fermentation agents have a significant impact on the binding and unfolding degree of gluten protein networks, but may not have an effect on the intermolecular interactions of gluten proteins. Correlation analysis demonstrated that the texture characteristics of the dough were significantly positively correlated with particle size and β-sheet content and negatively correlated with zeta potential, -SH content, β-turn, and α-helix. Therefore, the quality of whole wheat steamed buns can be controlled by regulating the heating temperature. This study provides a reference for the application of compound hair waking agent in whole wheat steamed buns and its processing technology.

CRediT authorship contribution statement

Siyu Zhang: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Huining Li: Resources, Project administration, Formal analysis. Yuchang Nie: Visualization, Validation, Supervision, Software. Lina Yang: Writing – original draft, Software, Resources, Project administration, Funding acquisition. Danshi Zhu: Writing – original draft, Validation, Funding acquisition.

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 the National Key Research and Development Program of China (Grant No. 2022YFD2100302).

Contributor Information

Lina Yang, Email: 398510872@qq.com.

Danshi Zhu, Email: zhudanshi1978@163.com.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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