Comparison of the effects of pH-shifting, acetic acid modification and TGase treatment on the physicochemical and functional properties of wheat gluten protein

Comparison of the effects of pH-shifting, acetic acid modification and TGase treatment on the physicochemical and functional properties of wheat gluten protein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of the effects of pH-shifting, acetic acid modification and TGase treatment on the physicochemical and functional properties of wheat gluten protein Mengxue Dong, Yusha Sun, Dandan Xiong, Qi Song, Jie jia, Xuebo Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2486190/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Wheat gluten protein (WGP) is a high-quality plant-based protein resource. However, due to its unique reticulation structure, the processing properties of WGP are extremely poor, limiting its application. To overcome these drawbacks, the aim of this study was to modify wheat gluten protein by three relatively novel and mainstream chemical modifications. The results showed that the pH-shifting treatment changed the apparent morphology of the protein, showing a uniform flocculent structure, leading to significant improvements in foaming capacity and emulsification property. After deamidation by acetic acid, the solubility of WGP was greatly improved (60.1%), which was nearly four times that of the control group (15.8%), and its foam stability was also significantly improved. The WGP had the highest thermal stability (deformation temperature up to 148 ℃) after TGase deamidation. These results indicate that the three modification methods improve the functional properties of WGP in different aspects and expand its application potential. wheat gluten protein pH-shifting acetic acid transglutaminase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Wheat gluten protein (commonly known as gluten meal, WGP) is a by-product of the wheat starch production process (Dong et al., 2022 ). It is widely used in the food and feed processing industry as a high-quality protein raw material due to its rich natural resources, high nutritional value, food safety and low price. However, due to their high molecular weight and a large amount of hydrophobic amino acids, wheat gluten molecules have a large hydrophobic intramolecular interaction area and a low solubility (Wang & Arntfield, 2016 ). In this case, it is difficult for native WGP to have multiple processing functional properties. Therefore, it is necessary to adopt appropriate modification methods to improve and broaden its functional properties to meet the needs of different sectors of industry. Previous modifications mainly include physical and chemical methods. Physical modifications are favored for their rapidity, greenness, and high safety factor (Morales, Santo, & Miranda, 2020). These methods are a targeted modification of proteins and generally do not involve the primary structure of the protein molecules (Zhang et al., 2020 ). Relatively speaking, chemical modification has countless advantages over other methods, including short reaction time, low cost, no need for specialized equipment, and highly visible modification effects. Therefore, chemical modification has become the mainstream approach for protein modification (Robertson et al., 2014 ). In order to give a comprehensive understanding of effects of chemical modification on WGP, this work chose three typically chemical approaches to modify WGP and compare their influences on structural and functional properties of WGP. Firstly, pH-shifting modification is a novel and simple method that is widely used to modify plant proteins. In a pH-shifting treatment, the pH of the protein solution is adjusted to a very acidic or basic pH to allow the protein molecules to unfold. Then the pH is adjusted back to neutral to refold the protein molecules (Jiang et al., 2017 ). This unfolding and refolding process significantly alters the structural and functional properties of proteins. At present, this method had been applied to vegetable proteins such as soy protein (Lee et al., 2016 ), pea protein (Jiang et al., 2017 ) and chickpea protein isolate (Wang et al., 2022 ). Our latest work demonstrated that the pH-shifting treated WGP possessed an enhanced emulsifying property and could be utilized in powdered oils (Xiong et al., 2023 ). This inspired us to further explore the different influence of this method with other chemical modifications. Secondly, the deamidation is the conversion of an amide group from glutamine (Gln) and asparagine (Asn) residues to carboxyl groups, including glutamic acid and aspartic acid. Numerous studies had attempted to catalyze the deamidation of protein by acid-bases and enzyme through various reaction mechanisms (Yong, Yamaguchi, & Matsumura, 2006 ). One of the most common methods of deamidation is hydrochloric acid treatment. However, substantial hydrolysis of peptide bonds is inevitable, producing bitter peptides and reducing processing properties of proteins (Liao et al., 2010 ). Carboxylic acid has been reported to be a better deamidation option, which reduces the potential risk for celiac disease patients and produces little protein hydrolysis (Qiu et al., 2013 ). Therefore, in this study, WGP was modified by deamidation with low concentration acetic acid (0.1 M) to observe the changes in the physicochemical properties of the protein. Thirdly, in addition to pH-shifting and acid hydrolysis deamidation, enzymatic treatment is another typical method to modify proteins. It is reported that the protein transglutaminase (TGase) can be used to improve the quality of WGP products because of its safe, healthy, and environmentally friendly properties (Wee and Jeyakumar Henry, 2019 ). Transglutaminase is an enzyme that forms covalent cross-links between gluten and gliadin, specifically, it catalyzes the reaction between the ammonia (NH 2 ) group of glutamine and lysine to form a covalent ε-(γ-glutamyl) lysine bridge (G-L bond) (Kuraishi, Yamazaki, & Susa, 2001 ). Accordingly, the processing properties of WGP would also significantly alter. Therefore, the purpose of this work is to compare the effects of three representatively chemical methods (pH-shifting treatment, acetate deamidation and enzymatic treatment) on structural and functional properties of WGP, providing a reference for subsequent WGP modification and applications. 2. Materials And Methods 2.1. Materials Commercially available WGP was purchased from Henan Danmire Trading Co. Transglutaminase was provided by Aladdin Biochemical Technology Co. Sodium hydroxide, hy-drochloric acid were supplied from Tianjin Damao Chemical Reagent Factory. All other chemical reagents (including so-dium dodecyl sulfate, 2-mercaptoethanol, trimethylolaminomethane, bromophenol blue, potassium bromide, phosphate buffer (PBS buffer), 8-aniline-1-naphthalenesulfonic acid (ANS)) were analytically pure. 2.2. pH-shifting, acetic acid, and enzymatic treatment Control: Dissolve 1% WGP (W/V) in distilled water and stir for 1 h. The suspension was centrifuged at 7000 g for 10 min and the supernatant was lyophilised. This group was the control group without modification treatment. pH-shifting treatment (pH): the pH of 1% WGP (W/V) suspension was adjusted to 12 using 1 M NaOH, which was placed in a water bath at 80°C for 30 min. The suspension was taken out immediately after the water bath and stirred magnetically at room temperature for 1 h. After stirring was completed, the pH of solution was adjusted to 7 with l M HCl and magnetically stirred again for 1 h. Next, the suspension was then dispensed into centrifuge tubes and centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside. Acetic acid treatment (AAT): AAT was based on the method of Liao et al. with minor modifications (Liao et al., 2010 ). The 1% WGP (W/V) was mixed with 0.1 M acetic acid to form a suspension, which was stirred in a water bath at room temperature for 2 h and then heated at 121℃ for 10 min. The suspension was removed and immediately placed in a cold-water bath for 5 min to stop the reaction and finally centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside. Enzymatic treatment (ET): The ET was based on the method of Lei et al. with minor modifications (Lei, & Ma, 2021 ). The 1% WGP (W/V) was dissolved in deionized water stirred for 0.5 h, after adding 20 U/g of TGase (transglutaminase), the solution was placed in a water bath (40°C) and shaked at 110 r/min for 24 h. After that, the suspension was cooled in an ice-water bath to inhibit enzyme activity. The resulting suspension was centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside. 2.3. Protein solubility Protein solubility was calculated as the percentage of soluble protein content in the supernatant to the total protein added to the dispersion. The mass of soluble protein in the lyophilised supernatant was m 1 and the total mass of WGP was m 0 . The protein solubility was shown in Eq. 1: Protein solubility (%) = m 1 /m 0 X 100 (1) 2.4. Particle size The samples were dispersed in PBS (10 mM, pH 7.0) and prepared as a 7% suspension, followed by vortexing with a vortexer to mix well. A wet method was used to determine the particle size of the samples, using the same concentration of PBS as the aqueous phase and setting the absorbance and refractive index at 0.001 and 1.414 respectively (Day, Xu, Lundin, & Wooster, 2009 ). The particle size was determined at room temperature and each sample was measured 3 times and averaged to obtain a volume-weighted average diameter. 2.5. Foaming properties The foaming capacity (FA) and foam stability (FS) of WGP were measured according to the method of Xiong et al. with minor modifications (Jia et al., 2021 ). Firstly, the WGP solution (2%) was prepared with deionised water, then 15 mL of the protein solution was vortexed and homogenised for 2 min and quickly transferred to a measuring cylinder. The volume of foam at t = 0 min was calculated as V 0 , and the volume of foam at t = 30 min was calculated as V 30 . The formula for FA and FS was as follows: FA (%) = V 0 /15 X 100 (2) FS (%) = V 30 /V 0 X 100 (3) 2.6. Emulsification properties The protein was dissolved in 0.2 M pH 8.2 phosphate buffer to make a 24 mL 2 mg/ mL protein solution, and 8 mL soybean oil was slowly added while stirring. A sample of 50 µL of the prepared emulsion was taken from the bottom of the solution at different times (0 and 10 min) and added to 5 mL of 0.1% SDS solution, and the absorbance at 500 nm (A 0 and A 10 ) was measured, using SDS solution as a blank. The emulsification activity index (EAI) and the emulsion stability index (ESI) were calculated using the following equations: Where: DF is the dilution multiple (DF = 100); C is the sample mass concentration (g/mL); φ is the light range (φ = 1 cm); θ is the proportion of oil phase in the emulsion (θ = 0.25); A 0 is the absorbance measured at 0 min; A 10 is the absorbance measured at 10 min (Tang, Hettiarachchy, Horax, & Eswaranandam, 2003 ). 2.7. Structure of WGP 2.7.1. SDS-PAGE of soluble WGP Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on soluble WGP following the procedure described by Jiang et al. (Jiang, Chen, & Xiong, 2009 ). SDS-PAGE was carried out using 12% separation gels (pH 8.8) and 5% stacking gels (pH 6.8). The electrophoresis voltage was stabilized at 80 V throughout the process. 2.7.2. FT-IR The sample was accurately weighed at 2 mg, added to potassium bromide at a mass ratio of 1:100, ground to a homogeneous powder using a mortar and pestle, pressed into thin slices and then scanned by fourier transform infrared spectrometer (Vetex70, Brooke, Germany) at full wavelength (400–4,000 cm − 1 ) for 32 times. 2.7.3. Surface hydrophobicity (H 0 ) The surface hydrophobicity of soluble protein was determined according to the method of Mu et al. with minor modifications (Dong et al., 2022 ). The protein sample of 100 mg was dispersed in 15 mL of 0.01 M phosphate buffer at pH 7.0 and centrifuged at 7 000 r/min for 10 min. ANS reagent (8.0 mM) was prepared from the above phosphate buffer. The supernatant was diluted 1.00, 0.75, 0.50, 0.25 and 0.125 times, and then 40 µL of ANS reagent was added to 4 mL of protein solution. The excitation wavelength of the fluorescence spectrophotometer (LS55, USA) was set to 365 nm and the emission wavelength was set to 484 nm, and then the fluorescence intensity of different mass concentrations of protein sample was measured. The slope of the curve was defined as the surface hydrophobicity H 0 of the sample being tested. 2.7.4. Free sulfhydryl content (SH) The free sulfhydryl content was determined according to previous research with slight modification (Xiong et al., 2023 ). The lyophilised samples were dissolved in Tris-Gly buffer (containing 0.09 M glycine, 0.086 M Tris and 4 mM NaEDTA) at pH 8.0 to obtain a 0.5% concentration of the sample solution. The solution sample was reacted in a water bath at 25 ℃ for 0.5 h and then centrifuged at 8000 r/min for 15 min. The mixture was prepared by adding 0.05 mL of DTNB to 5 mL of supernatant for 15 min and the absorbance was measured at 412 nm. The formula for calculating the free hydrophobic content was as described below: Where 1.36×10 4 is the molar extinction coefficient of the DTNB solution; A 412 is the absorbance value at 412 nm; D is the dilution multiple; and C is the concentration of the sample solution (mg/mL). 2.7.5. SEM The morphology of the modified WGP was observed at an accelerating voltage of 3.0 kV. Samples were plated with gold to avoid charge under the electron beam. Images were taken using a scanning electron microscope (SEM, Nano SEM-450, US) at ×10,000 magnification. 2.8. Apparent viscosity The apparent viscosity of the WGP samples was determined according to the method of Jia et al. with some modifications (Jia et al., 2021 ). The protein dispersion of 15 mg/mL was prepared using PBS buffer (10 mM, pH 7.4). The protein dispersions were loaded onto parallel plates. The gap height was set to 1 mm and the apparent viscosity of the dispersions was measured by shear scan (0.01–100 s − 1 ). Apparent viscosity measurement was carried out on a rotational rheometer (DHR-1, USA) and was repeated three times for each set of samples. 2.9. Thermal characterization The thermal property was measured according to the previous method (Dong et al., 2022 ) and the differential scanning calorimetry (DSC) measurement was carried out on a differential scanning calorimeter (TA Q2000, TA company, US). The lyophilised sample powder of 3 mg was placed in an aluminium tray, pressed and the empty tray was used as a control for thermal scanning. The heating rate was set to 10 ℃/min, the nitrogen flow rate was kept at 50 mg/mL, and the temperature scanning range was 20–200℃. The denaturation temperature (T d ) was obtained by analyzing the heat flow curve of the sample with the software Universal Analysis 2000. 2.10. Statistical analysis All measurements were repeated three times and the results were shown as average value ± standard deviation (SD). Analysis of variance (ANOVA) followed by Duncan’s multiple range test was performed using by SPSS 26.0. Results were considered statistically significant at p < 0.05. 3. Results And Discussion 3.1. Protein solubility Solubility is one of the important functional properties of protein, as it influences other functional properties of protein (Zhang et al., 2021 ). The solubility of WGP was evaluated after different treatments in this work, as shown in Fig. 1 A. The acetic acid treatment showed the highest protein solubility of 60.2%, while the control group showed the lowest (15.9%). The solubility of the pH-shifting sample was significantly higher than that of the control and ET groups, but significantly lower than that of the AAT group. In the process of pH shifting, WGP firstly unfolded and then began to fold, so that some polar groups were exposed and their flexibility increased, which enhanced the interaction between the protein and water and led to an increase in solubility (Jiang, Chen, & Xiong, 2009 ). The solubility of the AAT group (60.2%) was nearly four times higher than that of the control group (15.9%), and the acetic acid treated protein exposed more polar groups. Jiang et al. (Jiang, Chen & xiong, 2009 ) reported that when protein was exposed to extremely acidic or alkaline pH conditions, the increase in ionic strength in the medium led to partial unfolding of the protein, also known as "molten globule (MG)" structure, so that the protein solubility was highest under acetic acid conditions. Protein in the MG state may lose some side chain interactions and become flexible. For the ET group, the solubility was higher than that of the control group but significantly lower than that of the other two treatment groups. The significant increase in particle size and surface hydrophobicity led to the formation of larger molecular aggregates in aqueous solutions, which reduced the solubility of WGP. In addition, the increase in solubility for ET may also be due to an increase in charged protein-water ion interactions. 3.2. Particle size The particle size of protein has a significant effect on protein foaming capacity as it can influence protein adsorption at the gas-liquid interface. The particle size of the three groups of WGP was shown in Fig. 1 B. All treatments produced soluble WGP aggregates with sizes of > 1000 nm except for the AAT group. The average particle size of the control group was 1982.5 nm and pH-shifting treatment (1939.7 nm) slightly reduced the particle size of the WGP. The particle size of ET group was the largest, which was 5477.7 nm. This treatment was deamidated for 24 hours, as previously reported in the literature, which may be related to excessive deamidation causing protein aggregation (Liao et al., 2010 ). And the AAT group had the smallest particle size, which was mainly attributed to acetic acid stabilizing the folded protein structure of the disulphide bond within the WGP molecules (Sun et al., 2019 ). It also validated that solubility increases at smaller particle sizes. And protein particle size is also related to foaming capacity, which will be explored below. 3.3. Foaming properties In this work, foaming capacity (FA) and foam stability (FS) were determined by measuring the volume of the foam. The effect of the different treatments on FA and FS of WGP was shown in Fig. 2 A. Compared to the control protein sample, the FA and FS of WGP treated by pH-shifting increased from 128–138%, and from 76–80% respectively, which may be related to the increased β-sheet of the protein (Table 1 ). The β-sheet conformation could facilitate the formation and expansion of bubbles, thus improving the foaming properties (Mundi & Aluko, 2012 ). Also, the WGP partially unfolded at a very basic pH and then partially folded at a neutral pH, resulting in the WGP being in a molten state, followed by an increase in surface hydrophobicity, a decrease in particle size and an increase in foaming properties (Jiang, Wang, & Xiong, 2018 ). The increase of hydrophobic groups on the protein surface promoted the formation of a water-air interface, making it easier for protein to adsorb to that interface and formed more stable foams. For the AAT and ET groups, their FA was significantly lower, but the FS of the protein was significantly higher. Wheat gluten protein treated by acetic acid and enzyme showed an increase in FS, which may be due to their reticulate and lamellar structure (Fig. 5 ), whose surface structure allowed them to exist longer at the water-air interface. In addition, chemical and physical properties such as free sulfhydryl content, electrostatic interactions and molecular size can also influence the interfacial behaviour of protein (Han et al., 2019 ). Table 1 The α-helix, β-sheet, β-turn and random coil contents of control, pH, AAT and ET samples. Different superscript letters in the same column indicate significance at the p < 0.05. Treatment α-helix β-sheet β-turn random coil Control 26.99 ± 1.26 a 21.44 ± 0.32 c 21.87 ± 2.73 a 29.70 ± 1.24 a pH 25.77 ± 0.45 ab 24.32 ± 0.53 a 18.41 ± 0.29 a 31.51 ± 0.36 a AAT 26.46 ± 1.87 a 22.85 ± 0.64 b 20.51 ± 2.81 a 30.17 ± 1.56 a ET 24.52 ± 1.11 b 26.20 ± 0.35 a 21.62 ± 3.34 a 31.66 ± 2.53 a 3.4. Emulsification properties Emulsification properties are an indicator of the ability of a protein to absorb into the oil-water interface and can be assessed by the emulsion activity index (EAI) and the emulsion stability index (ESI) of the protein. EAI is a measure of how much oil can be emulsified per unit of protein and ESI is a measure of emulsion stability over a given time span (Boye et al., 2010 ). Figure 2 B showed the EAI and ESI of WGP with different treatments. The pH-shifting and AAT samples had higher EAI and ESI, while the ET group and the control group showed lower EAI and ESI. This could be explained that the AAT and pH-shifting groups had higher solubility. Compared with the control group, the EAI of sample after pH-shifting treatment increased from 1.04 to 1.44 m 2 /g, which may be due to the disulfide bond breakage and subsequent molecular rearrangement. Another reason for the phenomenon is the exposure of hydrophobic groups that increased hydrophobicity and oil binding capacity during pH-shifting treatment (Liu et al., 2021 ). However, the EAI of the AAT group (1.51 m 2 /g) was slightly higher than that of the pH-shifting group (1.44 m 2 /g), probably due to acid-induced protein degradation. And the reduced size of the protein allowed easier adsorption to the oil-water interface and improved emulsification activity. On the contrary, the pH-shifting treatment gave the best emulsion stability with the highest ESI values, which may be related to the homogeneous flocculent structure of its surface (Fig. 5 ), whose special structure made it more stable at the oil-water interface. 3.5. Structure of WGP 3.5.1. SDS-PAGE of soluble WGP Figure 3 showed the SDS-PAGE bands of WGP under non-reducing (A) and reducing (B) conditions. In Fig. 3 A, the control group was mainly protein of 14 kDa and 40 kDa. These subunit bands were clearly visible in the pH-shifting group, meaning the treatment retained a relatively complete molecular structure. In contrast, the AAT group showed increased intensity of the bands at ~ 34 kDa (LMW-GS + Gliadins, low molecular weight-glutenin subunits and gliadins) and 14 kDa (Liu et al., 2021 ). For the ET group, only the 14 kDa subunit band was shown, with the rest of the bands having reduced intensity. The reduce in molecular weight of the protein after enzymatic digestion suggested that enzymatic deamidation reduced the solubility of WGP in SDS solution. The control and AAT groups showed an increase in band intensity at 43 kDa in reducing conditions, which was predominantly a low molecular weight glutenin subunit, rich in β-turn structures (Tatham, Drake, & Shewry, 1989 ). This result was in line with previous findings that WGP was sulphur-containing protein and that the denaturation process reduced existing S-S bonds and exposed previously unexposed SH groups (Dong et al., 2022 ). 3.5.2. FT-IR In the FT-IR spectrum, the amide I region (1,700-1,600 cm − 1 ) was the characteristic band associated with the secondary structure of WGP. Figure 4 A and Table 1 showed the profiles and contents of the secondary structures of four types (α-helix, β-sheet, β-turn and random coil) of WGP. In Fig. 4 A, the absorption peak around 3,600-3,300 cm − 1 was caused by N-H stretching and O-H deformation vibrations (Guan et al., 2006 ). The pH group showed broader absorption peak in the 3,600-3,300 cm − 1 regions, indicating higher levels of -OH and -NH 2 groups. According to previous studies (Lei & Ma, 2021 ), all treatment groups showed a more pronounced absorption peak at 2,364 cm − 1 , which was attributed to NH 2 stretching vibrations. The absorption peak of the control group was much sharper, resulting that WGP had a high denaturation temperature (Fig. 4 B) due to the presence of many intramolecular or intermolecular hydrogen bonds within its molecules, possibly due to stretching vibrations. The results of the deconvolution of the amide I bands in the FT-IR spectrum, followed by second order derivative fitting, were used to calculate the proportion of the various secondary structures of WGP after different treatments for the attribution corresponding to each peak as shown in Table 1 . Typically, hydrogen bonding is the main force that maintains the α-helix, and the α-helix structure represents the stronger conformation of the protein interface (Jarpa-Parra et al., 2015 ). In Table 1 , the control group had the highest α-helix content, confirming our speculation above that there was more hydrogen bonding within the control protein. As the α-helix decreases, the ordered structure of the protein changed to a disordered structure (Liu et al., 2021 ). Compared to the control group, the three modified groups showed a decrease in α-helix, an increase in random coil and the WGP tended to be more disordered in structure. In addition, the decrease in β-sheet and β-turn indicated that all treatments weakened the ordered structure of the hydrogen bonds of the polypeptide chains within the WGP, which was detrimental to the foaming capacity (FA) of the protein, in agreement with previous findings (Wang et al., 2022 ). And the pH group had the lowest β-turn in Table 1 , and its emulsion stability was the best (Fig. 2 B). The more disordered the protein tends to be, the more favorable its presence at the oil-water interface and the better its emulsification properties, which confirmed the above-mentioned result that the modified groups had better emulsification than the control group. 3.5.3. Surface hydrophobicity (H 0 ) Protein surface hydrophobicity refers to the number of hydrophobic groups on the protein surface in polar environment. It is an index to evaluate the conformational change of protein, and is closely related to the emulsification, foaming and gel ability of protein. As shown in Fig. 1 C, the pH-shifting treatment significantly increased the surface hydrophobicity of WGP from 495 to 1,202 compared to the control. Compared with the acidic condition of AAT group, the alkaline condition was more conducive to the improvement of surface hydrophobicity, which was supported by previous research (Jiang, Zhu, Liu, & Xiong, 2014 ). This was because the unfolding and refolding processes in the acid treatment may not significantly alter the hydrophobic groups in the WGP (Lee et al., 2016 ). Whereas the alkaline treatment (pH 12) was further away from the isoelectric region of the WGP, and thus would unfold the WGP molecules more strongly. As a result, more of the hydrophobic part or non-polar amino acid residues initially buried within the protein were exposed to the protein surface and the balance between hydrophilic and hydrophobic groups was then altered (Jiang, Zhu, Liu, & Xiong, 2014 ). The increase in surface hydrophobicity indicated the exposure of more hydrophobic groups, which can affect protein adsorption at the water-gas and water-oil interfaces and further affect the emulsification and foaming properties of the protein. Surprisingly, the surface hydrophobicity of the AT group was as high as 1,356. This may be since the long enzymatic deamidation treatment exposed too many hydrophobic groups of the WGP and the hydrophobic interactions led to the aggregation of the protein, increasing the surface hydrophobicity and particle size of the protein molecules (Fig. 1 B). 3.5.4. Free sulfhydryl content (SH) Figure 1 D showed the changes in free sulfhydryl content of WGP after different treatments, which can be used to indicate the formation or breakage of disulfide bonds in protein and indirectly the tertiary structure of protein. Among all the treatments, the pH-shifting group had the highest SH content, the control and AAT groups had the lowest SH content, followed by the ET group. It has been reported that pH changed in both extremely acidic and alkaline conditions break disulfide bonds and increased the free sulfhydryl content of protein (Li et al., 2020 ). In addition, under alkaline pH conditions, thiol groups tend to be more reactive to form mercaptide ion species (S-), which accelerates SH oxidation (Jiang, Chen, & Xiong, 2009 ). The higher SH content of pH-shifting group indicated the exposure of internal SH groups due to protein unfolding or the cleavages of S-S bonds in native protein. Thus, surface SH content was closely associated with conformational changed and protein unfolding, indicating exposure of SH groups or disruption of disulfide bonds (Jiang et al., 2017 ). The SH content of the ET group was lower than that of pH-shifting group but higher than that of control group, which was attributed to prolonged enzymatic deamidation-induced protein unfolding and degradation, which exposed more internal sulfhydryl groups (Zhang et al., 2021 ). The AAT group had the lowest content of free sulfhydryl groups, which was consistent with previous findings that the reduction in the number of disulfide bonds after deamidation of WGP by acetic acid led to a decrease in the content of free sulfhydryl groups (Liao, Zhao et al., 2010 ), resulting in a significant increase in its foam stability (Fig. 2 A). 3.5.5. SEM The surface micrographs of the WGP lyophilised powder samples were shown in Fig. 5 . The microstructure of the modified samples changed significantly compared to the control samples. It was observed that the native protein particles were intact and smooth. The pH-shifting samples had a homogeneous flocculent structure, which was closely related to their excellent emulsification properties. For the AAT samples, the development of a network of WGP occurred, forming a three-dimensional mesh structure which wrapped the water molecules and increased AAT samples solubility in water. This network was associated with the interaction of alcoholic and WGP through covalent (SS) and non-covalent bonds (hydrogen, ionic and hydrophobic bonds) and the conformation of protein (Zhang et al., 2021 ). The ET samples were rough lamellar structures rich in small pores due to deamidation (Su et al., 2021 ), which was consistent with the fact that the protein particle was large. 3.6. Apparent viscosity As a water-insoluble protein, it is difficult to find relative studies on the rheological properties of WGP dispersions. In this work, the viscoelastic interval was determined, and it was found that there was no specific viscoelastic interval for the dispersion. Therefore, only apparent viscosity of samples was explored. The flow behaviour of the WGP sample dispersion was shown in Fig. 6 . As shown, the apparent viscosity of all samples decreased with increasing shear rate, exhibiting pseudoplastic behaviour (shear thinning) (Jiang et al., 2020 ). Although the above results showed that the treatment altered the secondary and tertiary structure of the WGP, they did not change the flow properties and remained pseudoplastic. Shear thinning was usually caused by rupture of protein aggregates, alignment of aggregates in the flow direction or disruption of covalent and non-covalent interaction forced (e.g. hydrogen bonds, hydrophobic interactions and electrostatic interactions), which may lead to modification of the WGP network structure (Song et al., 2013 ). This result can be clearly observed in the SEM image. In this case, the apparent viscosity of the AAT samples did not decrease steadily with shear rate, but fluctuates somewhat. This was probably due to the fact that there were more aggregates and the droplets were not homogeneous and stable, leading to an increase in viscosity in AAT dispersions (Wang et al., 2022 ). 3.7. Thermal characterization The thermal properties of the lyophilised samples were analysed by DSC. The denaturation temperatures (T d ), the temperature at which the molecular chain of the polymer reaches the point at which polymer chain degradation becomes apparent, was shown in Fig. 4 B. Surprisingly, the pH-shifting sample with heat treatment had the lowest T d (110°C). The ET group had the highest T d at 148°C, followed by the control and AAT group. From the SEM results, the surface of pH-shifting protein was the most homogeneous structure, but its small particle flocculent structure made its molecular chains more susceptible to degradation during the warming process. On the contrary, the lamellar structure of the ET sample gave it a high T d and made it less susceptible to degradation during the warming process. In addition, the excessive exposure of hydrophobic groups during the pH-shifting also suggested that the hydrophobic groups in WGP were not heat resistant and the structure may be altered when the temperature was increased to 110°C. 4. Conclusion This research investigated the effects of three representatively chemical modifications on the molecular structure, particle morphology and functional properties of WGP. The results showed that the pH-shifting treatment changed the apparent morphology of the protein, showing a homogeneous flocculent structure, leading to significant improvements in FA, EAI and ESI. After acetic acid deamidation treatment, the particle size of WGP was reduced and solubility was greatly increased, and the reduction of disulfide bonds led to a decrease in its free sulfhydryl content, which significantly improved the foam stability compared with the other two methods. Furthermore, the WGP treated by TGase has the highest thermal stability. Declarations Funding Declaration This work was supported by the Key Research & Development Program of Shaanxi Province (No. 2022NY-010) and National Natural Science Foundation of China (Nos. 32172205). Acknowledgements The authors would like to thank the instrument shared platform of the College of Food Science & Engineering of NWAFU, for the assistance in the nanometer particle size and zeta potentiometer, dynamic shear rheometer, fourier transform infrared spectrometer, and differential scanning calorimeter. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of Interest Statement There are no conflicts to declare. References Abedi, E., & Pourmohammadi, K. (2021). Chemical modifications and their effects on gluten protein: An extensive review. Food Chemistry, 343, 128398. Boye, J. I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth, E., & Rajamohamed, S. H. (2010). Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques. Food Research International, 43 (2), 537–546. Day, L., Xu, M., Lundin, L., & Wooster, T. J. (2009). Interfacial properties of deamidated wheat protein in relation to its ability to stabilise oil-in-water emulsions. Food Hydrocolloids, 23 (8), 2158–2167. Dong, M., Tian, L., Li, J., Jia, J., Dong, Y., Tu, Y., Liu, X., Tan, C., & Duan, X. (2022). 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Physicochemical Properties and Functionality of Rice Bran Protein Hydrolyzate Prepared from Heat-stabilized Defatted Rice Bran with the Aid of Enzymes. Journal of Food Science, 68 (1), 152–157. Tatham, A. S., Drake, A. F., & Shewry, P. R. (1989). Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein. Biochemical Journal, 259 (2), 471–476. Wang, K., & Arntfield, S. D. (2016). Modification of interactions between selected volatile flavour compounds and salt-extracted pea protein isolates using chemical and enzymatic approaches. Food Hydrocolloids, 61 , 567–577. Wang, Y., Wang, S., Li, R., Wang, Y., Xiang, Q., Li, K., & Bai, Y. (2022). Effects of combined treatment with ultrasound and pH shifting on foaming properties of chickpea protein isolate. Food Hydrocolloids, 124 , 107351. Wang, Y., Zhao, J., Zhang, S., Zhao, X., Liu, Y., Jiang, J., & Xiong, Y. L. (2022). Structural and rheological properties of mung bean protein emulsion as a liquid egg substitute: The effect of pH shifting and calcium. Food Hydrocolloids, 126 , 107485. Wee, M. S., & Jeyakumar Henry, C. (2019). Effects of Transglutaminase on the Protein Network and In Vitro Starch Digestibility of Asian Wheat Noodles. Foods, 8(12): 607. Xiong, D., Xu, Q., Tian, L., Bai, J., Yang, L., Jia, J., Liu, X., Yang, X., & Duan, X. (2023). Mechanism of improving solubility and emulsifying properties of wheat gluten protein by pH cycling treatment and its application in powder oils. Food Hydrocolloids, 135, 108132. Yong, Y. H., Yamaguchi, S., & Matsumura, Y. (2006). Effects of Enzymatic Deamidation by Protein-Glutaminase on Structure and Functional Properties of Wheat Gluten. Journal of Agricultural and Food Chemistry, 54(16), 6034–6040. Zhang, C., Yang, Y.-H., Zhao, X.-D., Zhang, L., Li, Q., Wu, C., Ding, X., & Qian, J.-Y. (2021). Assessment of impact of pulsed electric field on functional, rheological and structural properties of vital wheat gluten. LWT, 147 , 111536. Zhang, H., Chen, G., Liu, M., Mei, X., Yu, Q., & Kan, J. (2020). Effects of multi-frequency ultrasound on physicochemical properties, structural characteristics of gluten protein and the quality of noodle. Ultrasonics Sonochemistry, 67 , 105135. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 30 Jan, 2023 Editor assigned by journal 30 Jan, 2023 Submission checks completed at journal 29 Jan, 2023 First submitted to journal 17 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2486190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":171590958,"identity":"9325de5f-9cb1-4a73-adb0-c36b8e70f8d6","order_by":0,"name":"Mengxue Dong","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengxue","middleName":"","lastName":"Dong","suffix":""},{"id":171590959,"identity":"0da5b0a7-1d4c-41af-a1ad-0ebf6110a8fa","order_by":1,"name":"Yusha Sun","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yusha","middleName":"","lastName":"Sun","suffix":""},{"id":171590960,"identity":"6aa96bdb-0f6b-4f8f-b8b0-b6d3d02d819a","order_by":2,"name":"Dandan Xiong","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Xiong","suffix":""},{"id":171590961,"identity":"817f716b-eb56-4666-a16a-84067c4bc79a","order_by":3,"name":"Qi Song","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Song","suffix":""},{"id":171590962,"identity":"2b767381-9482-47a8-923d-285a40914d88","order_by":4,"name":"Jie jia","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"jia","suffix":""},{"id":171590963,"identity":"dc525641-3bc4-4d95-9d68-a4338fb0a213","order_by":5,"name":"Xuebo Liu","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuebo","middleName":"","lastName":"Liu","suffix":""},{"id":171590964,"identity":"e017e530-c840-4b6a-94ad-7c347c3d0ebf","order_by":6,"name":"Long Sheng","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Sheng","suffix":""},{"id":171590965,"identity":"b74ceb84-e0ed-4174-a536-9f1acd2d33cd","order_by":7,"name":"Xiang Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDCCAyDCAEQcbHzwwcDGjgQtjIcPG84oSEsmUgsIMB9Lk+b5cIixgZAOvhvJzx7zFNjlybudMZC2MTjAzMB++OgGfFokb6SZG84wSC42PHPGwDjH4A4fA09a2g18WgxuJJhJfDBgTtw444xBco7BM2YGCR4zAlrSv0kkGNQnbpz/xuCwhcFhxgbCWnJAthxOnM9wLLGZgRgtkmfelEnOMDieuIHh8GHGHoO0ZDZCfuE7nr5NmudPdeL8hoPtP378sbHjZz98DK8WhAsPQBlsRCkHAfkGopWOglEwCkbBSAMAqVVSZvm6tj0AAAAASUVORK5CYII=","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Duan","suffix":""}],"badges":[],"createdAt":"2023-01-17 05:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2486190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2486190/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32293044,"identity":"a48db7f3-fe27-44df-b115-7b24924a1333","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69136,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the protein solubility (A), particle size (B) and surface hydrophobicity H\u003csub\u003e0\u003c/sub\u003e (C), free sulfhydryl content (D) of different protein samples. Different superscript letters indicate significance at the \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. \u0026nbsp;\u003c/p\u003e","description":"","filename":"1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/e67c6d4a85ae998880c6523d.jpeg"},{"id":32293043,"identity":"60823875-561e-45e1-be4c-ce458caaf3a2","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48508,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different treatments on foaming properties (A) and emulsification properties (B) of wheat gluten protein (WGP). Different letters (a, b, c, d) indicate\u003c/p\u003e\n\u003cp\u003esignificant differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/207753b3dec3e15fc5c9bfc2.jpeg"},{"id":32293042,"identity":"5494d514-987d-40fd-a097-d3a15500cfca","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50780,"visible":true,"origin":"","legend":"\u003cp\u003eThe non-reducing (A) and reducing (B) SDS-PAGE analysis of different protein samples. Lanes: M, protein marker; (1), control: untreated protein; (2), pH-shifting treatment; (3), acetic acid treatment; (4), enzymatic treatment.\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/1aac7fc304a9588cbfa8b12f.jpeg"},{"id":32293047,"identity":"3578b325-fc95-476a-88a0-7371ff7f6372","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61108,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra (A) and DSC (B) curves of different protein samples. The FT-IR spectra were ranging from 400 to 4000 cm\u003csup\u003e-1\u003c/sup\u003e. The DSC curves were at 30-180°C.\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/ea2755576e31d8053400eca4.jpeg"},{"id":32293045,"identity":"d6a7f6f8-a34f-44bf-a9b4-7f27ceafa1c0","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":233398,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of the surface of control, pH-shifting (pH), acetic acid (AAT) and enzymatic (ET) treatment samples (Magnifications was 10,000×).\u003c/p\u003e","description":"","filename":"5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/28de9934256530ddd42ce989.jpeg"},{"id":32293046,"identity":"512c4ec8-f249-49fc-bfa6-c461695942fb","added_by":"auto","created_at":"2023-01-31 20:46:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75544,"visible":true,"origin":"","legend":"\u003cp\u003eThe apparent viscosity as a function of shear rate for control, pH-shifting (pH), acetic acid (AAT) and enzymatic (ET) treatment dispersion.\u003c/p\u003e","description":"","filename":"6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/f1e38095761542eed4e42b67.jpeg"},{"id":32293079,"identity":"c99ebdeb-ab5f-4a97-bdf1-05ff8d9e79f5","added_by":"auto","created_at":"2023-01-31 20:46:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":951113,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2486190/v1/c1d35640-1235-4514-83e5-f59ac09fcb93.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of the effects of pH-shifting, acetic acid modification and TGase treatment on the physicochemical and functional properties of wheat gluten protein","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWheat gluten protein (commonly known as gluten meal, WGP) is a by-product of the wheat starch production process (Dong et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is widely used in the food and feed processing industry as a high-quality protein raw material due to its rich natural resources, high nutritional value, food safety and low price. However, due to their high molecular weight and a large amount of hydrophobic amino acids, wheat gluten molecules have a large hydrophobic intramolecular interaction area and a low solubility (Wang \u0026amp; Arntfield, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this case, it is difficult for native WGP to have multiple processing functional properties. Therefore, it is necessary to adopt appropriate modification methods to improve and broaden its functional properties to meet the needs of different sectors of industry.\u003c/p\u003e \u003cp\u003ePrevious modifications mainly include physical and chemical methods. Physical modifications are favored for their rapidity, greenness, and high safety factor (Morales, Santo, \u0026amp; Miranda, 2020). These methods are a targeted modification of proteins and generally do not involve the primary structure of the protein molecules (Zhang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Relatively speaking, chemical modification has countless advantages over other methods, including short reaction time, low cost, no need for specialized equipment, and highly visible modification effects. Therefore, chemical modification has become the mainstream approach for protein modification (Robertson et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In order to give a comprehensive understanding of effects of chemical modification on WGP, this work chose three typically chemical approaches to modify WGP and compare their influences on structural and functional properties of WGP.\u003c/p\u003e \u003cp\u003eFirstly, pH-shifting modification is a novel and simple method that is widely used to modify plant proteins. In a pH-shifting treatment, the pH of the protein solution is adjusted to a very acidic or basic pH to allow the protein molecules to unfold. Then the pH is adjusted back to neutral to refold the protein molecules (Jiang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This unfolding and refolding process significantly alters the structural and functional properties of proteins. At present, this method had been applied to vegetable proteins such as soy protein (Lee et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), pea protein (Jiang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and chickpea protein isolate (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our latest work demonstrated that the pH-shifting treated WGP possessed an enhanced emulsifying property and could be utilized in powdered oils (Xiong et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This inspired us to further explore the different influence of this method with other chemical modifications.\u003c/p\u003e \u003cp\u003eSecondly, the deamidation is the conversion of an amide group from glutamine (Gln) and asparagine (Asn) residues to carboxyl groups, including glutamic acid and aspartic acid. Numerous studies had attempted to catalyze the deamidation of protein by acid-bases and enzyme through various reaction mechanisms (Yong, Yamaguchi, \u0026amp; Matsumura, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). One of the most common methods of deamidation is hydrochloric acid treatment. However, substantial hydrolysis of peptide bonds is inevitable, producing bitter peptides and reducing processing properties of proteins (Liao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Carboxylic acid has been reported to be a better deamidation option, which reduces the potential risk for celiac disease patients and produces little protein hydrolysis (Qiu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, in this study, WGP was modified by deamidation with low concentration acetic acid (0.1 M) to observe the changes in the physicochemical properties of the protein.\u003c/p\u003e \u003cp\u003eThirdly, in addition to pH-shifting and acid hydrolysis deamidation, enzymatic treatment is another typical method to modify proteins. It is reported that the protein transglutaminase (TGase) can be used to improve the quality of WGP products because of its safe, healthy, and environmentally friendly properties (Wee and Jeyakumar Henry, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Transglutaminase is an enzyme that forms covalent cross-links between gluten and gliadin, specifically, it catalyzes the reaction between the ammonia (NH\u003csub\u003e2\u003c/sub\u003e) group of glutamine and lysine to form a covalent ε-(γ-glutamyl) lysine bridge (G-L bond) (Kuraishi, Yamazaki, \u0026amp; Susa, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Accordingly, the processing properties of WGP would also significantly alter.\u003c/p\u003e \u003cp\u003eTherefore, the purpose of this work is to compare the effects of three representatively chemical methods (pH-shifting treatment, acetate deamidation and enzymatic treatment) on structural and functional properties of WGP, providing a reference for subsequent WGP modification and applications.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eCommercially available WGP was purchased from Henan Danmire Trading Co. Transglutaminase was provided by Aladdin Biochemical Technology Co. Sodium hydroxide, hy-drochloric acid were supplied from Tianjin Damao Chemical Reagent Factory. All other chemical reagents (including so-dium dodecyl sulfate, 2-mercaptoethanol, trimethylolaminomethane, bromophenol blue, potassium bromide, phosphate buffer (PBS buffer), 8-aniline-1-naphthalenesulfonic acid (ANS)) were analytically pure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. pH-shifting, acetic acid, and enzymatic treatment\u003c/h2\u003e \u003cp\u003eControl: Dissolve 1% WGP (W/V) in distilled water and stir for 1 h. The suspension was centrifuged at 7000 g for 10 min and the supernatant was lyophilised. This group was the control group without modification treatment.\u003c/p\u003e \u003cp\u003epH-shifting treatment (pH): the pH of 1% WGP (W/V) suspension was adjusted to 12 using 1 M NaOH, which was placed in a water bath at 80\u0026deg;C for 30 min. The suspension was taken out immediately after the water bath and stirred magnetically at room temperature for 1 h. After stirring was completed, the pH of solution was adjusted to 7 with l M HCl and magnetically stirred again for 1 h. Next, the suspension was then dispensed into centrifuge tubes and centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside.\u003c/p\u003e \u003cp\u003eAcetic acid treatment (AAT): AAT was based on the method of Liao et al. with minor modifications (Liao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The 1% WGP (W/V) was mixed with 0.1 M acetic acid to form a suspension, which was stirred in a water bath at room temperature for 2 h and then heated at 121℃ for 10 min. The suspension was removed and immediately placed in a cold-water bath for 5 min to stop the reaction and finally centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside.\u003c/p\u003e \u003cp\u003eEnzymatic treatment (ET): The ET was based on the method of Lei et al. with minor modifications (Lei, \u0026amp; Ma, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The 1% WGP (W/V) was dissolved in deionized water stirred for 0.5 h, after adding 20 U/g of TGase (transglutaminase), the solution was placed in a water bath (40\u0026deg;C) and shaked at 110 r/min for 24 h. After that, the suspension was cooled in an ice-water bath to inhibit enzyme activity. The resulting suspension was centrifuged at 7000 g for 10 min, the supernatant was lyophilised and set aside.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Protein solubility\u003c/h2\u003e \u003cp\u003eProtein solubility was calculated as the percentage of soluble protein content in the supernatant to the total protein added to the dispersion. The mass of soluble protein in the lyophilised supernatant was m\u003csub\u003e1\u003c/sub\u003e and the total mass of WGP was m\u003csub\u003e0\u003c/sub\u003e. The protein solubility was shown in Eq.\u0026nbsp;1:\u003c/p\u003e \u003cp\u003eProtein solubility (%) = m\u003csub\u003e1\u003c/sub\u003e/m\u003csub\u003e0\u003c/sub\u003e X 100 (1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Particle size\u003c/h2\u003e \u003cp\u003eThe samples were dispersed in PBS (10 mM, pH 7.0) and prepared as a 7% suspension, followed by vortexing with a vortexer to mix well. A wet method was used to determine the particle size of the samples, using the same concentration of PBS as the aqueous phase and setting the absorbance and refractive index at 0.001 and 1.414 respectively (Day, Xu, Lundin, \u0026amp; Wooster, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The particle size was determined at room temperature and each sample was measured 3 times and averaged to obtain a volume-weighted average diameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Foaming properties\u003c/h2\u003e \u003cp\u003eThe foaming capacity (FA) and foam stability (FS) of WGP were measured according to the method of Xiong et al. with minor modifications (Jia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Firstly, the WGP solution (2%) was prepared with deionised water, then 15 mL of the protein solution was vortexed and homogenised for 2 min and quickly transferred to a measuring cylinder. The volume of foam at t\u0026thinsp;=\u0026thinsp;0 min was calculated as V\u003csub\u003e0\u003c/sub\u003e, and the volume of foam at t\u0026thinsp;=\u0026thinsp;30 min was calculated as V\u003csub\u003e30\u003c/sub\u003e. The formula for FA and FS was as follows:\u003c/p\u003e \u003cp\u003eFA (%) = V\u003csub\u003e0\u003c/sub\u003e/15 X 100 (2)\u003c/p\u003e \u003cp\u003eFS (%) = V\u003csub\u003e30\u003c/sub\u003e/V\u003csub\u003e0\u003c/sub\u003e X 100 (3)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Emulsification properties\u003c/h2\u003e \u003cp\u003eThe protein was dissolved in 0.2 M pH 8.2 phosphate buffer to make a 24 mL 2 mg/ mL protein solution, and 8 mL soybean oil was slowly added while stirring. A sample of 50 \u0026micro;L of the prepared emulsion was taken from the bottom of the solution at different times (0 and 10 min) and added to 5 mL of 0.1% SDS solution, and the absorbance at 500 nm (A\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003e10\u003c/sub\u003e) was measured, using SDS solution as a blank. The emulsification activity index (EAI) and the emulsion stability index (ESI) were calculated using the following equations:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003cbr\u003e\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e \u003cp\u003eWhere: DF is the dilution multiple (DF\u0026thinsp;=\u0026thinsp;100); C is the sample mass concentration (g/mL); φ is the light range (φ\u0026thinsp;=\u0026thinsp;1 cm); θ is the proportion of oil phase in the emulsion (θ\u0026thinsp;=\u0026thinsp;0.25); A\u003csub\u003e0\u003c/sub\u003e is the absorbance measured at 0 min; A\u003csub\u003e10\u003c/sub\u003e is the absorbance measured at 10 min (Tang, Hettiarachchy, Horax, \u0026amp; Eswaranandam, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Structure of WGP\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. SDS-PAGE of soluble WGP\u003c/h2\u003e \u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on soluble WGP following the procedure described by Jiang et al. (Jiang, Chen, \u0026amp; Xiong, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). SDS-PAGE was carried out using 12% separation gels (pH 8.8) and 5% stacking gels (pH 6.8). The electrophoresis voltage was stabilized at 80 V throughout the process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. FT-IR\u003c/h2\u003e \u003cp\u003eThe sample was accurately weighed at 2 mg, added to potassium bromide at a mass ratio of 1:100, ground to a homogeneous powder using a mortar and pestle, pressed into thin slices and then scanned by fourier transform infrared spectrometer (Vetex70, Brooke, Germany) at full wavelength (400\u0026ndash;4,000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 32 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Surface hydrophobicity (H\u003csub\u003e0\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eThe surface hydrophobicity of soluble protein was determined according to the method of Mu et al. with minor modifications (Dong et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The protein sample of 100 mg was dispersed in 15 mL of 0.01 M phosphate buffer at pH 7.0 and centrifuged at 7 000 r/min for 10 min. ANS reagent (8.0 mM) was prepared from the above phosphate buffer. The supernatant was diluted 1.00, 0.75, 0.50, 0.25 and 0.125 times, and then 40 \u0026micro;L of ANS reagent was added to 4 mL of protein solution. The excitation wavelength of the fluorescence spectrophotometer (LS55, USA) was set to 365 nm and the emission wavelength was set to 484 nm, and then the fluorescence intensity of different mass concentrations of protein sample was measured. The slope of the curve was defined as the surface hydrophobicity H\u003csub\u003e0\u003c/sub\u003e of the sample being tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.4. Free sulfhydryl content (SH)\u003c/h2\u003e \u003cp\u003eThe free sulfhydryl content was determined according to previous research with slight modification (Xiong et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The lyophilised samples were dissolved in Tris-Gly buffer (containing 0.09 M glycine, 0.086 M Tris and 4 mM NaEDTA) at pH 8.0 to obtain a 0.5% concentration of the sample solution. The solution sample was reacted in a water bath at 25 ℃ for 0.5 h and then centrifuged at 8000 r/min for 15 min. The mixture was prepared by adding 0.05 mL of DTNB to 5 mL of supernatant for 15 min and the absorbance was measured at 412 nm. The formula for calculating the free hydrophobic content was as described below:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003cbr\u003e\u003c/p\u003e \u003cp\u003eWhere 1.36\u0026times;10\u003csup\u003e4\u003c/sup\u003e is the molar extinction coefficient of the DTNB solution; A\u003csub\u003e412\u003c/sub\u003e is the absorbance value at 412 nm; D is the dilution multiple; and C is the concentration of the sample solution (mg/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.7.5. SEM\u003c/h2\u003e \u003cp\u003eThe morphology of the modified WGP was observed at an accelerating voltage of 3.0 kV. Samples were plated with gold to avoid charge under the electron beam. Images were taken using a scanning electron microscope (SEM, Nano SEM-450, US) at \u0026times;10,000 magnification.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Apparent viscosity\u003c/h2\u003e \u003cp\u003eThe apparent viscosity of the WGP samples was determined according to the method of Jia et al. with some modifications (Jia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The protein dispersion of 15 mg/mL was prepared using PBS buffer (10 mM, pH 7.4). The protein dispersions were loaded onto parallel plates. The gap height was set to 1 mm and the apparent viscosity of the dispersions was measured by shear scan (0.01\u0026ndash;100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Apparent viscosity measurement was carried out on a rotational rheometer (DHR-1, USA) and was repeated three times for each set of samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Thermal characterization\u003c/h2\u003e \u003cp\u003eThe thermal property was measured according to the previous method (Dong et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the differential scanning calorimetry (DSC) measurement was carried out on a differential scanning calorimeter (TA Q2000, TA company, US). The lyophilised sample powder of 3 mg was placed in an aluminium tray, pressed and the empty tray was used as a control for thermal scanning. The heating rate was set to 10 ℃/min, the nitrogen flow rate was kept at 50 mg/mL, and the temperature scanning range was 20\u0026ndash;200℃. The denaturation temperature (T\u003csub\u003ed\u003c/sub\u003e) was obtained by analyzing the heat flow curve of the sample with the software Universal Analysis 2000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll measurements were repeated three times and the results were shown as average value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Analysis of variance (ANOVA) followed by Duncan\u0026rsquo;s multiple range test was performed using by SPSS 26.0. Results were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Protein solubility\u003c/h2\u003e \u003cp\u003eSolubility is one of the important functional properties of protein, as it influences other functional properties of protein (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The solubility of WGP was evaluated after different treatments in this work, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The acetic acid treatment showed the highest protein solubility of 60.2%, while the control group showed the lowest (15.9%). The solubility of the pH-shifting sample was significantly higher than that of the control and ET groups, but significantly lower than that of the AAT group. In the process of pH shifting, WGP firstly unfolded and then began to fold, so that some polar groups were exposed and their flexibility increased, which enhanced the interaction between the protein and water and led to an increase in solubility (Jiang, Chen, \u0026amp; Xiong, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The solubility of the AAT group (60.2%) was nearly four times higher than that of the control group (15.9%), and the acetic acid treated protein exposed more polar groups. Jiang et al. (Jiang, Chen \u0026amp; xiong, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that when protein was exposed to extremely acidic or alkaline pH conditions, the increase in ionic strength in the medium led to partial unfolding of the protein, also known as \"molten globule (MG)\" structure, so that the protein solubility was highest under acetic acid conditions. Protein in the MG state may lose some side chain interactions and become flexible. For the ET group, the solubility was higher than that of the control group but significantly lower than that of the other two treatment groups. The significant increase in particle size and surface hydrophobicity led to the formation of larger molecular aggregates in aqueous solutions, which reduced the solubility of WGP. In addition, the increase in solubility for ET may also be due to an increase in charged protein-water ion interactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Particle size\u003c/h2\u003e \u003cp\u003eThe particle size of protein has a significant effect on protein foaming capacity as it can influence protein adsorption at the gas-liquid interface. The particle size of the three groups of WGP was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. All treatments produced soluble WGP aggregates with sizes of \u0026gt;\u0026thinsp;1000 nm except for the AAT group. The average particle size of the control group was 1982.5 nm and pH-shifting treatment (1939.7 nm) slightly reduced the particle size of the WGP. The particle size of ET group was the largest, which was 5477.7 nm. This treatment was deamidated for 24 hours, as previously reported in the literature, which may be related to excessive deamidation causing protein aggregation (Liao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). And the AAT group had the smallest particle size, which was mainly attributed to acetic acid stabilizing the folded protein structure of the disulphide bond within the WGP molecules (Sun et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It also validated that solubility increases at smaller particle sizes. And protein particle size is also related to foaming capacity, which will be explored below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Foaming properties\u003c/h2\u003e \u003cp\u003eIn this work, foaming capacity (FA) and foam stability (FS) were determined by measuring the volume of the foam. The effect of the different treatments on FA and FS of WGP was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Compared to the control protein sample, the FA and FS of WGP treated by pH-shifting increased from 128\u0026ndash;138%, and from 76\u0026ndash;80% respectively, which may be related to the increased β-sheet of the protein (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The β-sheet conformation could facilitate the formation and expansion of bubbles, thus improving the foaming properties (Mundi \u0026amp; Aluko, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Also, the WGP partially unfolded at a very basic pH and then partially folded at a neutral pH, resulting in the WGP being in a molten state, followed by an increase in surface hydrophobicity, a decrease in particle size and an increase in foaming properties (Jiang, Wang, \u0026amp; Xiong, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The increase of hydrophobic groups on the protein surface promoted the formation of a water-air interface, making it easier for protein to adsorb to that interface and formed more stable foams. For the AAT and ET groups, their FA was significantly lower, but the FS of the protein was significantly higher. Wheat gluten protein treated by acetic acid and enzyme showed an increase in FS, which may be due to their reticulate and lamellar structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e), whose surface structure allowed them to exist longer at the water-air interface. In addition, chemical and physical properties such as free sulfhydryl content, electrostatic interactions and molecular size can also influence the interfacial behaviour of protein (Han et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe α-helix, β-sheet, β-turn and random coil contents of control, pH, AAT and ET samples. Different superscript letters in the same column indicate significance at the \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα-helix\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-sheet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-turn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003erandom coil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eET\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Emulsification properties\u003c/h2\u003e \u003cp\u003eEmulsification properties are an indicator of the ability of a protein to absorb into the oil-water interface and can be assessed by the emulsion activity index (EAI) and the emulsion stability index (ESI) of the protein. EAI is a measure of how much oil can be emulsified per unit of protein and ESI is a measure of emulsion stability over a given time span (Boye et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB showed the EAI and ESI of WGP with different treatments. The pH-shifting and AAT samples had higher EAI and ESI, while the ET group and the control group showed lower EAI and ESI. This could be explained that the AAT and pH-shifting groups had higher solubility. Compared with the control group, the EAI of sample after pH-shifting treatment increased from 1.04 to 1.44 m\u003csup\u003e2\u003c/sup\u003e/g, which may be due to the disulfide bond breakage and subsequent molecular rearrangement. Another reason for the phenomenon is the exposure of hydrophobic groups that increased hydrophobicity and oil binding capacity during pH-shifting treatment (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the EAI of the AAT group (1.51 m\u003csup\u003e2\u003c/sup\u003e/g) was slightly higher than that of the pH-shifting group (1.44 m\u003csup\u003e2\u003c/sup\u003e/g), probably due to acid-induced protein degradation. And the reduced size of the protein allowed easier adsorption to the oil-water interface and improved emulsification activity. On the contrary, the pH-shifting treatment gave the best emulsion stability with the highest ESI values, which may be related to the homogeneous flocculent structure of its surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e), whose special structure made it more stable at the oil-water interface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Structure of WGP\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. SDS-PAGE of soluble WGP\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e showed the SDS-PAGE bands of WGP under non-reducing (A) and reducing (B) conditions. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the control group was mainly protein of 14 kDa and 40 kDa. These subunit bands were clearly visible in the pH-shifting group, meaning the treatment retained a relatively complete molecular structure. In contrast, the AAT group showed increased intensity of the bands at ~\u0026thinsp;34 kDa (LMW-GS\u0026thinsp;+\u0026thinsp;Gliadins, low molecular weight-glutenin subunits and gliadins) and 14 kDa (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For the ET group, only the 14 kDa subunit band was shown, with the rest of the bands having reduced intensity. The reduce in molecular weight of the protein after enzymatic digestion suggested that enzymatic deamidation reduced the solubility of WGP in SDS solution. The control and AAT groups showed an increase in band intensity at 43 kDa in reducing conditions, which was predominantly a low molecular weight glutenin subunit, rich in β-turn structures (Tatham, Drake, \u0026amp; Shewry, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). This result was in line with previous findings that WGP was sulphur-containing protein and that the denaturation process reduced existing S-S bonds and exposed previously unexposed SH groups (Dong et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2. FT-IR\u003c/h2\u003e \u003cp\u003eIn the FT-IR spectrum, the amide I region (1,700-1,600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was the characteristic band associated with the secondary structure of WGP. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed the profiles and contents of the secondary structures of four types (α-helix, β-sheet, β-turn and random coil) of WGP. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the absorption peak around 3,600-3,300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was caused by N-H stretching and O-H deformation vibrations (Guan et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The pH group showed broader absorption peak in the 3,600-3,300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e regions, indicating higher levels of -OH and -NH\u003csub\u003e2\u003c/sub\u003e groups. According to previous studies (Lei \u0026amp; Ma, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), all treatment groups showed a more pronounced absorption peak at 2,364 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was attributed to NH\u003csub\u003e2\u003c/sub\u003e stretching vibrations. The absorption peak of the control group was much sharper, resulting that WGP had a high denaturation temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) due to the presence of many intramolecular or intermolecular hydrogen bonds within its molecules, possibly due to stretching vibrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the deconvolution of the amide I bands in the FT-IR spectrum, followed by second order derivative fitting, were used to calculate the proportion of the various secondary structures of WGP after different treatments for the attribution corresponding to each peak as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Typically, hydrogen bonding is the main force that maintains the α-helix, and the α-helix structure represents the stronger conformation of the protein interface (Jarpa-Parra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the control group had the highest α-helix content, confirming our speculation above that there was more hydrogen bonding within the control protein. As the α-helix decreases, the ordered structure of the protein changed to a disordered structure (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Compared to the control group, the three modified groups showed a decrease in α-helix, an increase in random coil and the WGP tended to be more disordered in structure. In addition, the decrease in β-sheet and β-turn indicated that all treatments weakened the ordered structure of the hydrogen bonds of the polypeptide chains within the WGP, which was detrimental to the foaming capacity (FA) of the protein, in agreement with previous findings (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And the pH group had the lowest β-turn in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and its emulsion stability was the best (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The more disordered the protein tends to be, the more favorable its presence at the oil-water interface and the better its emulsification properties, which confirmed the above-mentioned result that the modified groups had better emulsification than the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3. Surface hydrophobicity (H\u003csub\u003e0\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eProtein surface hydrophobicity refers to the number of hydrophobic groups on the protein surface in polar environment. It is an index to evaluate the conformational change of protein, and is closely related to the emulsification, foaming and gel ability of protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the pH-shifting treatment significantly increased the surface hydrophobicity of WGP from 495 to 1,202 compared to the control. Compared with the acidic condition of AAT group, the alkaline condition was more conducive to the improvement of surface hydrophobicity, which was supported by previous research (Jiang, Zhu, Liu, \u0026amp; Xiong, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This was because the unfolding and refolding processes in the acid treatment may not significantly alter the hydrophobic groups in the WGP (Lee et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Whereas the alkaline treatment (pH 12) was further away from the isoelectric region of the WGP, and thus would unfold the WGP molecules more strongly. As a result, more of the hydrophobic part or non-polar amino acid residues initially buried within the protein were exposed to the protein surface and the balance between hydrophilic and hydrophobic groups was then altered (Jiang, Zhu, Liu, \u0026amp; Xiong, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The increase in surface hydrophobicity indicated the exposure of more hydrophobic groups, which can affect protein adsorption at the water-gas and water-oil interfaces and further affect the emulsification and foaming properties of the protein. Surprisingly, the surface hydrophobicity of the AT group was as high as 1,356. This may be since the long enzymatic deamidation treatment exposed too many hydrophobic groups of the WGP and the hydrophobic interactions led to the aggregation of the protein, increasing the surface hydrophobicity and particle size of the protein molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.5.4. Free sulfhydryl content (SH)\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD showed the changes in free sulfhydryl content of WGP after different treatments, which can be used to indicate the formation or breakage of disulfide bonds in protein and indirectly the tertiary structure of protein. Among all the treatments, the pH-shifting group had the highest SH content, the control and AAT groups had the lowest SH content, followed by the ET group. It has been reported that pH changed in both extremely acidic and alkaline conditions break disulfide bonds and increased the free sulfhydryl content of protein (Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, under alkaline pH conditions, thiol groups tend to be more reactive to form mercaptide ion species (S-), which accelerates SH oxidation (Jiang, Chen, \u0026amp; Xiong, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The higher SH content of pH-shifting group indicated the exposure of internal SH groups due to protein unfolding or the cleavages of S-S bonds in native protein. Thus, surface SH content was closely associated with conformational changed and protein unfolding, indicating exposure of SH groups or disruption of disulfide bonds (Jiang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The SH content of the ET group was lower than that of pH-shifting group but higher than that of control group, which was attributed to prolonged enzymatic deamidation-induced protein unfolding and degradation, which exposed more internal sulfhydryl groups (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The AAT group had the lowest content of free sulfhydryl groups, which was consistent with previous findings that the reduction in the number of disulfide bonds after deamidation of WGP by acetic acid led to a decrease in the content of free sulfhydryl groups (Liao, Zhao et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), resulting in a significant increase in its foam stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.5.5. SEM\u003c/h2\u003e \u003cp\u003eThe surface micrographs of the WGP lyophilised powder samples were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The microstructure of the modified samples changed significantly compared to the control samples. It was observed that the native protein particles were intact and smooth. The pH-shifting samples had a homogeneous flocculent structure, which was closely related to their excellent emulsification properties. For the AAT samples, the development of a network of WGP occurred, forming a three-dimensional mesh structure which wrapped the water molecules and increased AAT samples solubility in water. This network was associated with the interaction of alcoholic and WGP through covalent (SS) and non-covalent bonds (hydrogen, ionic and hydrophobic bonds) and the conformation of protein (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The ET samples were rough lamellar structures rich in small pores due to deamidation (Su et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which was consistent with the fact that the protein particle was large.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Apparent viscosity\u003c/h2\u003e \u003cp\u003eAs a water-insoluble protein, it is difficult to find relative studies on the rheological properties of WGP dispersions. In this work, the viscoelastic interval was determined, and it was found that there was no specific viscoelastic interval for the dispersion. Therefore, only apparent viscosity of samples was explored. The flow behaviour of the WGP sample dispersion was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As shown, the apparent viscosity of all samples decreased with increasing shear rate, exhibiting pseudoplastic behaviour (shear thinning) (Jiang et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although the above results showed that the treatment altered the secondary and tertiary structure of the WGP, they did not change the flow properties and remained pseudoplastic. Shear thinning was usually caused by rupture of protein aggregates, alignment of aggregates in the flow direction or disruption of covalent and non-covalent interaction forced (e.g. hydrogen bonds, hydrophobic interactions and electrostatic interactions), which may lead to modification of the WGP network structure (Song et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This result can be clearly observed in the SEM image. In this case, the apparent viscosity of the AAT samples did not decrease steadily with shear rate, but fluctuates somewhat. This was probably due to the fact that there were more aggregates and the droplets were not homogeneous and stable, leading to an increase in viscosity in AAT dispersions (Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Thermal characterization\u003c/h2\u003e \u003cp\u003eThe thermal properties of the lyophilised samples were analysed by DSC. The denaturation temperatures (T\u003csub\u003ed\u003c/sub\u003e), the temperature at which the molecular chain of the polymer reaches the point at which polymer chain degradation becomes apparent, was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Surprisingly, the pH-shifting sample with heat treatment had the lowest T\u003csub\u003ed\u003c/sub\u003e (110\u0026deg;C). The ET group had the highest T\u003csub\u003ed\u003c/sub\u003e at 148\u0026deg;C, followed by the control and AAT group. From the SEM results, the surface of pH-shifting protein was the most homogeneous structure, but its small particle flocculent structure made its molecular chains more susceptible to degradation during the warming process. On the contrary, the lamellar structure of the ET sample gave it a high T\u003csub\u003ed\u003c/sub\u003e and made it less susceptible to degradation during the warming process. In addition, the excessive exposure of hydrophobic groups during the pH-shifting also suggested that the hydrophobic groups in WGP were not heat resistant and the structure may be altered when the temperature was increased to 110\u0026deg;C.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis research investigated the effects of three representatively chemical modifications on the molecular structure, particle morphology and functional properties of WGP. The results showed that the pH-shifting treatment changed the apparent morphology of the protein, showing a homogeneous flocculent structure, leading to significant improvements in FA, EAI and ESI. After acetic acid deamidation treatment, the particle size of WGP was reduced and solubility was greatly increased, and the reduction of disulfide bonds led to a decrease in its free sulfhydryl content, which significantly improved the foam stability compared with the other two methods. Furthermore, the WGP treated by TGase has the highest thermal stability.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key Research \u0026amp; Development Program of Shaanxi Province (No. 2022NY-010) and National Natural Science Foundation of China (Nos. 32172205).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe authors would like to thank the instrument shared platform of the College of Food Science \u0026amp; Engineering of NWAFU, for the assistance in the nanometer particle size and zeta potentiometer, dynamic shear rheometer, fourier transform infrared spectrometer, and differential scanning calorimeter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbedi, E., \u0026amp; Pourmohammadi, K. 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Ultrasonics Sonochemistry, \u003cem\u003e67\u003c/em\u003e, 105135.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"wheat gluten protein, pH-shifting, acetic acid, transglutaminase","lastPublishedDoi":"10.21203/rs.3.rs-2486190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2486190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWheat gluten protein (WGP) is a high-quality plant-based protein resource. However, due to its unique reticulation structure, the processing properties of WGP are extremely poor, limiting its application. To overcome these drawbacks, the aim of this study was to modify wheat gluten protein by three relatively novel and mainstream chemical modifications. The results showed that the pH-shifting treatment changed the apparent morphology of the protein, showing a uniform flocculent structure, leading to significant improvements in foaming capacity and emulsification property. After deamidation by acetic acid, the solubility of WGP was greatly improved (60.1%), which was nearly four times that of the control group (15.8%), and its foam stability was also significantly improved. The WGP had the highest thermal stability (deformation temperature up to 148 ℃) after TGase deamidation. These results indicate that the three modification methods improve the functional properties of WGP in different aspects and expand its application potential.\u003c/p\u003e","manuscriptTitle":"Comparison of the effects of pH-shifting, acetic acid modification and TGase treatment on the physicochemical and functional properties of wheat gluten protein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-31 20:45:59","doi":"10.21203/rs.3.rs-2486190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-01-30T10:14:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-01-30T10:13:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-01-30T01:46:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2023-01-17T05:55:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f5a6493d-f6c3-425d-8e64-737546df7878","owner":[],"postedDate":"January 31st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-05-18T07:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-31 20:45:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2486190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2486190","identity":"rs-2486190","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}