Method for preparing soybean protein isolate gel from amino acid compound TG enzyme

Abstract

The invention provides a method for preparing soybean protein isolate gel by using amino acid composite TG enzyme. Preparing a protein solution, and stirring at room temperature; 40 U / g TG enzyme is added and fully mixed with the protein solution, then acidic amino acid with the concentration of 0.2%-1.0% (w / v) is added, the mixture is subjected to water bath for 1 h at the temperature of 40 DEG C and heated for 30 min in the water bath at the temperature of 95 DEG C, and the mixture is taken out, cooled in an ice-water bath for 10 min and stored overnight at the temperature of 4 DEG C. The specific amino acid is added into a soybean protein isolate system, and the cross-linking effect of the TG enzyme is combined, so that the elasticity and the hardness of the gel are remarkably enhanced, the water-retaining property and the water-holding capacity of the gel are improved, and the gel strength of the TG enzyme cross-linked soybean protein isolate gel is improved, so that the quality of the plant protein gel is improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and particularly relates to the field of plant protein gel preparation technology. Specifically, it relates to a method for preparing soybean protein isolate gel with high gelation and water-holding properties using a complex TG enzyme with different amino acids. Background Technology

[0002] In the food industry, soy protein isolate has become an important plant protein resource due to its rich nutritional components and diverse functional properties. Furthermore, as a sustainable plant protein source, soy protein isolate can effectively replace animal protein, reducing cholesterol and saturated fatty acid intake while having a smaller environmental impact, thus meeting the requirements of sustainable development. These advantages enable soy protein isolate to not only meet the food industry's demand for high-quality protein but also provide important support for promoting the development of healthy foods and building a sustainable food system. However, traditional soy protein isolate gels still have shortcomings in terms of texture, elasticity, and water retention, limiting their application in food processing.

[0003] Transglutaminase (TG enzyme) is an enzyme that catalyzes acyl transfer reactions. It efficiently catalyzes these reactions, a process crucial for protein cross-linking and modification. During catalysis, TG enzyme uses the γ-carboxamide group of glutamine residues in proteins and peptide bonds as an acyl donor. Through reaction with an acyl acceptor, covalent bonds are formed, thereby achieving cross-linking between protein molecules or the binding of proteins to other molecules. TG enzyme cross-linking can significantly enhance the hardness, elasticity, and water-holding capacity of soy protein isolate gels. Although TG enzyme can form a gel network structure during cross-linking, the uniformity and stability of this structure still require further optimization.

[0004] The addition of amino acids has a significant impact on the structure and functional properties of proteins. Through interactions with protein molecules, amino acids can alter the secondary structure and surface properties of proteins, thereby affecting their gelation properties. The side-chain characteristics of specific amino acids (such as hydrophobicity, charge, and hydrogen bond formation ability) directly influence the three-dimensional structure and stability of proteins.

[0005] However, current research on the effects of amino acids on the gel properties of TG enzyme-crosslinked soy protein isolate is still limited. Therefore, developing a method to enhance the gel properties of TG enzyme-crosslinked soy protein isolate through different amino acids has significant scientific and practical value. Summary of the Invention

[0006] This invention aims to provide a method for preparing novel soy protein isolate gels using an acidic amino acid complex TG enzyme, which effectively improves the gelation and water-holding properties of the gel. By adding aspartic acid and glutamic acid, the strength and elasticity of the gel are significantly improved, and its water-holding capacity is enhanced. The objective of this invention is achieved through the following technical solutions:

[0007] A method for preparing soybean protein isolate gel using an amino acid complex TG enzyme includes the following steps:

[0008] Step 1: Prepare a protein solution with a mass-volume ratio (w / v) of 11.5% to 12.5% ​​and stir at room temperature for 2 hours to fully dissolve the soy protein isolate and form a homogeneous solution.

[0009] Step 2: Add 40 U / g TG enzyme to the protein solution and stir to mix it thoroughly with the protein solution; so that the TG enzyme is evenly dispersed in the solution system;

[0010] Step 3: Add acidic amino acids to the mixture at a concentration of 0.2% to 1.0% (w / v); the acidic amino acids are aspartic acid or glutamic acid; after adding the amino acids, stir again to ensure that the amino acids are fully and evenly mixed with the solution;

[0011] Step 4: Seal the solution after adding amino acids and react it in a 40°C water bath for 1 hour; keep the water bath temperature stable during this process.

[0012] Step 5: Transfer the solution after the first water bath reaction to another water bath at 95°C, and heat it in a 95°C water bath for 30 minutes to further promote gel formation and cross-linking reaction.

[0013] Step 6: After heating is complete, remove the gel and cool it in an ice water bath for 10-15 minutes to allow the gel to cool and set rapidly.

[0014] Step 7: Finally, store at 4°C overnight to further stabilize and optimize the gel structure, obtaining soy protein isolate gel.

[0015] Further optimization involves using a soy protein isolate solution with a w / v ratio of 12%.

[0016] Further optimization involved adding aspartic acid as the acidic amino acid in step 3, at a concentration of 0.4% to 0.6%.

[0017] Further optimization involved adding glutamic acid as the acidic amino acid in step 3, at a concentration of 0.4%.

[0018] Furthermore, in step 1, the stirring speed at room temperature is 500 rpm / min.

[0019] Furthermore, in step 2, after adding the TG enzyme, the method of thorough mixing is stirring for 30 minutes.

[0020] Furthermore, in step 4, during the reaction in a 40°C water bath for 1 hour, the solution in the water bath container is stirred at a speed of 500 rpm / min.

[0021] Furthermore, in step 5, during the 30-minute heating process in a 95°C water bath, the power of the heating equipment is 800W.

[0022] Furthermore, in step 6, during the ice-water bath cooling, the mass ratio of ice to water is 1:2.

[0023] Mechanism of Action Elucidated: This invention, through a systematic study of the effects of different amino acids on gel properties, reveals the interaction mechanism between amino acids, soy protein isolate, and TG enzyme. Previous experimental studies have shown that the effect of basic amino acids on gel strength is also related to the type and concentration of amino acids, as well as the initial properties of the protein. Under certain conditions, the addition of basic amino acids weakens the electrostatic interactions between protein molecules, thereby reducing gel strength. Acidic amino acids carry a negative charge; their addition increases the negative charge density on the surface of soy protein isolate molecules, thereby enhancing the electrostatic repulsion between protein molecules. In this case, the interaction between protein molecules and water molecules is enhanced, resulting in more bound water and relatively less free water in the gel. The secondary structure of SPI is mainly maintained by β-sheet structures. When aspartic acid and glutamic acid are added, the content of β-sheet structures in soy protein isolate molecules increases significantly. This structural change alters the interactions between proteins, promoting the cross-linking reaction of soy protein isolate by TG enzyme. The increase in β-sheet structures makes the arrangement of soy protein isolate molecules more compact, thus providing more cross-linking sites for TG enzyme and enhancing the stability and strength of the gel network. This discovery provides an important theoretical basis for further optimizing gel preparation processes and helps to develop gel products with better performance.

[0024] In summary, the addition of acidic amino acids alters the microstructure of soy protein isolate gel, particularly increasing the content of β-sheet structures and decreasing the content of α-helical and β-turn structures, thereby promoting the cross-linking reaction of soy protein isolate by TG enzymes. Simultaneously, acidic amino acids influence water distribution by regulating the electrostatic interactions between protein molecules, resulting in increased bound water content and decreased free water content in the soy protein isolate gel. This further optimizes the gel's microstructure and provides important technical support and theoretical basis for the widespread application of soy protein isolate gel in various fields.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects:

[0026] 1) Improved gel strength: By adding specific amino acids, the hardness, elasticity, and other strength indicators of the gel were significantly improved. Compared with the control group without added amino acids, the addition of aspartic acid at a concentration of 0.6% (w / v) increased the gel hardness by 10.67 N and the elasticity by 7.37 mm, better meeting the requirements for gel strength in food processing and material applications.

[0027] 2) Enhanced water-holding capacity: Adding specific concentrations of aspartic acid and glutamic acid significantly improved the water-holding capacity of the gel. When aspartic acid was added at a concentration of 0.4% (w / v), the water-holding capacity of the gel increased to 100% compared to the control group. Similarly, adding 0.4% glutamic acid also increased the water-holding capacity to 100%. This result indicates that the addition of both amino acids effectively improves the water-holding properties of the gel, reducing moisture loss during storage and use. This improved water-holding capacity not only extends the product's shelf life but also enhances the gel's texture and quality. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 Microstructure of soy protein isolate gel prepared by combining TG enzyme at optimal concentrations of aspartic acid and glutamic acid. Electron beam acceleration voltage 5.00 kV, magnification 500x, demonstrates the effect of different amino acid concentrations on the microstructure of the soy protein isolate gel. By comparing the scanning electron microscope images of Examples 3, 7, and Comparative Example 1, the significant changes in the microstructure of the soy protein isolate gel due to the addition of different amino acids can be visually observed.

[0030] Figure 2 MRI images of soy protein isolate gels prepared with a combined TG enzyme at the optimal concentrations of aspartic acid and glutamic acid. At 3.76x magnification, the differences in MRI images of soy protein isolate gels prepared with the addition of specific amino acids are shown. By comparing the MRI images of Examples 3, 7, and Comparative Example 1, the significant effects of the addition of different amino acids on the internal structure and moisture distribution of the soy protein isolate gels can be clearly observed.

[0031] Figure 3 Relaxation curve analysis of soy protein isolate gel prepared by a compound TG enzyme at optimal concentrations of aspartic acid and glutamic acid. The measurements were performed using a low-frequency nuclear magnetic resonance (NMR) analyzer and CPMG pulse sequence analysis. The relaxation time range was 0.01 ms to 10000 ms.

[0032] Figure 4 Analysis of the moisture distribution of soy protein isolate gels prepared by a compound TG enzyme at the optimal concentrations of aspartic acid and glutamic acid. In the figure, aspartic acid is from Example 3, and glutamic acid is from Example 7.

[0033] Figure 5 The Zeta potential of soy protein isolate gels prepared using a combined TG enzyme at the optimal concentrations of aspartic acid and glutamic acid is shown in the figure. Aspartic acid is from Example 3, and glutamic acid is from Example 7. Different lowercase letters indicate significant differences (P<0.05) between groups with different types of amino acids added. The same applies below.

[0034] Figure 6 The particle size of soy protein isolate gel prepared by compound TG enzyme at the optimal addition concentrations of aspartic acid and glutamic acid.

[0035] Figure 7 Surface hydrophobicity analysis of soy protein isolate gel prepared by compound TG enzyme at the optimal addition concentrations of aspartic acid and glutamic acid.

[0036] Figure 8 Rheological properties analysis of soy protein isolate gel prepared by compound TG enzyme at optimal concentrations of aspartic acid and glutamic acid. Solid symbol represents G', hollow symbol represents G".

[0037] Figure 9 Fourier transform infrared (FTIR) spectra of soybean protein isolate gels prepared with a compound TG enzyme at optimal concentrations of aspartic acid and glutamic acid were obtained. The spectrum was scanned 32 times in the range of 400–4000 cm⁻¹ using a Fourier transform infrared spectrometer with a resolution of 4 cm⁻¹. Peak fitting of the amide I band in the 1700–1600 cm⁻¹ range was performed using Omnic data acquisition software and Peakfit 4.12 peak processing software to identify the correspondence between the positions of each sub-peak and secondary structure components. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments.

[0039] Example 1

[0040] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme, the method comprising the following steps:

[0041] Step 1: Dissolve soy protein isolate in water to prepare a 12% (w / v) solution and stir at 500 rpm / min for 2 hours at room temperature;

[0042] Step 2: Add 40 U / g TG enzyme to the above solution and stir for 30 min to ensure that the TG enzyme is fully mixed with the protein solution;

[0043] Step 3: Add aspartic acid to the protein solution to a concentration of 0.2% (w / v) and stir thoroughly;

[0044] Step 4: Dispense the obtained solution into 50mL beakers, seal them with plastic wrap, and place them in a 40℃ water bath for 1 hour; the solution in the water bath should be stirred at a speed of 500rpm / min.

[0045] Step 5: Transfer the beaker to a 95°C water bath and heat for 30 minutes; the heating device should have a power of 800W.

[0046] Step 6: Remove the beaker and place it in an ice-water bath to cool for 15 minutes. The mass ratio of ice to water should be 1:2.

[0047] Step 7: Then store overnight at 4°C to obtain aspartic acid-crosslinked TG enzyme soy protein isolate gel.

[0048] The gel prepared in this embodiment was subjected to performance testing, and the gel strength was 3.90 g / cm³. 2 It has a water holding capacity of 99.95%, a hardness of 7.92N, and an elasticity of 4.98mm.

[0049] Example 2

[0050] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0051] Step 3: Add aspartic acid to the protein solution to a concentration of 0.4% (w / v) and stir thoroughly. The gel prepared in this example was tested for performance; the gel strength was 8.59 g / cm³. 2 It has a water holding capacity of 100.00%, a hardness of 10.09N, and an elasticity of 6.50mm.

[0052] Example 3

[0053] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0054] Step 3: Add aspartic acid to the protein solution to achieve a concentration of 0.6% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 10.67 g / cm³. 2It has a water holding capacity of 99.92%, a hardness of 14.07 N, and an elasticity of 7.37 mm.

[0055] Example 4

[0056] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0057] Step 3: Add aspartic acid to the protein solution to achieve a concentration of 0.8% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 5.25 g / cm³. 2 It has a water holding capacity of 99.82%, a hardness of 13.80N, and an elasticity of 5.75mm.

[0058] Example 5

[0059] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0060] Step 3: Add aspartic acid to the protein solution to achieve a concentration of 1.0% (w / v) and stir thoroughly. The gel prepared in this example was tested for performance; the gel strength was 4.89 g / cm³. 2 It has a water holding capacity of 99.91%, a hardness of 13.62N, and an elasticity of 5.30mm.

[0061] Example 6

[0062] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0063] Step 3: Add glutamic acid to the protein solution to a concentration of 0.2% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.60 g / cm³. 2 It has a water holding capacity of 99.96%, a hardness of 5.99N, and an elasticity of 4.92mm.

[0064] Example 7

[0065] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0066] Step 3: Add glutamic acid to the protein solution to a concentration of 0.4% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 3.12 g / cm³.2 It has a water holding capacity of 100.00%, a hardness of 6.32N, and an elasticity of 4.86mm.

[0067] Example 8

[0068] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0069] Step 3: Add glutamic acid to the protein solution to a concentration of 0.6% (w / v) and stir thoroughly. The gel prepared in this example was then tested for performance; the gel strength was 2.10 g / cm³. 2 It has a water holding capacity of 92.91%, a hardness of 6.11 N, and an elasticity of 3.30 mm.

[0070] Example 9

[0071] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0072] Step 3: Add glutamic acid to the protein solution to a concentration of 0.8% (w / v) and stir thoroughly. The gel prepared in this example was then tested for performance; the gel strength was 1.96 g / cm³. 2 It has a water holding capacity of 99.27%, a hardness of 6.15N, and an elasticity of 3.58mm.

[0073] Example 10

[0074] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0075] Step 3: Add glutamic acid to the protein solution to a concentration of 1.0% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.86 g / cm³. 2 It has a water holding capacity of 92.53%, a hardness of 6.19 N, and an elasticity of 3.53 mm.

[0076] Comparative Example 1

[0077] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0078] Step 3: Do not add amino acids to the protein solution.

[0079] The gel prepared in this embodiment was subjected to performance testing, and the gel strength was 1.56 g / cm³. 2 It has a water holding capacity of 89.60%, a hardness of 4.18N, and an elasticity of 3.00mm.

[0080] Comparative Example 2

[0081] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0082] Step 3: Add arginine to the protein solution to achieve a concentration of 0.2% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.99 g / cm³. 2 It has a water holding capacity of 80.06%, a hardness of 2.76N, and an elasticity of 0.81mm.

[0083] Comparative Example 3

[0084] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0085] Step 3: Add arginine to the protein solution to achieve a concentration of 0.4% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.88 g / cm³. 2 It has a water holding capacity of 80.51%, a hardness of 3.61N, and an elasticity of 1.49mm.

[0086] Comparative Example 4

[0087] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0088] Step 3: Add arginine to the protein solution to achieve a concentration of 0.6% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.67 g / cm³. 2 It has a water holding capacity of 96.64%, a hardness of 3.50N, and an elasticity of 1.60mm.

[0089] Comparative Example 5

[0090] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0091] Step 3: Add arginine to the protein solution to achieve a concentration of 0.8% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.73 g / cm³. 2 It has a water holding capacity of 93.84%, a hardness of 3.60N, and an elasticity of 2.03mm.

[0092] Comparative Example 6

[0093] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0094] Step 3: Add arginine to the protein solution to achieve a concentration of 1.0% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.95 g / cm³. 2 It has a water holding capacity of 97.86%, a hardness of 3.43N, and an elasticity of 2.13mm.

[0095] Comparative Example 7

[0096] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0097] Step 3: Add lysine to the protein solution to a concentration of 0.2% (w / v) and stir thoroughly. The gel prepared in this example was then tested for performance; the gel strength was 0.98 g / cm³. 2 It has a water holding capacity of 98.94%, a hardness of 2.71N, and an elasticity of 0.92mm.

[0098] Comparative Example 8

[0099] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0100] Step 3: Add lysine to the protein solution to a concentration of 0.4% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.90 g / cm³. 2 It has a water holding capacity of 95.97%, a hardness of 3.10N, and an elasticity of 1.28mm.

[0101] Comparative Example 9

[0102] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0103] Step 3: Add lysine to the protein solution to achieve a concentration of 0.6% (w / v) and stir thoroughly. The gel prepared in this example was then tested for performance; the gel strength was 0.85 g / cm³. 2 It has a water holding capacity of 97.80%, a hardness of 3.21N, and an elasticity of 0.77mm.

[0104] Comparative Example 10

[0105] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0106] Step 3: Add lysine to the protein solution to achieve a concentration of 0.8% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 0.84 g / cm³. 2 It has a water holding capacity of 98.31%, a hardness of 3.27 N, and an elasticity of 1.33 mm.

[0107] Comparative Example 11

[0108] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0109] Step 3: Add lysine to the protein solution to a concentration of 1.0% (w / v) and stir thoroughly. The gel prepared in this example was then tested for performance; the gel strength was 0.82 g / cm³. 2 It has a water holding capacity of 85.32%, a hardness of 2.24 N, and an elasticity of 1.01 mm.

[0110] Comparative Example 12

[0111] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0112] Step 3: Add histidine to the protein solution to achieve a concentration of 0.2% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.31 g / cm³. 2 It has a water holding capacity of 90.99%, a hardness of 3.25N, and an elasticity of 2.93mm.

[0113] Comparative Example 13

[0114] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0115] Step 3: Add histidine to the protein solution to achieve a concentration of 0.4% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.27 g / cm³. 2 It has a water holding capacity of 91.61%, a hardness of 3.52N, and an elasticity of 2.73mm.

[0116] Comparative Example 14

[0117] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0118] Step 3: Add histidine to the protein solution to achieve a concentration of 0.6% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.46 g / cm³. 2 It has a water holding capacity of 94.09%, a hardness of 3.25N, and an elasticity of 1.15mm.

[0119] Comparative Example 15

[0120] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0121] Step 3: Add histidine to the protein solution to achieve a concentration of 0.8% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.33 g / cm³. 2 It has a water holding capacity of 92.93%, a hardness of 3.38N, and an elasticity of 2.20mm.

[0122] Comparative Example 16

[0123] This embodiment provides a method for preparing soybean protein isolate gel using an amino acid complex TG enzyme. The raw materials used are the same as in Example 1, but the preparation method differs from that in Example 1 in the following ways:

[0124] Step 3: Add histidine to the protein solution to achieve a concentration of 1.0% (w / v) and stir thoroughly. The gel prepared in this example was then subjected to performance testing; the gel strength was 1.53 g / cm³. 2 It has a water holding capacity of 94.73%, a hardness of 3.62N, and an elasticity of 2.47mm.

[0125] Examples, comparative examples, results and analysis:

[0126] As shown in Tables 1, 2, and 3, the addition of aspartic acid and glutamic acid effectively enhances the structural stability of TG enzyme-crosslinked soy protein isolate gels and promotes gel network formation. The soy protein isolate gel with added aspartic acid showed significant differences compared to other experimental groups. Example 3 exhibited the best gel strength among all gels, indicating that Example 3 (SPI gel with added aspartic acid) has higher crosslinking efficiency and stronger network formation ability in the gel system. This high-strength gel structure not only better retains moisture but also maintains a stable morphology under external pressure, exhibiting good mechanical properties. Similarly, Example 7 (gel with added glutamic acid) also showed some improvement in gel texture. The water retention rates of Examples 2 and 7 both reached 100%, indicating that these two amino acids have good water retention capabilities in the gel system, showing a significant difference compared to Control Group 1 (untreated SPI gel sample).

[0127] like Figure 1 The microstructural features of Comparative Example 1, as well as Examples 3 and 7, are shown in the figure. Using scanning electron microscopy (SEM), the differences in the three-dimensional network gel structures formed after cross-linking with TG enzyme in different samples can be clearly observed. Comparative Example 1 exhibits large pores in its microstructure, with a rough surface lacking smoothness and uniformity. The presence of these pores makes the entire gel structure relatively loose, lacking tight network connections. Example 3 shows a significant reduction in surface pores, a denser structure, and a smooth, uniform, and dense surface, exhibiting good microscopic uniformity. These changes indicate that the addition of aspartic acid effectively promotes the unfolding of the protein structure, exposing more active groups on the protein surface. This structural change enhances the surface hydrophobicity of the gel, thus providing more favorable conditions for the cross-linking reaction of TG enzyme. Under the catalysis of TG enzyme, the cross-linking between protein molecules is more complete, ultimately forming a denser and more stable gel structure. While Example 7 shows a reduction in surface pores and a denser structure, the gel surface remains relatively rough, lacking smoothness and uniformity.

[0128] like Figure 2As shown, the gel samples of Example 3, Example 7, and Comparative Example 1 were analyzed by low-field nuclear magnetic resonance (LF-NMR) and nuclear magnetic resonance imaging (MRI). The MRI of Example 3 shows the internal structure and moisture distribution of the soy protein isolate gel prepared with the addition of 0.6% (w / v) aspartic acid, indicating that the addition of amino acids effectively improved the uniformity and stability of the gel. The MRI of Example 7 shows the internal structure and moisture distribution of the soy protein isolate gel prepared with the addition of 0.4% (w / v) glutamic acid. Compared with Example 7, the gel of Example 3 has a finer internal structure and a more uniform moisture distribution. The MRI image of Comparative Example 1 shows the internal structure of the soy protein isolate gel without added amino acids. It can be seen from the image that the internal structure of the gel is relatively loose and the moisture distribution is uneven, indicating that the stability and uniformity of the gel are poor without the participation of amino acids.

[0129] like Figure 3 , Figure 4 As shown, four peaks appeared in the relaxation time distribution from 0.01 to 10000 ms, corresponding to three states of water distribution in soy protein isolate gel: bound water, stagnant water (water that is not easily moved), and free water. The proportion of bound water in the soy protein isolate gel without added amino acids was relatively small, at 4%. This proportion increased with the addition of acidic amino acids. When the protein gel strength increased, the relaxation curve showed an increase in bound water content and a decrease in free water content, indicating a tighter binding between water and protein. Simultaneously, the water distribution diagram showed a denser gel network structure, with water more effectively fixed within the protein network. This structural change not only enhanced the mechanical strength of the gel but also improved its water-holding capacity, further illustrating the synergistic effect of enhanced intermolecular protein interactions and water distribution. Free water accounted for the largest proportion; this portion corresponds to water in the soy protein isolate gel that is not bound to the gel network structure. Most of this water adheres to the gel surface, has high fluidity, and is easily lost. This proportion significantly decreased with the addition of acidic amino acids. The "retained water" portion corresponds to the part of the soy protein isolate gel trapped by the gel network structure. In this portion, protein and water molecules are bound together with relatively low bond energies, resulting in a low degree of binding. In soy protein isolate gel without added amino acids, this portion accounts for only 1%, while in soy protein isolate gel prepared with 0.6% (w / v) aspartic acid and 0.4% (w / v) glutamate combined with TG enzyme, it accounts for 36% and 35%, respectively. Combined with the water-holding capacity experiment results, the addition of acidic amino acids is beneficial for forming a stable protein gel network, improving the water-retaining capacity of the soy protein isolate gel. Consequently, the water content remaining in the gel network structure increases, enhancing the water-holding capacity.

[0130] like Figure 5 , Figure 6 , Figure 7 The image shows the Zeta potential, particle size, and surface hydrophobicity analysis of soy protein isolate gels prepared with a combined TG enzyme at the optimal concentrations of aspartic acid and glutamic acid. All samples showed negative Zeta potentials. With the addition of acidic amino acids at the optimal concentrations, the absolute values ​​of the Zeta potentials increased, indicating improved stability of the soy protein isolate gel system. When the aspartic acid content was 0.6% (w / v), the absolute value of the gel potential significantly increased to 13.80, the charge density on the gel surface decreased, and the electrostatic repulsion weakened. This may be due to the decreased surface hydrophobicity of the protein, increased intermolecular aggregation tendency, resulting in more hydrophobic groups being encapsulated within the molecule. Furthermore, the decrease in Zeta potential may be related to the refolding of the protein structure, which hides hydrophobic regions, further reducing surface hydrophobicity. Meanwhile, from... Figure 6 It can be seen that the addition of 0.6% (w / v) aspartic acid can increase the particle size, indicating that the cross-linking between protein molecules is enhanced, thereby improving the stability of soy protein isolate gel.

[0131] Depend on Figure 8 The rheological properties of the TG enzyme-crosslinked soy protein isolate gels prepared in Examples 3, 7, and Comparative Example 1 were analyzed. The results showed that, across the entire frequency range, the storage modulus (elastic modulus) G' of all gel samples was greater than the loss modulus (viscous modulus) G”, indicating that these gels possess strong elastic characteristics, exhibit gel-like behavior, and all gels exhibited a near-solid state. Furthermore, both G' and G” of the prepared gels increased with increasing frequency, showing a strong frequency dependence, indicating that the gel system prepared in this experiment belongs to a "weak gel" system. When comparing Examples 3 and 7, the gel of Example 3 was found to exhibit higher elasticity.

[0132] Depend on Figure 9 The content of different secondary structures in each protein gel sample was determined by Fourier transform infrared spectroscopy (FT-IR). With the addition of aspartic acid and glutamic acid, the β-sheet structure in the sample significantly increased, while the content of α-helices, β-turns, and random coil structures decreased accordingly. This structural change is beneficial for improving the gel performance and water-holding capacity of soy protein isolate. In the amide I band of the protein secondary structure, 1650–1660 cm⁻¹ represents α-helical structures, 1600–1640 cm⁻¹ represents β-sheet structures, 1660–1670 cm⁻¹ represents β-turn structures, and 1640–1650 cm⁻¹ represents random coil structures. Based on this, the relative content of each part of the secondary structure in the sample can be calculated, as shown in Table 6. Among them, α-helices and β-sheets are tightly ordered structures, β-turns are relatively loosely ordered structures, and random coils are loosely disordered structures. Figure 6It was found that the addition of 0.6% (w / v) aspartic acid and 0.4% (w / v) glutamic acid caused the amide I band of soy protein isolate to shift blue from 1641.82 cm⁻¹ to 1633.88 cm⁻¹ and 1639.77 cm⁻¹, respectively. In Example 3, the amide II band shifted blue from 1537.01 cm⁻¹ to 1529.07 cm⁻¹, and in Example 7, it shifted red to 1538.20 cm⁻¹. These intensity changes and spectral shifts in these regions are helpful in determining the interaction between amino acids and soy protein isolate, indicating that the addition of amino acids altered the secondary structure of soy protein isolate. This may be due to the binding of different amino acids to the C=O, CN, and NH groups of soy protein isolate through hydrogen bonds and hydrophobic interactions. The characteristic absorption peaks of the amide A region are mainly related to the hydrogen bonds of the polypeptide backbone, exhibiting stretching vibrations of N-H and O-H bonds. The addition of 0.6% (w / v) aspartic acid and 0.4% (w / v) glutamic acid resulted in a blue shift of the absorption peaks from 3285.39 cm⁻¹ to 3279.05 cm⁻¹ and 3284.19 cm⁻¹, respectively, due to a decrease in the number or strength of hydrogen bonds in the protein molecules. This indicates that the addition of amino acids led to a conformational rearrangement of the protein molecules. This conformational change may enhance the intermolecular interactions of protein molecules, thereby promoting protein aggregation and cross-linking, forming a tighter gel network. At the molecular level, the stability of the α-helix conformation mainly depends on the intermolecular hydrogen bonds formed between the carbonyl oxygen (CO) and amino hydrogen (NH). Therefore, the addition of aspartic acid may have promoted the formation of these hydrogen bonds, thereby enhancing the stability of the protein structure. Further comparative analysis showed that the sample with added glutamic acid had a more significant relative increase in β-sheet content compared to the sample with added aspartic acid. This phenomenon suggests that the addition of glutamic acid may disrupt the original rigid structure of soy protein isolate gel to a greater extent, increasing its flexibility and causing the protein structure to stretch, transforming the ordered molecular structure into a disordered one. This structural unfolding leads to an increase in the β-sheet content of amino acids participating in the formation of soy protein isolate gel, which in turn affects the gel strength, making the gel network more compact and thus improving the mechanical properties and water-holding capacity of soy protein isolate gel.

[0133] Table 1

[0134]

[0135]

[0136] Note: Different lowercase letters indicate significant differences between groups with different types of amino acids added (P < 0.05).

[0137] Table 2

[0138]

[0139] Note: Different lowercase letters indicate significant differences (P<0.05) between groups with different types of amino acids added. Table 3

[0140]

[0141] Note: Different lowercase letters indicate significant differences between groups with different types of amino acids added (P<0.05).

[0142] Table 4

[0143]

[0144] Table 5

[0145]

[0146] Note: Different lowercase letters indicate significant differences between groups with different types of amino acids added (P<0.05).

[0147] The content of this invention is not limited to the embodiments exemplified in the examples. Any conventional modifications and equivalent substitutions made by those skilled in the art to the technical solutions of this invention by reading this specification are within the scope of the specific embodiments of this invention.

Claims

1. A method for preparing soy protein isolate gel using amino acid composite TG enzyme, characterized in that: The steps include: Step 1, prepare a protein solution with a mass volume ratio of 11.5% to 12.5%, and stir at room temperature for 2 h; Step 2, adding 40 U / g TG enzyme to the protein solution and mixing it with the protein solution thoroughly; Step 3, adding an acidic amino acid to the mixed solution at a concentration of 0.2% to 1.0%; the acidic amino acid is aspartic acid or glutamic acid; the concentration is a mass-to-volume ratio; Step 4, the solution after adding the amino acid is sealed and reacted in a 40° C. water bath for 1 h; Step 5, heat in a 95°C water bath for 30 min; Step 6: After heating is completed, take out and cool in an ice water bath for 10 to 15 minutes; Step 7, finally storing at 4° C. overnight to obtain soy protein isolate gel.

2. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: The acidic amino acid added in step 3 is aspartic acid, and the added concentration is 0.4% to 0.6%.

3. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: The acidic amino acid added in step 3 is glutamic acid, and the added concentration is 0.4%.

4. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: Step 1, the protein solution is a soy protein isolate solution, and the mass volume ratio of the soy protein isolate solution is 12%.

5. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: Step 1, the stirring speed during the stirring at room temperature is 500 rpm / min.

6. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: Step 2: After adding the TG enzyme, the mixture is fully mixed by stirring for 30 min.

7. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: Step 4, during the reaction in a water bath at 40° C. for 1 hour, the solution in the water bath container is in a stirring state at a stirring speed of 500 rpm / min.

8. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: In step 5, during the heating in a 95° C. water bath for 30 min, the power of the heating device is 800 W.

9. The method for preparing soy protein isolate gel using amino acid composite TG enzyme according to claim 1, characterized in that: Step 6, when cooling in the ice-water bath, the mass ratio of ice to water is 1:

2.

10. A soy protein isolate gel obtained by the preparation method according to any one of claims 1 to 9.

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