Small ice wheat dough and gluten protein modified by enzyme

Abstract

The application discloses a kind of small ice wheat dough and gluten protein by enzyme modification, comprising the following steps: different adding amount Xyn, β-Glu small ice wheat dough preparation;Xyn, β-Glu and Xyn+β-Glu different adding amount on the texture determination of small ice wheat dough;Xyn, β-Glu and Xyn+β-Glu different adding amount on the determination and analysis of free thiol and disulfide bond of small ice wheat dough;Xyn, β-Glu and Xyn+β-Glu on the determination and analysis of thermal characteristics of small ice wheat dough;Xyn, β-Glu and Xyn+β-Glu on the determination and analysis of dynamic rheology and creep-recovery of small ice wheat dough;Xyn, β-Glu and Xyn+β-Glu on the influence analysis of small ice wheat gluten;Three kinds of different adding amount of enzyme all have significant difference (P<0.05) on the texture parameter of dough, the influence law of different adding amount of Xyn, β-Glu and Xyn+β-Glu on dough is basically identical, and hardness and chewiness present the trend of first falling and then rising with the increase of adding amount, wherein the hardness of 4mg / kg Xyn+40mg / kg β-Glu decreases most obviously, is 776.873±31.969g, and is reduced by 24.89% compared with small ice wheat dough without adding enzyme.

Description

Technical Field

[0001] This invention belongs to the field of mini iced wheat technology, specifically relating to a mini iced wheat dough and gluten protein modified by enzymes. Background Technology

[0002] Little Ice Wheat is a high-quality new wheat variety bred through distant hybridization of wheat and ice grass. It combines the excellent processing characteristics of wheat with the stress resistance and disease resistance of ice grass. The plant grows vigorously, with plump and nutritious grains. Not only are the protein, dietary fiber, and various mineral contents higher than those of ordinary wheat, but it also contains unique bioactive components. It has wide adaptability and can be stably planted in cold and arid northern regions. Compared with ordinary wheat, this wheat variety has advantages such as strong Connie characteristics and high nutritional quality. However, research on its subsequent processing, especially in the preparation of bread and steamed buns, is relatively scarce. Therefore, the dough and finished products of Little Ice Wheat have broad research prospects.

[0003] Adding improvers to dough to enhance its quality has been widely studied in recent years. Emulsifiers, hydrocolloids, and other chemical additives are commonly added to bread. However, with consumers increasingly favoring natural ingredients and foods free of chemical additives, enzymes are becoming more widely used commercially. For consumers, enzyme preparations represent a natural and clean image compared to other chemical additives. Replacing chemical additives with enzymes in the baking industry is significant due to their higher safety and efficiency. Various enzymes have been applied to bread quality improvement. Enzymes primarily influence flavor by producing precursors directly or indirectly related to the flavor formation process. Studies have shown that xylanase (Xyn) can hydrolyze insoluble arabinoxylan, releasing bound water and optimizing the gluten network structure; β-glucanase (β-Glu) can specifically degrade β-glucan, reducing its adverse effects on dough processing. Both enzymes have shown good results in wheat dough improvement. However, the regulatory effects of these two enzymes and their combined use on the quality and gluten protein structure of wheat dough have not yet been systematically studied.

[0004] Based on the above research background, this invention aims to explore the effects of different amounts of xylanase, β-glucanase, and their complex enzymes on the texture, thermal properties, and rheological properties of wheat flour. Simultaneously, it seeks to analyze the regulatory mechanism of enzyme preparations on wheat gluten structure from the perspectives of free sulfhydryl groups, disulfide bonds, non-covalent bonds, and secondary structures, and to screen the optimal enzyme addition combination. This will provide theoretical support and technical reference for the application of enzyme preparations in wheat processing, and promote the high-value utilization of wheat resources. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution: an enzyme-modified wheat flour dough and gluten protein, comprising the following steps: Preparation of small ice wheat doughs with different amounts of Xyn and β-Glu; Determination of the textural effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on small ice wheat dough; Analysis of the effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on the free thiol groups and disulfide bonds in small ice wheat dough; Determination and analysis of the thermal properties of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Analysis of dynamic rheology and creep-recovery of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on wheat gluten protein; Preparation of gluten protein: The prepared dough was rinsed with 20g / L NaCl solution to form coarse gluten. Then the starch was washed with deionized water until the starch was washed away. The gluten protein was freeze-dried and used for the determination of physicochemical properties. Determination and analysis of non-covalent bonds in wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Determination and analysis of the secondary structure of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Determination and analysis of fluorescence spectrometry of Xyn, β-Glu and Xyn+β-Glu in wheat gluten protein; Determination and analysis of the surface hydrophobicity of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Data processing: All experiments were repeated at least three times, and the results were expressed as mean ± standard deviation. One-way ANOVA was used to analyze the differences between multiple groups of data.

[0006] As a preferred technical solution for enzyme-modified mini ice wheat dough and gluten protein of the present invention, the preparation method of mini ice wheat dough with different addition amounts of Xyn and β-Glu is as follows: First, Xyn and β-Glu are added to mini ice wheat flour (200g) according to different addition amounts (Xyn: 2mg / kg, 4mg / kg, 6mg / kg, 8mg / kg; β-Glu: 20mg / kg, 40mg / kg, 80mg / kg, 160mg / kg). Then, salt (1.6g), sugar (5g), dehydrated yeast (1g), and water (100g) are added and mixed. The dough without added enzyme is used as a blank control group. All ingredients are mixed in a mixer at low speed for 5 minutes to form dough and then mixed at high speed until the dough forms a smooth and transparent gluten film that can be stretched. After the dough is shaped, it is placed in a fermentation box (35℃, 75% humidity) for 45 minutes. After being taken out and cooled to room temperature, one batch is freeze-dried, and another batch is used for subsequent parameter determination.

[0007] As a preferred technical solution for enzyme-modified wheat dough and gluten protein of the present invention, the method for determining the texture of wheat dough with different addition amounts of Xyn, β-Glu, and Xyn+β-Glu is as follows: After fermentation in a fermentation chamber and cooling to room temperature, the dough was allowed to stand at 20°C for 30 minutes. Before testing, the dough was made into cubes with a side length of 50 mm and a height of 25 mm using a self-made mold. The texture analyzer probe used in the experiment was a P / 36R type. The test operation mode was: pressure measurement; operation type: TPA; compression rate 50.0%; the speed before, during, and after the test was set to 3 mm / s, 1 mm / s, and 3 mm / s, respectively; the initial sensing force was 5 g; the trigger type was set to: Auto; the data acquisition rate was 200 pps; the interval between two compressions was 5.0 s. The texture of dough with different addition amounts was selected for PCA analysis.

[0008] As a preferred technical solution for enzyme-modified wheat dough and gluten protein of the present invention, the determination and analysis of free sulfhydryl groups and disulfide bonds in wheat dough by different addition amounts of Xyn, β-Glu, and Xyn+β-Glu are as follows: Prepared freeze-dried dough is pulverized through a 100-mesh sieve, 0.075g is weighed, placed in a 10mL centrifuge tube, 1mL of buffer solution and 4.7g of guanidine hydrochloride are added and mixed, and the volume is adjusted to 10mL with buffer solution. For free sulfhydryl group (SHF) content, 1mL of sample solution, 4mL of urea-guanidine hydrochloride solution, and 0.04mL of DTNB reagent (4mg / mL) are placed in a 10mL test tube, mixed, and the absorbance is measured at 412nm using a UV spectrophotometer. Each sample is measured three times and the average value is taken. The SHF content is calculated according to the formula.

[0009] As a preferred technical solution for enzyme-modified wheat dough and gluten protein of the present invention, dynamic rheological determination is performed as follows: Wheat dough with different enzymes is placed at 25°C and allowed to cool to room temperature. Then, a certain amount of sample is taken from the center of the dough, spread flat on the test platform of the rheometer, and excess dough is cut off with scissors. To prevent moisture loss, silicone oil is applied to the edge of the dough. Next, dynamic flow testing is performed in oscillation mode, with the following settings: plate diameter 40mm, slit distance 1mm, temperature 25°C, strain 0.1%, scanning frequency range 0.1~10Hz, number of points set to 20, and all tests are repeated three times. As a preferred technical solution of enzyme-modified small ice wheat dough and gluten protein of the present invention, creep-recovery test: small ice wheat dough with different enzymes is placed at 25°C and left to stand until cooled to room temperature for testing. The stress is kept constant at 50Pa. The dough is pressed for 150s, and then the external force is removed. The sample recovers for 150s.

[0010] As a preferred technical solution for enzyme-modified wheat gluten dough and gluten protein of the present invention, the determination and analysis of the secondary structure of wheat gluten protein is carried out by Fourier transform infrared spectroscopy: 1 mg of freeze-dried samples of wheat dough with different added enzymes is accurately weighed and placed together with 120-150 mg of solid potassium bromide, also ground into powder, in an agate mortar. The mixture is thoroughly ground until the powder adheres completely to the mortar wall, with a particle diameter of approximately 2 μm. The powder is then poured into a tablet press, forming a pile to fill the mold completely without gaps, and then tableted. The pressure of the instrument is about 1 ton for 1 minute. The sample after being compressed by the tablet press should be transparent or translucent. After checking that it is correct, the infrared operation can begin. Carefully place the sample in the instrument. The scanning conditions are: wavenumber range of 4000~500 cm⁻¹, interval of 4 cm⁻¹, and scanning frequency of 32. The infrared absorption wave is measured. The Fourier transform infrared spectrum of the sample is collected and analyzed using Omnic 8.0 software. The main analysis is in the 1600~1700 cm⁻¹ band. Each experiment is repeated three times to ensure accuracy and consistency.

[0011] As a preferred technical solution for enzyme-modified wheat dough and gluten protein of the present invention, the fluorescence spectral analysis of wheat gluten protein was performed using a fluorescence spectrophotometer. 100 mg of lyophilized gluten protein sample was extracted with 20 mL of 0.5 mol / L acetic acid solution at room temperature for 2 h. After centrifugation (4000 g, 15 min), the supernatant was diluted with 0.5 mol / L acetic acid solution to 1 mg / mL. The fluorescence intensity was measured using a fluorescence spectrometer at an excitation wavelength of 280 nm, an emission wavelength of 290 nm to 410 nm, and a slit width of 5 nm.

[0012] As a preferred technical solution for enzyme-modified wheat dough and gluten protein of the present invention, the determination and analysis of the surface hydrophobicity of wheat gluten protein involves using 8-aniline-1-naphthalenesulfonate (ANS) as a fluorescent probe to measure the surface hydrophobicity of gluten protein and its components. The extract is diluted at different concentrations, and 50 μL of ANS solution (8 mmol / L, pH 5.8) is added to 10 mL of sample solution. The reaction is carried out in the dark for 20 min. The fluorescence intensity of the sample is measured by a fluorescence spectrometer at an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 5 nm. The initial slope of the fluorescence intensity versus protein concentration graph is used as an indicator of surface hydrophobicity.

[0013] Compared with the prior art, the beneficial effects of the present invention are: Compared with the unenzyme-added wheat dough and the enzyme-added group, all different amounts of the three added enzymes had significant differences in the texture parameters of the dough (P<0.05). The effects of different amounts of Xyn, β-Glu, and Xyn+β-Glu on the dough were basically consistent, showing a trend of first decreasing and then increasing in hardness and chewiness with increasing addition amount. Among them, the hardness of 4 mg / kg Xyn+40 mg / kg β-Glu decreased the most significantly, by 776.873±31.969 g, which was 24.89% lower than that of the unenzyme-added wheat dough. This may be due to the specific degradation of non-starch polysaccharides in the wheat dough by the enzyme. Compared with the control, the addition of 40 mg / kg β-Glu and 4 mg / kg Xyn can reduce the hardness of the wheat dough, increase its elasticity, and significantly reduce its chewiness. As the amount of β-glucanase added exceeds 40 mg / kg, the dough's hardness, stickiness, and chewiness gradually increase, while its elasticity decreases. This is because excessive β-glucanase leads to damage to the starch structure. Appropriate damage is more conducive to yeast fermentation and improves water retention; however, excessive damage results in sticky dough. The addition of appropriate amounts of β-glucanase is positively correlated with gluten network structure. Xylanase functions by breaking down insoluble arabinoxylan. The arabinoxylan releases water bound to xylan and redistributes it to the gluten network, thereby improving dough elasticity. The combined use of β-glucanase and xylanase brings more complex and unique changes to the dough texture. Among them, the reduction in hardness and chewiness is most obvious with 40β-Glu + 4Xyn. In the regulation of dough hardness, the two work together. Xylanase continuously optimizes the gluten network and reduces hardness, thus improving the initial softness of the dough. β-glucanase further decomposes β-Glu in the dough. Compared with the use of single enzymes, the complex enzyme can better resist moisture loss and structural aging, thus having a positive effect on dough hardness. The regulatory effect of Xyn, β-Glu and complex enzyme on the structure of wheat dough, and the effect of complex enzyme on the balance of disulfide bonds and free sulfhydryl groups due to the synergistic effect of hydrolyzing hemicellulose, are stronger than those of single enzymes. With the addition of enzymes, the dough G′ and G′′ both decrease and the frequency dependence decreases, the creep recovery rate increases, and the dough exhibits higher elastic behavior in dough preparation. Xyn increases the α-helix content in gluten protein, indicating that gluten protein has a more stable and ordered structure; the addition of enzymes makes the gluten protein structure more compact, causing tryptophan to be buried; the addition of enzymes exposes the hydrophobic regions inside the protein, leading to enhanced hydrophobic interactions between gluten molecules, thereby promoting protein aggregation. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1The effect of Xyn, β-Glu, and Xyn+β-Glu on free thiol groups and disulfide bonds in fascia protein is shown in the figure. Figure 2 The graph shows the effects of Xyn, β-Glu, and Xyn+β-Glu on the thermal properties of dough. Figure 3 Figure 1 shows the effect of Xyn, β-Glu and Xyn+β-Glu on the rheological properties of small ice wheat dough. Figure 4 The effect of Xyn, β-Glu and Xyn+β-Glu on the non-covalent bonds of fascia protein is shown in the figure. Figure 5 The effect of Xyn, β-Glu and Xyn+β-Glu on the secondary structure of glutenin; Figure 6 Figure showing the effect of enzyme treatment on the endogenous fluorescence characteristic peaks of glutenin; Figure 7 The effect of enzyme treatment on the hydrophobicity of gluten protein surface is shown in the figure. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0016] Please see Figure 1-7 ,and Figure 2 In the table, (A) represents the mass loss rate, and (B) represents the thermal degradation temperature. Figure 3 In the equation (A) G′, elastic modulus; (B) G′′, viscous modulus; (C) tanδ, loss tangent; (D) creep-recovery characteristics, and Figure 4 The present invention provides the following technical solutions: (A) ionic bonds, (B) hydrogen bonds, and (C) hydrophobic interactions. Preparation of small ice wheat doughs with different amounts of Xyn and β-Glu: Before conducting the experiment, Xyn and β-Glu were added to 200g of wheat flour at different dosages (Xyn: 2mg / kg, 4mg / kg, 6mg / kg, 8mg / kg; β-Glu: 20mg / kg, 40mg / kg, 80mg / kg, 160mg / kg). Salt (1.6g), sugar (5g), dehydrated yeast (1g), and water (100g) were then added and mixed. Dough without added enzymes served as a blank control group. All ingredients were mixed in a mixer at low speed for 5 minutes to form a dough, and then mixed at high speed until a smooth, transparent gluten film could be stretched. The dough was then shaped and placed in a fermentation chamber (35℃, 75% humidity) for 45 minutes. After cooling to room temperature, one batch was freeze-dried, and another batch was used for subsequent parameter measurements.

[0017] Determination of the textural effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on small ice wheat dough; After fermentation in a fermentation chamber and cooling to room temperature, the dough was allowed to stand at 20°C for 30 minutes. Before testing, the dough was shaped into cubes with sides of 50 mm and a height of 25 mm using a homemade mold. The texture analyzer probe used in the experiment was a P / 36R type. The test operation mode was: pressure measurement; operation type: TPA; compression rate 50.0%; the speed before, during, and after the test was set to 3 mm / s, 1 mm / s, and 3 mm / s, respectively; the initial sensing force was 5 g; the trigger type was set to: Auto; the data acquisition rate was 200 pps; and the interval between two compressions was 5.0 s. The texture of dough with different additive amounts in each group was selected for PCA analysis.

[0018] Determination of free thiol groups and disulfide bonds in small ice wheat dough by different addition amounts of Xyn, β-Glu and Xyn+β-Glu; The prepared freeze-dried dough was pulverized through a 100-mesh sieve, and 0.075g was weighed and placed in a 10mL centrifuge tube. 1mL of buffer solution and 4.7g of guanidine hydrochloride were added and mixed, then the volume was adjusted to 10mL with buffer solution. To determine the free thiol (SHF) content, 1mL of sample solution, 4mL of urea-guanidine hydrochloride solution, and 0.04mL of DTNB reagent (4mg / mL) were placed in a 10mL test tube, mixed, and the absorbance was measured at 412nm using a UV spectrophotometer. Each sample was measured three times, and the average value was taken. The SHF content was calculated using the formula: F(μmol / g) = 73.53 × A⁴¹² × D / C In the formula: 73.53 is the molar absorptivity of DTNB; A412 is the absorbance of the solution at 412 nm; D is the dilution factor; C is the sample mass concentration, mg / mL.

[0019] For the total mercaptohydride (SH) content, 1 mL of sample solution, 0.05 mL of β-mercaptoethanol, and 4 mL of urea-guanidine hydrochloride solution were mixed and stored at 25 °C for 1 h. Then, 10 mL of 12% trichloroacetic acid (TCA) was added to the mixture, and the mixture was stored at 25 °C for 1 h, followed by centrifugation at 5000 × g for 10 min. The precipitate was washed twice with 5 mL of 12% TDA, and finally dissolved in 10 mL of 8 mol / L urea. 0.05 mL of Ellman's reagent was added to the treatment solution, and the absorbance value at 412 nm was measured. Each sample was measured three times, and the average value was taken. The disulfide bond (SS) content was calculated using the formula: SS (μmol / g) = (SH – SHF) / 2 In the formula: SS is the disulfide bond content, μmol / g.

[0020] Determination of the thermal properties of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Add 5.0 mg of freeze-dried dough to a thermogravimetric analyzer (TGA) to determine the thermal stability of the enzyme-added wheat dough. The test temperature ranged from 30 to 600 °C, with a heating rate of 20 °C. Each sample was tested in triplicate. The thermal denaturation temperature (Tp) and thermal degradation temperature (Td) of the sample can be obtained from the TGA curve and the first derivative curve.

[0021] Determination of dynamic rheology and creep-recovery of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Dynamic rheological determination: Small iced wheat doughs with different enzymes were placed at 25°C and allowed to cool to room temperature. Then, a sample was taken from the center of the dough, spread evenly on the test platform of the rheometer, and excess dough was trimmed off with scissors. To prevent moisture loss, silicone oil was applied to the edges of the dough. Next, dynamic flow testing was performed using the oscillation mode, with the following settings: plate diameter 40 mm, slit distance 1 mm, temperature 25°C, strain 0.1%, scanning frequency range 0.1–10 Hz, and 20 test points. All tests were performed in triplicate.

[0022] Creep-recovery assay: Small ice wheat dough with different enzymes was placed at 25°C and allowed to stand until it cooled to room temperature for measurement. The stress was kept constant at 50 Pa. The dough was pressed for 150 s, and then the external force was removed. The sample recovered for 150 s.

[0023] The effects of Xyn, β-Glu and Xyn+β-Glu on wheat gluten protein; Preparation of gluten protein: The prepared dough was rinsed with a 20 g / L NaCl solution to form coarse gluten. The starch was then washed with deionized water until it was completely removed. The gluten protein was freeze-dried and used for the determination of its physicochemical properties.

[0024] Determination of non-covalent bonds in wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Non-covalent bonds were disrupted using the following different buffer solutions (dissolved in 0.05 mol / L phosphate buffer, pH 7.0): (1) 0.05 mol / L NaCl (S1); (2) 0.6 mol / L NaCl (S2); (3) 0.6 mol / L NaCl + 1.5 mol / L urea (S3); (4) 0.6 mol / L NaCl + 8 mol / L urea (S4). The powder (200 mg) was dissolved in 10 mL of each buffer solution and extracted at 25 °C for 1 h. After centrifugation (10000 g, 15 min), the protein content of the supernatant was determined using a BCA kit. Ionic bonds were represented by the difference in protein content between S2 and S1. Hydrogen bonds were represented by the difference between S3 and S2. Hydrophobic interactions were represented by the difference between S4 and S3.

[0025] Determination of the secondary structure of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Fourier Transform Infrared (FTIR) Determination: 1 mg of freeze-dried samples of wheat dough with different added enzymes was accurately weighed and placed together with 120-150 mg of solid potassium bromide, also ground into powder, in an agate mortar. The mixture was ground thoroughly until all the powder adhered to the mortar walls, with a particle diameter of approximately 2 μm. The powder was then poured into a tablet press to form a pellet. The mold was filled completely, without any gaps, and tablets were pressed. The machine pressure was approximately 1 tonne (T) for 1 minute. The tableted sample should be transparent or translucent. After verification, the FTIR operation could begin. The sample was carefully placed in the instrument. Scanning conditions were: wavenumber range 4000-500 cm⁻¹, interval 4 cm⁻¹, and scan frequency 32. The infrared absorption wave was measured. The Fourier Transform Infrared spectra of the samples were collected and analyzed using Omnic 8.0 software. The 1600-1700 cm⁻¹ band was the primary focus of analysis. Each experiment was repeated three times to ensure accuracy and consistency.

[0026] Determination of Xyn, β-Glu and Xyn+β-Glu in the fluorescence spectroscopic analysis of wheat gluten protein in small ice cream; Analysis was performed using a fluorescence spectrophotometer (Fluorommax-4, HORIBA, France). 100 mg of lyophilized gluten protein was extracted with 20 mL of 0.5 mol / L acetic acid solution at room temperature for 2 h. After centrifugation (4000 g, 15 min), the supernatant was diluted to 1 mg / mL with 0.5 mol / L acetic acid solution. The fluorescence intensity was measured using a fluorescence spectrometer at an excitation wavelength of 280 nm, an emission wavelength of 290 nm–410 nm, and a slit width of 5 nm.

[0027] Determination of the surface hydrophobicity of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; The surface hydrophobicity of gluten protein and its components was investigated using 8-aniline-1-naphthalenesulfonate (ANS) as a fluorescent probe. The extract was diluted to different concentrations. 50 μL of ANS solution (8 mmol / L, pH 5.8) was added to 10 mL of sample solution, and the reaction was carried out in the dark for 20 min. The fluorescence intensity of the sample was measured using a fluorescence spectrometer at an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 5 nm. The initial slope of the fluorescence intensity versus protein concentration graph was used as an indicator of surface hydrophobicity.

[0028] Data processing: All experiments were repeated at least three times, and results are expressed as mean ± standard deviation. One-way ANOVA was used to analyze differences among multiple groups. p-values ​​were calculated using ANOVA; a p-value less than 0.05 indicated statistical significance, and less than 0.01 indicated highly significant significance. All graphs and calculations were performed using Origin 2022 software.

[0029] in conclusion Analysis of the effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on the texture of wheat flour dough; Studies have shown that dough texture has a significant correlation with the production of later products and is an important indicator for evaluating wheat products. Table 2-2 shows the effects of different amounts of added enzymes on the texture of wheat dough. Compared with wheat dough without added enzymes and the added enzyme group, different amounts of the three added enzymes all showed significant differences in the texture parameters of the dough (P<0.05). The effects of different amounts of Xyn, β-Glu, and Xyn+β-Glu on the dough were basically consistent, with the hardness and chewiness showing a trend of first decreasing and then increasing with the increase of the added amount. Among them, the hardness of 4 mg / kg Xyn+40 mg / kg β-Glu decreased the most significantly, by 776.873±31.969 g, which was 24.89% lower than that of wheat dough without added enzymes. This may be due to the specific degradation of non-starch polysaccharides in wheat dough by enzymes. Compared with the blank, the addition of 40 mg / kg β-Glu and 4 mg / kg Xyn can reduce the hardness of wheat dough, increase its elasticity, and significantly reduce its chewiness. As the amount of β-glucanase added exceeds 40 mg / kg, the dough's hardness, stickiness, and chewiness gradually increase, while its elasticity decreases. This is because excessive β-glucanase leads to damage to the starch structure. Appropriate damage is more conducive to yeast fermentation and improves water retention; however, excessive damage results in sticky dough. The addition of appropriate amounts of β-glucanase is positively correlated with gluten network structure. Xylanase functions by breaking down insoluble arabinoxylan. The arabinoxylan releases water bound to xylan and redistributes it to the gluten network, thereby improving dough elasticity. The combined use of β-glucanase and xylanase brings more complex and unique changes to dough texture, with the most significant decrease in hardness and chewiness observed at 40 β-Glu + 4 Xyn. In terms of dough hardness control, the two work together. Xylanase continuously optimizes the gluten network and reduces hardness, thus improving the initial softness of the dough. β-glucanase further breaks down β-Glu in the dough. Compared with using enzymes alone, the compound enzyme can better resist moisture loss and structural aging, thereby having a positive effect on dough hardness.

[0030] The table below shows the effects of different amounts of Xyn, β-Glu and Xyn+β-Glu on the hardness, chewiness, elasticity and cohesion of small ice wheat dough; Enzyme dosage (mg / kg) hardness elasticity cohesion chewing 0 <![CDATA[1034.433±67.587 b ]]> <![CDATA[0.853±0.170 b ]]> <![CDATA[0.706±0.003 b ]]> <![CDATA[661.247±21.121 ab ]]> 2Xyn 962.853±69.012a 0.864±0.287b 0.709±0.004a 579.099±39.734b 4Xyn <![CDATA[834.373±65.347 b ]]> <![CDATA[0.942±0.295 a ]]> <![CDATA[0.712±0.002 ab ]]> <![CDATA[517.142±44.274 b ]]> 6Xyn <![CDATA[987.609±64.039 a ]]> <![CDATA[0.861±0.111 b ]]> <![CDATA[0.715±0.004 a ]]> <![CDATA[658.207±31.825 a ]]> 8Xyn <![CDATA[1030.181±70.198 a ]]> <![CDATA[0.864±0.125 b ]]> <![CDATA[0.709±0.003 b ]]> <![CDATA[663.981±45.161 b ]]> 20β-Glu <![CDATA[979.154±49.549 a ]]> <![CDATA[0.854±0.125 a ]]> <![CDATA[0.708±0.002 a ]]> <![CDATA[628.207±31.825 a ]]> 40β-Glu <![CDATA[870.880±68.223 c ]]> <![CDATA[0.955±0.150 a ]]> <![CDATA[0.714±0.003 ab ]]> <![CDATA[502.980±43.035 c ]]> 80β-Glu <![CDATA[969.912±64.948 bc ]]> <![CDATA[0.873±0.126 b ]]> <![CDATA[0.711±0.004 ab ]]> <![CDATA[572.168±41.969 bc ]]> 120β-Glu <![CDATA[1054.960±61.692 b ]]> <![CDATA[0.865±0.160 b ]]> <![CDATA[0.717±0.004 a ]]> <![CDATA[614.418±57.513 ab ]]> 20β-Glu+2Xyn <![CDATA[833.959±46.615 bc ]]> <![CDATA[0.893±0.005 cd ]]> <![CDATA[0.709±0.002 bc ]]> <![CDATA[615.882±33.904 ab ]]> 30β-Glu+3Xyn <![CDATA[828.011±50.563 bc ]]> <![CDATA[0.934±0.007 b ]]> <![CDATA[0.713±0.001 b ]]> <![CDATA[566.917±26.315 bcd ]]> 40β-Glu+4Xyn <![CDATA[776.873±31.969 c ]]> <![CDATA[0.969±0.008 a ]]> <![CDATA[0.718±0.002 a ]]> <![CDATA[482.019±58.1.274 d ]]> 50β-Glu+5Xyn <![CDATA[858.295±44.569 bc ]]> <![CDATA[0.916±0.011 bc ]]> <![CDATA[0.711±0.003 b ]]> <![CDATA[523.717±44.313 cd ]]> 60β-Glu6+Xyn <![CDATA[904.000±61.357 b ]]> <![CDATA[0.890±0.008 b ]]> <![CDATA[0.705±0.003 a ]]> <![CDATA[602.897±71.776 a ]]> Analysis of the effects of different addition amounts of Xyn,β-Glu and Xyn+β-Glu on free thiol groups and disulfide bonds in wheat flour; Disulfide bonds are crucial chemical bonds for stabilizing the dough network structure. They are formed by the oxidation of two free thiol groups. In protein networks, higher disulfide bond content contributes to structural stability. However, dough decomposition disrupts these disulfide bonds, leading to an increase in free thiol groups. Compared to the control group (CK), the addition of Xyn, β-Glu, or a combination of Xyn and β-Glu to the wheat flour dough resulted in a significant increase in the free thiol content of gluten proteins, while the disulfide bond content decreased accordingly. The Xyn+β-Glu complex enzyme treatment group showed the most significant change. Mechanistically, disulfide bonds, as key covalent bonds maintaining the stability of the gluten protein network structure, directly influence the integrity of the gluten network. Xyn hydrolyzes xylan in the dough, and β-Glu hydrolyzes β-glucan. Their enzymatic hydrolysis of hemicellulose in the wheat flour dough disrupts the interaction between gluten proteins and hemicellulose, thereby causing the disulfide bonds between gluten protein molecules to break and convert into free thiol groups. Further studies at the molecular bonding level confirmed the regulatory effects of Xyn, β-Glu, and the complex enzyme on the structure of wheat dough. Moreover, the complex enzyme had a stronger effect on the balance between disulfide bonds and free sulfhydryl groups than the single enzyme due to its synergistic effect on the hydrolysis of hemicellulose.

[0031] Analysis of the effects of Xyn,β-Glu and Xyn+β-Glu on the thermal properties of wheat dough; Thermogravimetric analysis (TG) can be used to obtain the cumulative weight loss curve of small wheat samples during heating, i.e., the TG curve. The DTG curve is the first derivative of the TG curve with respect to temperature (or time), and it is usually peak-shaped. The peak point corresponds to the inflection point of the TG curve, which is the maximum point of the weight loss rate. Compared with the blank control group (CK), the gluten protein mass loss rate of the Xyn group, β-Glu group, and Xyn+β-Glu complex enzyme group all showed a significant decreasing trend, and the decrease in the complex enzyme group was greater than that of the single enzyme group. This indicates that the addition of enzymes weakened the thermal stability of gluten protein, because Xyn can hydrolyze xylan in dough and β-Glu can hydrolyze β-glucan, resulting in damage to the integrity of the gluten protein network structure, making it more prone to the loss of small molecules during heating, thus manifesting as a decrease in the mass loss rate. Figure 2The thermal degradation temperature curves in group (B) show the opposite trend. Compared with the CK group, the thermal degradation temperatures of gluten proteins in the Xyn group, β-Glu group, and complex enzyme group are all significantly higher, with the complex enzyme group exhibiting the highest thermal degradation temperature. The mechanism is that enzymatic hydrolysis weakens the steric hindrance effect of xylan and β-Glu on gluten protein molecules, making it easier for gluten protein molecules to form hydrogen bonds and hydrophobic interactions, thus enhancing the heat resistance of the protein molecules themselves and pushing the thermal degradation temperature to a higher range. The results of the two sub-figures corroborate each other, jointly indicating that Xyn, β-Glu, and their complex enzymes can bidirectionally affect the thermal properties of wheat gluten proteins in small-batch wheat dough by regulating the action system of hemicellulose in dough, and the complex enzymes show a certain synergistic enhancement effect in the regulation of thermal properties.

[0032] Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on the dynamic rheology and creep-recovery of small ice wheat dough; The rheological properties of dough are determined by the content and structure of its components, and have a significant impact on subsequent product manufacturing. The elastic modulus (G'), also called the storage modulus, characterizes the elasticity of the dough and corresponds to its elasticity; the viscous modulus (G"), also called the loss modulus, characterizes the viscosity of the dough and corresponds to its flowability and viscosity. The loss tangent is the ratio of viscosity to elasticity in the measured dough, i.e., tanδ = G" / G'. A smaller tanδ indicates a higher proportion of elasticity in the dough, weaker flowability, and a higher proportion of polymers or a higher degree of polymerization; conversely, a larger tanδ indicates a higher proportion of viscosity, better flowability, and a greater number of low-polymerization components.

[0033] Figure 3 As shown in Figure A, G′ reflects the dough's resistance to elastic deformation. G′ increases with increasing oscillation frequency in all groups, with the blank group consistently showing the highest G′. G′ decreases significantly in the Xyn and β-Glu groups, and the Xyn+β-Glu complex enzyme group shows the largest decrease in G′. Figure 3 As shown in Figure B, the G′′ values ​​of each group increased with increasing frequency, and the G′′ values ​​after enzyme addition were all lower than those of the blank group. The Xyn+β-Glu group showed the most significant decrease, indicating that after the enzyme destroyed the hemicellulose in the dough, it tended to reduce both its elastic modulus and viscous modulus. Figure 3 As shown in Figure C, all tanδ values ​​are less than 1, indicating that all doughs are solid. Furthermore, the addition of enzymes reduced the dough's resistance to deformation and increased its fluidity. The complex enzymes regulate dough viscoelasticity by hydrolyzing hemicellulose, with the complex enzymes showing the most significant effects in decreasing G′ and G′′ and increasing tanδ.

[0034] The creep-recovery curve of the dough sample is as follows: Figure 3As shown in Figure C, the maximum creep strain characterizes the strength of the dough. The creep recovery test reflects the changing trend of the dough under constant stress and the dynamic recovery process of the dough after stress removal. Compared with the CK group, the creep curves (both during the continuous application of external force and the rebound stage after the removal of external force) of the Xyn group, β-Glu group, and Xyn+β-Glu complex enzyme group are generally above the CK group, with the complex enzyme group showing the largest deviation. During the creep stage, the deformation of the dough in the enzyme-treated groups is significantly greater than that in the CK group, indicating that the addition of enzymes reduces the dough's ability to resist deformation under external force. This is because hemicellulase hydrolyzes polysaccharides, making it easier for protein molecules to form reversible hydrogen bonds and hydrophobic interactions. These interactions can be rapidly reconstructed after the removal of external force, driving the dough's deformation and rebound. The complex enzyme group has the best recovery effect because the reconstruction efficiency of the intermolecular interactions of dough is higher.

[0035] Both dynamic frequency scanning and creep recovery test results indicate that both the wheat flour dough and the enzyme-treated dough exhibit primarily elastic solid behavior. With the addition of enzymes, both G′ and G′′ of the dough decreased, and the frequency dependence decreased, while the creep recovery rate increased, resulting in higher elastic behavior during dough preparation.

[0036] The effects of Xyn, β-Glu and Xyn+β-Glu on wheat gluten protein; Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on the non-covalent bonds of wheat gluten protein; Non-covalent interactions, including ionic bonds, hydrogen bonds, and hydrophobic interactions, play a crucial role in maintaining the three-dimensional network structure of gluten. The figure shows the changes in ionic bonds, hydrogen bonds, and hydrophobic interactions in gluten and its components after enzyme treatment. With increasing NaCl concentration, the solubility of gluten and its components changed slightly, indicating that the ionic bonds in gluten and its components are relatively weak. Xyn and β-Glu increased the ionic bond strength of gluten. Therefore, we hypothesize that Xyn and β-Glu reduced the steric hindrance effect of non-starch polysaccharides, leading to an increase in the content of small peptides and salt-soluble proteins, thereby increasing the content of ionizable amino acids and forming more ionic bonds. The synergistic effect of the complex enzyme resulted in the most significant change in ionic bonds. Hydrogen bonds and hydrophobic interactions were disrupted by urea, causing the protein chain to unfold, thus increasing the solubility in S3 and S4. Figure 4 (B) and Figure 4 As shown in (C), enzyme treatment has little effect on the hydrogen bonds of glutenin and its components, and there is no significant difference in the hydrophobic interaction force of glutenin. This indicates that enzyme treatment has little effect on hydrogen bonds and no significant difference in the hydrophobic interaction force of glutenin. This suggests that Xyn reduces the steric hindrance effect of AX, and β-Glu may release charged groups or change the local pH through hydrolysis of polysaccharides.

[0037] Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on the secondary structure of wheat gluten protein in small ice cream; Infrared spectroscopy is a commonly used method for analyzing the secondary structure of macromolecules. Different functional groups exhibit different characteristic frequencies in infrared spectra. This characteristic can be used to determine whether enzyme addition affects the secondary structure of wheat gluten dough. Different vibrational modes of C=O, CN, and NH in proteins are related to secondary structure. The amide I band (mainly the stretching vibration of C=O, 1600-1700 cm⁻¹) contains information on β-turns, α-helices, random coils, and β-sheets. α-helices and β-sheets play a crucial role in dough stability. α-helices are primarily supported by hydrogen bonds, while β-sheets are layered structures formed under hydrogen bonding. Relatively higher levels of these two structures indicate a denser gluten network and better viscoelasticity. Conversely, β-turns and random coils are disordered structures; a higher proportion of these in proteins results in a poorer gluten network. Therefore, we analyzed the effect of enzymes on the secondary structure of wheat gluten proteins by calculating the peak areas corresponding to different structures.

[0038] Secondary structure is an important characteristic of proteins, and its structure and composition are closely related to the degree of protein polymerization. The contents of α-helices, β-sheets, β-turns, and random coils in proteins were calculated using the absorption bands of amide I at 1650 cm⁻¹–1659 cm⁻¹, 1610 cm⁻¹–1640 cm⁻¹, 1660 cm⁻¹–1699 cm⁻¹, and 1640 cm⁻¹–1650 cm⁻¹. The effects of two enzymes and a complex enzyme on the secondary structure content of gluten proteins are shown in the figure. Xyn increased the α-helix content in gluten proteins, indicating that gluten proteins possess a more stable and ordered structure.

[0039] Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on the fluorescence spectroscopic analysis of wheat gluten protein in small ice cream; Intrinsic fluorescence spectroscopy provides information about changes in the polarity of the microenvironment of fluorescent aromatic amino acids (such as tryptophan and tyrosine), and therefore it is often used to characterize changes in protein conformation. In natural proteins, nonpolar amino acid residues are generally embedded within the protein molecule. Typically, the unfolding of the protein structure exposes these amino acid residues to a polar microenvironment, causing the fluorescence absorption peak to shift to a longer wavelength. The main fluorescent groups in proteins are tyrosine (Tyr) residues and tryptophan (Trp) residues, with fluorescence peaks at 303 nm and 348 nm, respectively. Figure 6As shown, the maximum emission peak of wheat gluten protein appears at 350 nm, indicating that the intrinsic fluorescence of wheat gluten protein mainly originates from tryptophan residues. Compared with the blank, the three enzyme treatments did not significantly affect the maximum fluorescence absorption wavelength of wheat gluten protein, but the fluorescence intensity decreased. This may be due to protein aggregation, which reduces the amount of tryptophan exposed on the surface. This suggests that the addition of enzymes makes the wheat gluten protein structure more compact, thus burying the tryptophan.

[0040] Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on the surface hydrophobicity of wheat gluten protein; Surface hydrophobicity is an indicator of the ability of protein molecules to interact and is largely related to the aggregation and unfolding of protein molecules. ANS, as a hydrophobic fluorescent probe, specifically binds to samples with exposed hydrophobic regions. Figure 7 The effects of three enzyme treatments on the surface hydrophobicity of gluten protein were shown. The enzymes added to the Xyn and β-Glu and Xyn+β-Glu groups increased the surface hydrophobicity of gluten protein by 15.88%, 13.95%, and 22.34%, respectively, indicating that the addition of enzymes exposed the hydrophobic regions inside the protein, leading to enhanced hydrophobic interactions between gluten molecules and thus promoting protein aggregation.

[0041] Finally, it should be noted that, in this invention, unless otherwise explicitly specified and limited, the terms "installation," "setting," "connection," "fixing," "screw connection," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Unless otherwise explicitly limited, those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of enzyme-modified wheat flour dough and gluten protein, comprising the following steps: Preparation of small ice wheat doughs with different amounts of Xyn and β-Glu; Determination of the textural effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on small ice wheat dough; Analysis of the effects of different addition amounts of Xyn, β-Glu and Xyn+β-Glu on the free thiol groups and disulfide bonds in small ice wheat dough; Determination and analysis of the thermal properties of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Analysis of dynamic rheology and creep-recovery of small ice wheat dough by Xyn, β-Glu and Xyn+β-Glu; Analysis of the effects of Xyn, β-Glu and Xyn+β-Glu on wheat gluten protein; Preparation of gluten protein: The prepared dough was rinsed with 20g / L NaCl solution to form coarse gluten. Then the starch was washed with deionized water until the starch was washed away. The gluten protein was freeze-dried and used for the determination of physicochemical properties. Determination and analysis of non-covalent bonds in wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Determination and analysis of the secondary structure of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Determination and analysis of fluorescence spectrometry of Xyn, β-Glu and Xyn+β-Glu in wheat gluten protein; Determination and analysis of the surface hydrophobicity of wheat gluten protein by Xyn, β-Glu and Xyn+β-Glu; Data processing: All experiments were repeated at least three times, and the results were expressed as mean ± standard deviation. One-way ANOVA was used to analyze the differences between multiple groups of data.

2. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The preparation method of small ice wheat dough with different addition amounts of Xyn and β-Glu is as follows: First, Xyn and β-Glu are added to small ice wheat flour (200g) according to different addition amounts (Xyn: 2mg / kg, 4mg / kg, 6mg / kg, 8mg / kg; β-Glu: 20mg / kg, 40mg / kg, 80mg / kg, 160mg / kg). Then, salt (1.6g), sugar (5g), dehydrated yeast (1g), and water (100g) are added and mixed. The dough without added enzyme is used as a blank control group. All ingredients are mixed in a mixer at low speed for 5 minutes to form a dough, and then mixed at high speed until the dough forms a smooth and transparent gluten film that can be stretched. After shaping the dough, it is placed in a fermentation box (35℃, 75% humidity) for 45 minutes. After cooling to room temperature, one batch is freeze-dried, and another batch is used for subsequent parameter determination.

3. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The method for determining the texture of small ice wheat dough with different addition amounts of Xyn, β-Glu, and Xyn+β-Glu was as follows: After fermentation in a fermentation chamber and cooling to room temperature, the dough was allowed to stand at 20°C for 30 minutes. Before testing, the dough was shaped into cubes with sides of 50 mm and a height of 25 mm using a self-made mold. The texture analyzer probe used in the experiment was a P / 36R type. The operating mode was: pressure measurement; operation type: TPA; compression rate 50.0%; the speed before, during, and after the test was set to 3 mm / s, 1 mm / s, and 3 mm / s, respectively; the initial sensing force was 5 g; the trigger type was set to: Auto; the data acquisition rate was 200 pps; the interval between two compressions was 5.0 s. The texture of dough with different addition amounts was selected for PCA analysis.

4. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The determination and analysis of free thiol groups and disulfide bonds in small ice wheat dough by different addition amounts of Xyn, β-Glu, and Xyn+β-Glu were performed by pulverizing the prepared freeze-dried dough through a 100-mesh sieve, weighing 0.075 g, placing it in a 10 mL centrifuge tube, adding 1 mL of buffer solution and 4.7 g of guanidine hydrochloride, and adjusting the volume to 10 mL with buffer solution. For free thiol (SHF) content, 1 mL of sample solution, 4 mL of urea-guanidine hydrochloride solution, and 0.04 mL of DTNB reagent (4 mg / mL) were placed in a 10 mL test tube, mixed, and the absorbance was measured at 412 nm using a UV spectrophotometer. Each sample was measured three times, and the average value was taken. The SHF content was calculated using a formula.

5. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: Dynamic rheological determination: Small iced wheat doughs with different enzymes were placed at 25°C and allowed to cool to room temperature. Then, a certain amount of sample was taken from the center of the dough, spread evenly on the test platform of the rheometer, and excess dough was cut off with scissors. To prevent moisture loss, silicone oil was applied to the edges of the dough. Next, dynamic flow tests were performed in oscillation mode with the following settings: plate diameter 40 mm, slit distance 1 mm, temperature 25°C, strain 0.1%, scanning frequency range 0.1~10 Hz, number of points set to 20, and all tests were repeated three times. Creep-recovery assay: Small ice wheat dough with different enzymes was placed at 25°C and allowed to stand until it cooled to room temperature for measurement. The stress was kept constant at 50 Pa. The dough was pressed for 150 s, and then the external force was removed. The sample recovered for 150 s.

6. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The secondary structure of wheat gluten protein was determined using Fourier transform infrared spectroscopy (FTIR). 1 mg of freeze-dried wheat dough samples with different enzymes was accurately weighed and placed together with 120-150 mg of powdered solid potassium bromide in an agate mortar. The mixture was thoroughly ground until all the powder adhered to the mortar wall, with a particle diameter of approximately 2 μm. The powder was then poured into a tablet press, forming a pile to completely fill the mold without gaps. The tablets were then pressed at approximately 1 tonne for 1 minute. The pressed samples should be transparent or translucent. After verification, the infrared spectroscopy was performed. The sample was carefully placed in the instrument, and the scanning conditions were: wavenumber range of 4000-500 cm⁻¹, interval of 4 cm⁻¹, and scanning frequency of 32 Hz. The Fourier transform infrared spectra of the samples were collected and analyzed using Omnic 8.0 software, primarily focusing on the 1600-1700 cm⁻¹ band. Each experiment was repeated three times to ensure accuracy and consistency.

7. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The fluorescence spectrometry analysis of wheat gluten protein was performed using a fluorescence spectrophotometer. 100 mg of lyophilized gluten protein sample was extracted with 20 mL of 0.5 mol / L acetic acid solution at room temperature for 2 h. After centrifugation (4000 g, 15 min), the supernatant was diluted with 0.5 mol / L acetic acid solution to 1 mg / mL. The fluorescence intensity was measured using a fluorescence spectrometer at an excitation wavelength of 280 nm, an emission wavelength of 290 nm to 410 nm, and a slit width of 5 nm.

8. The enzyme-modified wheat flour dough and gluten protein according to claim 1, characterized in that: The surface hydrophobicity of wheat gluten protein was determined by using 8-aniline-1-naphthalenesulfonate (ANS) as a fluorescent probe. The extract was diluted at different concentrations, and 50 μL of ANS solution (8 mmol / L, pH 5.8) was added to 10 mL of sample solution. The reaction was carried out in the dark for 20 min. The fluorescence intensity of the sample was measured by fluorescence spectroscopy at an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 5 nm. The initial slope of the fluorescence intensity versus protein concentration graph was used as an indicator of surface hydrophobicity.

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