Analysis of influence mechanism of grinding on the gel properties of soybean protein isolate based on the changes of its structure and solubility

doi:10.21203/rs.3.rs-2470116/v1

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Analysis of influence mechanism of grinding on the gel properties of soybean protein isolate based on the changes of its structure and solubility

https://doi.org/10.21203/rs.3.rs-2470116/v1

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The compact spherical structure of soy protein isolate results in low protein flexibility and poor functional properties. In this study, the influence mechanism of grinding on its gel properties was analyzed according to the changes of soy protein isolate structure and solubility after grinding. The results indicated that at 15 min of grinding, β-sheet of the soy protein isolate decreased from 38.54% to 29.15%, the particle size became smaller, and the original solid sphere became a hollow sphere, the protein had a more disordered secondary structure, and the solubility increased to 28.24%. The glucono-δ-lactone-induced cold-set gel of this protein exhibited higher water holding capacity (76.37%), storage modulus (G') and loss modulus (G″). Creep recovery test showed that this gel had better elasticity and rigid structure at 15 min of grinding. This study could lay a theoretical foundation for research on improving the gel properties of soy protein isolate by mechanical modification in future.

Soy protein isolate

Grinding

Structure

Gel properties

As one of the most important commercial protein product, soy protein isolate (SPI) is widely used in food industry due to its nutritional and functional advantages (Guo et al., 2022). As one of the most important functional properties of SPI, gel properties would determine whether SPI could be used in the production of tofu products or improve the texture properties of sausages, yogurt, etc (Li et al., 2021a,b). The protein content (dry basis) of SPI reaches 85%-90%, and glycinin (11S) and β-conglycinin (7S) account for more than 80% of the protein (Yu et al., 2021). However, the spherical structure of these two proteins is very compact, which makes SPI less flexible and less functional (Liu et al., 2022). Therefore, its functional characteristics are improved by using a certain processing method to change the structure of SPI, which is the direction of research on SPI in future.

Due to different processing methods, the functional characteristics of SPI would be improved to varying degrees, which is conducive to its application in specific foods. Hu et al. (2013a) found that after high-intensity ultrasonic treatment of SPI, the protein particle size decreased, the surface hydrophobicity was enhanced, and more soluble aggregates formed. These changes effectively improved the strength, hardness and water holding capacity of acid-induced gels. Bi et al. (2014) found that after high shear homogenization of SPI dispersions, the particle size decreased, the polydispersity index lowered. These changes reduced the voids in SPI gel microstructure and increased the gel compactness. At present, the modification method of SPI is mainly wet modification such as high pressure homogenization or ultrasonic (Xu et al., 2018; de Oliveira et al., 2020; O’Sullivan et al., 2015). However, there are few studies on dry modification of SPI.

Grinding is a physical means of denaturing materials through repeated high-energy impact and friction to produce chemical effects (Liu et al., 2020). As a new technology, the ultrafine powder prepared by grinding treatment could not only enhance the surface properties, but also improve some processing properties of the food compared with the coarse powder (Zhang et al., 2020). In recent years, grinding technology has attracted a great attention in the field of food. It was mainly due to its advantages of saving time, solvent and energy (Zhang et al., 2014). Huang et al. (2018) found that the physical and chemical properties of dietary fiber significantly changed after ultrafine grinding, leading to increasing the binding ability to bile acid. Our group found that mung bean starch granules were constantly deformed like elastic balls during grinding, and the starch molecules constantly moved and rearranged, which led to significant changes in their structure, properties and chemical activity (Zhang et al., 2019). Therefore, the flexibility of SPI might be improved if SPI molecules could rearranged under grinding, which might improve the relevant properties of its gel.

In this paper, SPI was modified by grinding, and acid-induced gel was prepared by this modified SPI to explore the structural changes of SPI and its gel properties. The internal relationship between structure of the SPI and its gel properties was analyzed by using protein chemistry and other theories, and the influence mechanism of grinding on gel properties of the SPI was discussed in depth.

Materials

Commercial SPI (> 90% protein) was purchased from Shandong Yuwang Ecological Food Co., Ltd. Glucono-δ-lactone (GDL) was purchased from Jiangxi Xinhuanghai Food Co., Ltd., and bovine serum albumin (BSA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All chemicals used in the experiments were analytical grade.

Grinding SPI

SPI was ground at 25°C using Speediness grind machine (ST-Q, Beijing Xuxin Instrument and Devices Co., Ltd., Beijing, China). The mass ratio of SPI and grinding balls was 1:5. The SPI was ground for different time (0, 3, 6, 9, 12 or 15 min). In order to prevent the temperature of SPI from rising during long-term grinding, the grinding tank was cooled to 25°C after grinding every 3 min. The ground protein powder was placed in a ziplock bag and stored at 4°C for later use.

Preparation of the GDL-induced SPI Cold-set Gel (GSCG)

In order to fully hydrate the protein solution, 10% (w/v) SPI solution was stood overnight at 4°C. The hydrated protein solution was heated in a water bath at 95°C for 20 min, and then immediately cooled to room temperature in an ice-water bath. 0.8% (w/v) GDL was added at the completion of cooling, magnetic stirring was required for 2 min after GDL addition, the solutions were incubated for 5 h at room temperature to form gel samples. Then the gel samples were stored overnight at 4°C for subsequent analysis (Zhao et al., 2021).

Measurement of Structure and Solubility of SPI

Scanning Electron Microscopy (SEM)

The morphology characteristics of SPI was observed by scanning electron microscope (QUANTA FEG250, FEI, USA). The SPI was fixed on the holder under the action of double-sided scotch tape. The surface of the SPI was coated with a platinum film on the ion sputtering apparatus. Observed and took pictures after selecting the best field of view by adjusting the scanning electron microscope (Zhao et al., 2020).

Fourier Transform Infrared Spectroscopy (FT-IR)

The SPI and KBr were thoroughly mixed and then pressed into tablets. At 25°C, Fourier transform infrared spectrometer (Nicolet iS5, ThermoFisher, USA) was used to scan 64 times with a resolution of 4 cm^− 1 to acquire spectra for the SPI between 4000 and 400 cm^− 1. The secondary structure changes of SPI were assessed by calculating the amide I band (1700 − 1600 cm^− 1) region, and the content percentage of α-helix, β-sheet, β-turn, random coil region was obtained (Hu & Xiong, 2022).

Dynamic and Static Light Scattering (DLS and SLS)

The determination method was slightly modified according to the method of Liu et al. (2020). Firstly, 0.1 mg/mL protein solution was prepared. The supernatant was filtered after standing, and then 3.5 mL of the protein solution was injected into a clean light scattering bottle. Measurements were performed with a laser scatterometer (BI-200SM, Brookhaven Instrument Company, USA). For dynamic light scattering (DLS), the scanning angle was set to 30–120°, the time was 3 min, and the interval step was 10°. For static light scattering (SLS), the scanning angle was set to 30–120° using the enrichment method. The interval step of SLS was set to 15°. During the test, the temperature was controlled at 25°C.

Free Sulfhydryl and Disulfide Bond Content

The determination method was slightly modified according to the method of Shen et al. (2017). For the determination of free SH, 0.5 mL of 1% (w/v) SPI solution and 2.5 mL of Tris-Gly buffer solution were mixed and centrifuged at 4000 rpm (LXJ-IIB, Shanghai Anting Scientific Instrument Factory, Shanghai, China) for 15 min. For the determination of total SH, 0.5 mL of 1% (w/v) SPI solution and 2.5 mL of Tris-Gly buffer solution containing 8 M urea were mixed and centrifuged at 4000 rpm (LXJ-IIB, Shanghai Anting Scientific Instrument Factory, Shanghai, China) for 15 min. Then 0.02 mL of Ellman's reagent was added to the supernatant. The mixed samples were incubated at room temperature for 20 min, and the absorbance was measured at 412 nm. Tris-Gly buffer solution containing Ellman's reagent was used as blank control. The content of free sulfhydryl and disulfide bond was determined according to the following formulas (Eqs. (1) and (3)).

$${\text{S}\text{H}}_{\text{f}\text{r}\text{e}\text{e}} \left({\mu }\text{m}\text{o}\text{l}/\text{g}\right)=\frac{73.53\times {\text{A}}_{412}\times \text{D}}{\text{c}}$$

$${\text{S}\text{H}}_{\text{t}\text{o}\text{t}\text{a}\text{l}} \left({\mu }\text{m}\text{o}\text{l}/\text{g}\right)=\frac{73.53\times {\text{A}}_{412}\times \text{D}}{\text{c}}$$

$$\text{S}\text{S} \left({\mu }\text{m}\text{o}\text{l}/\text{g}\right)=\frac{{\text{S}\text{H}}_{\text{t}\text{o}\text{t}\text{a}\text{l}}-{\text{S}\text{H}}_{\text{f}\text{r}\text{e}\text{e}}}{2}$$

where, SH_free (µmol/g), SH_total (µmol/g), SS (µmol/g) were the concentration of free sulfhydryl, total sulfhydryl, disulfide bond. A₄₁₂ was the absorbance at 412 nm, C was the SPI solution concentration (mg/mL), D was the dilution factor, the factor 73.53 was from 10⁶⁄(1.36 × 10⁴), 1.36 × 10⁴ was the molar absorptivity constant (M^− 1 cm^− 1).

Solubility

The determination method was slightly modified according to the method of Rezaei et al. (2022). 1.0% (w/v) SPI solution was centrifuged at 4000 rpm (LXJ-IIB, Shanghai Anting Scientific Instrument Factory, Shanghai, China) for 30 min. The supernatant was collected for subsequent experiments. The protein content of the supernatant was determined by the Bradford method with BSA as the standard. The percentage of protein content in the supernatant to total SPI was defined as the solubility of protein.

Measurement of Properties of GSCG

Rheological Properties of GSCG

The rheological properties of GSCG were measured by a modular intelligent rheometer (MCR102, Anton Paar, Graz, Austria). The parallel-plate titanium alloy rotor (diameter 60 mm, gap 1 mm) was selected for frequency sweep test and creep recovery test at 25°C. According to the strain scan test, the strain was set at 1%.

The frequency sweep test was performed by taking logarithmic points in the range of 0.1–100 rad/s. The variation of storage modulus (G') and loss modulus (G") with angular frequency could be represented by a power law function (Eqs. (4) and (5)) (Bi et al., 2020).

$${G}^{{\prime }}={K}^{{\prime }}\times {\omega }^{\text{n}{\prime }}$$

$$G"=K"\times {\omega }^{\text{n}"}$$

where, K' and K" were the power law constants, n' and n" were the frequency indices, ω was the angular frequency.

A constant stress of 10 Pa was applied to the gel samples and the imposed constant stress was withdrawn after 5 min. The amount of deformation during stress application and stress withdrawal was recorded as the creep and recovery curves of the gel. The quantitative relationship between stress, strain and the creep compliance in the creep phase could be obtained by the following formula (Eq. (6)).

$$\text{J}=\frac{{\gamma }}{{\sigma }}$$

where, J was the creep compliance (1/Pa), γ was the strain, σ was the stress (Pa).

The creep compliance curve could be fitted by the Burger's model (Eq. (7)) (Wang et al., 2019).

$$\text{J}\left(\text{t}\right)=\frac{1}{{\text{G}}_{0}}+\frac{1}{{\text{G}}_{1}}\times \left(1-\text{e}\text{x}\text{p}(-\frac{\text{t}}{{\lambda }})\right)+\frac{\text{t}}{{{\mu }}_{0}}$$

where, J was the creep compliance (1/Pa), G₀ was the instantaneous elastic coefficient (Pa), G₁ was the delayed elastic coefficient (Pa), µ₀ was the viscosity coefficient related to the viscous flow (Pa·s), and λ was the hysteresis time (s).

Water Holding Capacity (WHC) of GSCG

The determination method was slightly modified according to the method of Li et al. (2020). The 10 g gel was weighed and centrifuged at 4000 rpm (LXJ-IIB, Shanghai Anting Scientific Instrument Factory, Shanghai, China) for 30 min. And the weight of th sediment was measured after inversion on the filter paper for 20 min. The WHC (%) was determined according to the following formula (Eq. (8)).

$$\text{W}\text{H}\text{C} \left(\text{%}\right)=\frac{{\text{W}}_{2}-{\text{W}}_{0}}{{\text{W}}_{1}-{\text{W}}_{0}}\times 100$$

where, W₀ was the weight of empty centrifuge tube, W₁ was the weight of centrifuge tube plus SPI gel before centrifugation, W₂ was the weight of centrifuge tube plus sediment after centrifugation.

Statistical Analysis

The graphic presentations were performed by ORIGIN 8.0 software (OriginLab Inc, USA). The statistical analysis was performed with one-way analysis of variance (ANOVA) by SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), Ducan’s test was used for the existence of differences between different samples, and differences were considered significant at p < 0.05. The results are presented as mean ± standard deviation (n = 3).

Effect of Grinding on the Structure and Solubility of SPI

Morphological Characteristics of SPI

SEM is able to measure the changes in the morphological characteristics of the SPI before and after grinding. As shown in Fig. 1, the untreated commercial SPI showed a highly uniform, smooth-surfaced spherical particle structure (Zhao et al., 2020). At 3 min of grinding, the spherical structure of some protein particles was concaved. This indicated that the molecules inside protein particles were rearranged during the grinding process. At 6 min of grinding, the spherical structure of most protein particles was destroyed, and there were a few aggregates formed between the protein fragments. After long-time grinding (> 6 min), the spherical structure of protein particles was completely destroyed, the particle size of particle fragments became smaller and more uniform. This observation suggested that the compact spherical structure of protein particle was destroyed by violent collision and friction of the grinding balls, resulting in various shapes of the protein particles after grinding (Sun et al., 2016; Li et al., 2019).

Secondary Structure of SPI

FT-IR spectroscopy could characterize the secondary structure of protein. As shown in Fig. 2, the characteristic peak intensities and positions of the amide I band (1658 cm^− 1), the amide II band (1535 cm^− 1), and the amide A band (3295 cm^− 1) of the SPI were changed after grinding. This indicated that grinding could change specific groups or chemical bonds of SPI, which resulted in changes in its secondary structure (Zhao et al., 2021; Huang et al., 2012).

Amide I band is the most dominant absorption band in the protein infrared spectroscopy, and it is very sensitive to changes in protein secondary structure (Sun et al., 2018). As shown in Table 1, with the increase in grinding time, the β-sheet content of SPI gradually decreased, while the β-turn content gradually increased. The α-helix and random coil contents had the lowest values at 6 min of grinding. At 15 min of grinding, the content of β-sheet decreased from 38.54–29.15%, while the content of β-turn increased from 37.05–42.09%. This indicated that grinding could change the secondary structure of SPI, the protein molecules became more disordered, and the intermolecular structure had greater flexibility (Li et al., 2021a; Lian et al., 2022).

Table 1

Amide I band curve fitting results and light scattering results
Time (min)	β-sheet (%)	Random (%)	α-helix (%)	β-turn (%)	Rh (nm)	Rg (nm)	ρ	Structure
0	38.54 ± 0.39^a	9.23 ± 1.04^d	15.18 ± 1.22^c	37.05 ± 0.21^c	127.26 ± 2.75^b	100.50 ± 1.98^b	0.79	solid sphere
3	34.16 ± 0.09^b	12.31 ± 0.25^a	16.05 ± 0.02^ab	37.48 ± 0.32^c	148.89 ± 1.51^a	124.55 ± 1.91^a	0.84	solid sphere
6	34.39 ± 0.19^b	10.56 ± 0.17^c	14.83 ± 0.21^c	40.22 ± 0.57^b	76.40 ± 0.26^d	88.90 ± 3.54^c	1.16	hollow sphere
9	32.58 ± 0.01^c	11.01 ± 0.02^bc	15.36 ± 0.10^ab	41.05 ± 0.07^b	86.16 ± 1.90^c	77.95 ± 0.64^d	0.90	hollow sphere
12	30.97 ± 0.44^d	11.84 ± 0.36^ab	16.18 ± 0.21^ab	41.01 ± 0.14^b	66.41 ± 0.79^e	65.75 ± 0.35^e	0.99	hollow sphere
15	29.15 ± 0.03^e	12.07 ± 0.24^ab	16.68 ± 0.20^a	42.09 ± 0.41^a	65.50 ± 2.62^e	69.50 ± 2.12^e	1.06	hollow sphere
The values with different letters in the graph indicate significant differences between different samples obtained using Duncan's method (p < 0.05)

DLS and SLS of SPI

Light scattering techniques, including DLS and SLS, are an fast and efficient method for measuring the physical properties of polymers (Feng et al., 2018; Zhao et al., 2019a). The Rg obtained by SLS refers to the actual space occupied by the polymer chain. As shown in Table 1, with the increase in grinding time, Rg of the SPI first increased and then decreased. At 3 min of grinding, Rg increased from 100.50 nm to 124.55 nm. This indicated that the original spherical dense structure of protein began to dissociate and unfold, exposed some hydrophobic groups and polar groups, and the electrostatic repulsion between the protein molecules was weak, resulting in the formation of large soluble protein polymers. At 12 min of grinding, Rg decreased to 65.75 nm. This indicated that the long-term grinding made most hydrophobic groups of the protein molecules expose, and the electrostatic repulsion increased, so only smaller protein polymers could form (Zhao et al., 2018). The Rh obtained by DLS refers to the radius of an equivalent hard sphere with an identical diffusion coefficient as the polymer chain in the solution (Wang et al., 2021). It could be seen from Table 1 that Rh of the unground SPI was 127.26 nm. At 3 min of grinding, the Rh increased to 148.89 nm, and then decreased with the increase in grinding time. The ratio of Rg/Rh (ρ) reflects the morphological changes of protein molecules in solution and is an important parameter to characterize polymers and colloids (Semenova et al., 2007; Wang et al., 2005). It could be seen from Table 1 that ρ of the unground SPI was 0.79, which was a solid sphere structure. At 3 min of grinding, the ρ became 0.84, indicating that grinding made the spherical structure unfold, and the distribution width of protein molecules increased. At 6–15 min of grinding, the ρ increased, and the structure changed to a hollow sphere. This indicated that grinding dissociated and unfolded the protein molecules, the protein molecules exposed more hydrophobic groups and polar groups and formed polymers with different ρ in aqueous solution (Liu et al., 2020).

Free Sulfhydryl and Disulfide Bonds of SPI

The changes of free sulfhydryl and disulfide bond contents are directly related to the extension and aggregation of protein molecules. Generally speaking, there are two possibilities for the increase of free sulfhydryl content, one is the breaking of disulfide bonds, and the other is that the protein structure extends. There are also two possibilities for the reduction of free sulfhydryl content, one is the aggregation of proteins, and the other is the formation of disulfide bonds (Hu et al., 2013b; Peng et al., 2016). It could be seen from Fig. 3a that the content of free sulfhydryl first decreased and then increased, but the content of disulfide bond first increased and then decreased with the increase in grinding time. At 6 min of grinding, the content of free sulfhydryl was the lowest, but the content of disulfide bond was the highest. The reason was that at 3–6 min of grinding, the free sulfhydryl on protein surface were converted into disulfide bonds (Sun et al., 2015). At 9–15 min of grinding, the disulfide bond was broken by the strong mechanical force of grinding, and the protein structure had greater flexibility (Zhu et al., 2020). At the same time, the reduction of protein particle size was also conducive to the exposure of internal free sulfhydryl, thereby increasing its content (Hu et al., 2013c).

Solubility of SPI

The solubility of protein is a prerequisite for their use in high-moisture applied foods and is a key factor in determining functional properties such as emulsifying propertie and gelling propertie (Gao et al., 2020). As shown in Fig. 3b, the solubility of protein increased to 30.64% at 3 min of grinding. The reason was that proper grinding changed the structure of protein, and formed soluble aggregates that maintains the structure by non-covalent and covalent interactions, e.g. hydrophobic and electrostatic interactions and disulfide bonds, thereby affecting the solubility of protein (Tang et al., 2009). At 6 min of grinding, the solubility of protein showed the lowest value (23.90%). The reason was that more disulfide bonds led to the formation of more insoluble protein aggregates (Hu et al., 2013b). At 9–15 min of grinding, the solubility increased due to the breakage of the disulfide bonds (Fig. 3b).

Effect of Grinding on the Rheological Properties of GSCG and its WHC

Frequency Sweep Test of GSCG

As shown in Fig. 4, the G' and G" of all GSCG increased with the increase in the angular frequency, and the G' was always greater than G", showing the typical characteristics of weak physical gels formed by globular proteins (Chihi et al., 2018). At 3 min of grinding, both G' and G" of GSCG increased. This indicated that the expanded globular structure (Table 1), the enhanced disorder in the secondary structure (Table 1) (Tan et al., 2021), and the improved solubility (Fig. 3b) were helpful for the construction of gel network structure. At 9–15 min of grinding, the Rg and Rh of protein decreased (Table 1) and the solubility increased (Fig. 3b) due to the enhanced grinding effect. At the same time, the content of α-helix and random coil in the protein secondary structure also increased (Table 1) (Gu et al., 2013; Su et al., 2015). These all helped to prepare GSCG with higher elasticity and viscosity, manifested by a significant rise in G' (Fig. 4a) and G" (Fig. 4b).

Power-law function fitting analysis was performed on the frequency dependence of G' and G" of GSCG. Table 2 shows the K' and K" of GSCG were significantly improved after grinding, which indicated that grinding could significantly improve the stability of GSCG. The n' and n" of GSCG were the largest at 15 min of grinding, which indicated that grinding for 15 min could increase the disorder of the gel network structure (Bi et al., 2014).

Table 2

Parameters of power-law function for the frequency sweep, parameters of Burger's model for the creep and WHC of the glucono-δ-lactone-induced gels (GSCG) prepared by SPI with different grinding time
Time (min)	K'	n' (×10^− 2)	K"	n" (×10^− 2)	G₀ (Pa)	G₁ (Pa)	µ₀ ( Pa·s)	R²	WHC (%)
0	345.05 ± 21.74^d	8.55 ± 0.35^a	75.66 ± 4.49^e	6.65 ± 0.78^b	222.16 ± 2.97^e	270.15 ± 4.43^d	58663 ± 1928^c	0.995	71.64 ± 0.69^e
3	477.92 ± 17.75^c	9.56 ± 0.07^ab	111.32 ± 1.51^c	7.80 ± 0.04^ab	323.61 ± 27.53^c	335.18 ± 13.68^c	70858 ± 1387^b	0.994	74.57 ± 0.19^c
6	389.06 ± 19.23^d	8.07 ± 0.74^a	83.59 ± 5.30^e	6.30 ± 0.06^b	241.42 ± 3.44^e	274.75 ± 5.67^d	60781 ± 2309^c	0.993	73.49 ± 0.14^d
9	437.18 ± 20.90^c	8.45 ± 0.92^a	96.06 ± 7.18^d	6.45 ± 0.35^b	278.08 ± 10.49^d	318.61 ± 10.07^c	62779 ± 3203^c	0.993	75.07 ± 0.10^bc
12	525.37 ± 24.44^b	10.13 ± 0.83^b	122.90 ± 6.72^b	8.30 ± 0.82^a	368.15 ± 9.89^b	388.09 ± 12.59^b	77783 ± 2849^b	0.993	75.51 ± 0.25^b
15	573.77 ± 16.77^a	10.65 ± 0.50^b	134.49 ± 2.31^a	9.05 ± 0.64^a	436.05 ± 16.86^a	471.28 ± 24.62^a	85830 ± 5397^a	0.995	76.37 ± 0.30^a
The values with different letters in the graph indicate significant differences between different samples obtained using Duncan's method (p < 0.05)

Creep Recovery Test of GSCG

Figure 4c shows that in the initial phase, the strain value instantly increased to a certain value, because this phase was dominated by elasticity. The strain rate was then slowed down due to viscoelastic effects. After about 150 s, the strain curve increased in linear, and viscous flow dominated in this phase. Due to some irreversible strain of the viscous flow, the strain could not be fully recovered after the shear stress was removed (Bi et al., 2020). The rigidity of gel could be expressed in terms of the value of maximum creep strain (Bi et al., 2014). As shown in Fig. 4c, the value of maximum creep strain of the GSCG became smaller after grinding, which indicated that grinding helped to enhance the rigidity of the gel.

The creep compliance shown in Fig. 4d was analyzed by Burger model fitting to further characterize the viscoelasticity of the gel samples. It could be seen from Table 2 that G₀ and G₁ of the GSCG increased after grinding, which indicated that grinding could improve the cohesive force and deformation resistance of the gel (Wang et al., 2019). As shown in Table 2, an inflection point occurred at 6 min of grinding. The reason was that less disorder of the protein (Table 1), decreased solubility (Fig. 3b), and increased insoluble aggregates (Fig. 3a) led to the formation of a rougher network structure in the gels (Malik & Saini, 2017). At 15 min of grinding, the G₀ and G₁ of GSCG were the largest. The reason was that the SPI ground for 15 min had a more disordered secondary structure (Table 1) and a less compact tertiary structure (Fig. 3a), which were beneficial to expose hydrophobic groups to build the gel network structure. At the same time, the reduction in particle size (Fig. 1) and the increase in solubility of protein (Fig. 3b) also contributed to the construction of a dense network structure. The viscous behavior of gel could be reflected by the viscosity coefficient (µ₀). It could be seen from Table 2 that the µ₀ of GSCG increased after grinding, indicating that the fluidity of water in the gel decreased and the flow resistance increased (Gaspar & de Góes-Favoni, 2015).

WHC of GSCG

WHC refers to the ability to capture and fix water by capillary action of the matrix in the gel network structure, which is an important index to evaluate the gel properties (Zhao et al., 2019b; Ma et al., 2022).

As shown in Table 2, the WHC of GSCG was enhanced after grinding, which further proved that grinding could make the SPI form a denser network structure in the GSCG (Li et al., 2022). At 6 min of grinding, the WHC decreased because the increase of insoluble aggregates made the prepared gel network structure not dense (Liang et al., 2021). This was consistent with the analysis of the rheological properties of the gel.

SEM showed that the continuous grinding could make the internal molecules of SPI constantly move and rearrange. FT-IR spectroscopy showed that grinding increased the disorder of protein molecules, and the SPI had a more flexible secondary structure. At 3 min of grinding, Rg and Rh of the protein molecules increased. At 6 min of grinding, the SPI changed from a solid sphere to a hollow sphere. Prolonged grinding (9-15 min) made the disulfide bonds break, and the SPI had a more loose tertiary structure. These changes contributed to the formation of dense network structures in the GSCG, and improved the WHC of SPI gel. It could be seen that moderate grinding treatment of SPI was helpful to improve the WHC and rheological properties of GSCG. This study could lay a theoretical foundation for research on improving the gel properties of SPI by mechanical modification in future.

Author Contribution Yue Liu: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing. Yangyong Dai: Supervision, Reading of draft and final manuscript, Funding acquisition. Jian Cao: Supervision, Reading of draft and final manuscript, Conceptualization. Hanxue Hou: Supervision, Reading of draft and final manuscript. Wentao Wang: Visualization, Writing - review & editing. Hui Zhang: Conceptualization, Data curation. Xiuzhen Ding: Visualization, Writing - review & editing. Cheng Li: Conceptualization, Data curation.

Funding This study was funded by the National Natural Science Foundation of China (No. 32272268) and Natural Science Foundation of Shandong Province (No. ZR2022MC185).

Data Availability The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of Interest The authors declare no competing interests.

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Analysis of influence mechanism of grinding on the gel properties of soybean protein isolate based on the changes of its structure and solubility

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Abstract

Figures

Introduction

Materials And Methods

Materials

Grinding SPI

Preparation of the GDL-induced SPI Cold-set Gel (GSCG)

Measurement of Structure and Solubility of SPI

Scanning Electron Microscopy (SEM)

Fourier Transform Infrared Spectroscopy (FT-IR)

Dynamic and Static Light Scattering (DLS and SLS)

Free Sulfhydryl and Disulfide Bond Content

Solubility

Measurement of Properties of GSCG

Rheological Properties of GSCG

Water Holding Capacity (WHC) of GSCG

Statistical Analysis

Results And Discussion

Effect of Grinding on the Structure and Solubility of SPI

Morphological Characteristics of SPI

Secondary Structure of SPI

DLS and SLS of SPI

Free Sulfhydryl and Disulfide Bonds of SPI

Solubility of SPI

Effect of Grinding on the Rheological Properties of GSCG and its WHC

Frequency Sweep Test of GSCG

Creep Recovery Test of GSCG

WHC of GSCG

Conclusion

Declarations

References

Additional Declarations

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