Ternary functional protein complex and its preparation method and application

Abstract

The present invention discloses a ternary functional protein complex and a preparation method and application thereof, belonging to the technical field of functional protein complexes. The method of the present invention is to obtain a ternary composite system by using a pH-driven method with a water-soluble protein solution, zein and sodium alginate, wherein the mass ratio of the water-soluble protein solution to zein is 10:(1-9), and the mass ratio of zein to sodium alginate is (1:9)-(9:1). The ternary protein complex prepared by the method of the present invention exhibits excellent thermal stability and functional properties. The preparation method of the ternary protein complex of the present invention is more environmentally friendly, safe and efficient.

Description

Technical Field

[0001] The present invention relates to the field of preparation of functional protein complexes, and in particular to a ternary functional protein complex and a preparation method and application thereof. Background Art

[0002] Protein complexes are complex structures composed of multiple proteins. These proteins are bound together by specific interactions, such as hydrogen bonds, hydrophobic interactions, etc., to form a functional whole. Each protein has its specific function and position in the complex, which together maintain the stability and function of the complex. They can act inside or outside the cell and participate in a variety of biochemical reactions and cellular activities. Functional protein complexes can be prepared and used as stable carriers for the delivery of functional factors. Biopolymer assembly is one of the methods for constructing qualified delivery carriers, among which proteins and polysaccharides are more attractive because they are surface-active substances.

[0003] Water-soluble protein (whey protein, abbreviated as WPI) (from milk) is a by-product of cheese processing, mainly composed of β-lactoglobulin (β-Lg), α-water-soluble protein (whey protein) (α-La), bovine serum albumin (BSA), immunoglobulins (IGs) and lactoferrin (LF). Water-soluble protein (whey protein) has the advantages of high nutritional value, easy availability, and excellent technical and functional properties. Based on these advantages, nutrient delivery systems constructed with water-soluble protein (whey protein) as the main component have been widely studied to deliver functional ingredients such as astaxanthin, 3,3'-diindolylmethane, vitamin D3, resveratrol and β-carotene.

[0004] Zein (abbreviated as Z) is the main storage protein in corn kernels. Zein has poor water solubility because it contains a high percentage of hydrophobic amino acid residues (more than 50%), such as proline, leucine and alanine. It can be dissolved in 60-90% ethanol aqueous solution. The unique dissolution characteristics of zein can be used to prepare nanoparticles that encapsulate non-polar bioactive molecules. In recent years, the study of the interaction between proteins and polyphenolic flavonoids has become a hot topic, and the study of non-covalent interactions has also become a key research object in the food field. Most non-covalent interactions are caused by hydrogen bonds, hydrophobic interactions, electrostatic interactions and van der Waals forces, and are usually reversible. This method can cause the structure of proteins to aggregate or unfold, resulting in changes in their physical and chemical properties, functional properties, nutritional value, etc.

[0005] Sodium alginate (SA for short) is a natural polysaccharide, a linear binary copolymer composed of β-D-mannuronic acid and α-L-guluronic acid residues linked by (1,4) glycosidic bonds. It has good stability and acid and alkali resistance, can form nanoparticles in a variety of pH ranges, and is widely used in the food industry. It is a generally recognized as safe (GRAS) food material.

[0006] Currently, most methods for preparing protein complexes use organic solvents, such as antisolvent precipitation and antisolvent co-precipitation. However, their application has certain limitations. Some organic solvents are toxic and irritating and cannot be used on the human body.

[0007] The pH-shift method, sometimes also called the pH-driven method, has recently been used to incorporate functional factors into different types of edible nanoparticles without the use of organic solvents. The pH-driven method takes advantage of the fact that the solubility of functional factors in aqueous solutions strongly depends on the pH value. However, in the prior art, the modification of proteins using the pH-driven method usually requires heating reactions (such as heating to 60-80°C), and too high a temperature can easily lead to protein denaturation and inactivation. Summary of the invention

[0008] The purpose of the present invention is to propose a new ternary functional protein complex and its preparation method and application, which does not use any organic solvent and does not require heating. It is a new method for obtaining a green, safe and functional protein complex that is environmentally friendly and efficient.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows:

[0010] In the first aspect, the present invention proposes a method for preparing a ternary functional protein complex, in which a water-soluble protein solution, zein and sodium alginate are subjected to a pH-driven method to obtain a ternary composite system, wherein the mass ratio of the water-soluble protein solution to zein is (10:1) to 9, and the mass ratio of zein to sodium alginate is (1:9) to (9:1).

[0011] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0012] S1: Using deionized water as solvent, add water-soluble protein into deionized water at a volume-mass ratio of 10:9, overnight to obtain a water-soluble protein solution;

[0013] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0014] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h under a stirrer, and zein was added according to the mass ratio of water-soluble protein solution to zein of 10:(1-9), and stirred for 30 min under a stirrer; sodium alginate was added according to the mass ratio of zein to sodium alginate of (1:9)-(9:1), and stirred for 30 min under a stirrer; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex; the parameters of the stirrer were set as follows: stirring speed of 800 r / min, ambient temperature of 25°C.

[0015] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0016] S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water overnight to obtain a water-soluble protein solution;

[0017] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0018] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h under a stirrer, 0.001 g of zein was added, and stirred for 30 min under a stirrer; 0.009 g of sodium alginate was added, and stirred for 30 min under a stirrer; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex, which was recorded as WPI-Z-SA 10:1:9 .

[0019] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0020] S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water overnight to obtain a water-soluble protein solution;

[0021] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0022] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h under a stirrer, 0.003 g of zein was added, and stirred for 30 min under a stirrer; 0.007 g of sodium alginate was added, and stirred for 30 min under a stirrer; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex, which was recorded as WPI-Z-SA 10:3:7 .

[0023] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0024] S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water overnight to obtain a water-soluble protein solution;

[0025] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0026] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h under a stirrer, 0.005 g of zein was added, and stirred for 30 min under a stirrer; 0.005 g of sodium alginate was added, and stirred for 30 min under a stirrer; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex, which was recorded as WPI-Z-SA 10:5:5 .

[0027] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0028] S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water overnight to obtain a water-soluble protein solution;

[0029] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0030] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, 0.007 g of zein was added, and stirred for 30 min; 0.003 g of sodium alginate was added, and stirred for 30 min; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex, which was recorded as WPI-Z-SA 10:7:3 .

[0031] Preferably, the method for preparing the above-mentioned ternary functional protein complex comprises the following steps:

[0032] S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water overnight to obtain a water-soluble protein solution;

[0033] S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution;

[0034] S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, stirred for 2 h, 0.009 g of zein was added, and stirred for 30 min; 0.001 g of sodium alginate was added, and stirred for 30 min; the complex solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex, which was recorded as WPI-Z-SA 10:9:1 .

[0035] Preferably, the water-soluble protein is whey protein.

[0036] In a second aspect, the present invention also provides a ternary functional protein complex prepared by any of the above methods.

[0037] In a third aspect, the present invention further proposes the use of the above-mentioned ternary functional protein complex in the preparation of edible nutritional factor delivery products.

[0038] Compared with the prior art, the technical effects of the present invention are:

[0039] The ternary functional protein complex proposed in the present invention has excellent solubility and strong thermal stability compared with binary complexes and pure protein complexes. It can withstand gastrointestinal temperature and be suitable for gastrointestinal absorption when people eat it, and can be applied to edible nutritional factor delivery products. At the same time, the protein complex prepared by the pH-driven method of the present invention does not use any organic solvents and does not require heating. It is a new method for obtaining green, safe, functional protein complexes that is environmentally friendly and efficient. Compared with pure protein complexes, the heat resistance and particle size of the complexes with added polysaccharides are relatively excellent, further ensuring its application in the field of functional foods and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a scanning electron micrograph of a protein complex provided in an embodiment of the present invention.

[0041] Figure 2 Fourier infrared spectrum provided by the embodiment of the present invention Figure 1 .

[0042] Figure 3 Fourier infrared spectrum provided by the embodiment of the present invention Figure 2 .

[0043] Figure 4 This is a DSC diagram provided for an embodiment of the present invention.

[0044] Figure 5 This is a particle size diagram provided for an embodiment of the present invention.

[0045] Figure 6 A potential diagram provided for an embodiment of the present invention.

[0046] Figure 7 This is a molecular dynamics RMSD analysis diagram provided by an embodiment of the present invention.

[0047] Figure 8 This is a molecular dynamics RMSF analysis diagram provided by an embodiment of the present invention.

[0048] Fig. 9 The molecular dynamics thermodynamics provided by the embodiment of the present invention Figure 1 .

[0049] Fig.10 The molecular dynamics thermodynamics provided by the embodiment of the present invention Figure 2 . DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention.

[0051] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0052] The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Sodium alginate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., zein was purchased from Beijing Bailingwei Technology Co., Ltd., and water-soluble protein (whey protein) was purchased from Yingxin Laboratory.

[0053] Example 1

[0054] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.001g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.009g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：1：9 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0055] Example 2

[0056] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.002g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.008g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：2：8 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0057] Example 3

[0058] Using deionized water as solvent, 0.045g of water-soluble protein (whey protein) was added to 50 mL of deionized water, hydrated overnight at 4°C, adjusted to pH 12 with 2 M NaOH, and stirred thoroughly at room temperature (25±2°C) for 2 h. 0.003g of zein was added to the water-soluble protein (whey protein) solution and stirred rapidly for 30 min under a stirrer. 0.007g of sodium alginate was added to the water-soluble protein (whey protein)-zein complex solution and stirred rapidly for 30 min under a stirrer. The pH was adjusted to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：3：7 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0059] Example 4

[0060] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.004g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.006g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：4：6 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0061] Example 5

[0062] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.005g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.005g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：5：5 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0063] Example 6

[0064] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.006g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.004g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：6：4 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0065] Example 7

[0066] Using deionized water as solvent, 0.045g of water-soluble protein (whey protein) was added to 50 mL of deionized water, hydrated overnight at 4°C, adjusted to pH 12 with 2 M NaOH, and stirred thoroughly at room temperature (25±2°C) for 2 h. 0.007g of zein was added to the water-soluble protein (whey protein) solution and stirred rapidly for 30 min under a stirrer. 0.003g of sodium alginate was added to the water-soluble protein (whey protein)-zein complex solution and stirred rapidly for 30 min under a stirrer. The pH was adjusted to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA10：7：3 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0067] Example 8

[0068] Using deionized water as solvent, 0.045g of water-soluble protein (whey protein) was added to 50 mL of deionized water, hydrated overnight at 4°C, adjusted to pH 12 with 2 M NaOH, and stirred thoroughly at room temperature (25±2°C) for 2 h. 0.008g of zein was added to the water-soluble protein (whey protein) solution and stirred rapidly for 30 min under a stirrer. 0.002g of sodium alginate was added to the water-soluble protein (whey protein)-zein complex solution and stirred rapidly for 30 min under a stirrer. The pH was adjusted to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate ternary functional protein complex, denoted as WPI-Z-SA 10：8：2 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0069] Example 9

[0070] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir thoroughly at room temperature (25±2°C) for 2 h. Add 0.009g zein to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Add 0.001g sodium alginate to the water-soluble protein (whey protein)-zein complex solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein-sodium alginate protein functional protein complex, denoted as WPI-Z-SA 10：9：1 The parameters of the agitator are as follows: the stirring speed is 800 r / min and the ambient temperature is 25±2℃.

[0071] Comparative Example 1

[0072] Using deionized water as solvent, 0.045g of water-soluble protein (whey protein) was added to 50 mL of deionized water, hydrated overnight at 4°C, adjusted to pH 12 with 2 M NaOH, and stirred for 2 h at room temperature (25±2°C). 0.01g of zein was added to the water-soluble protein (whey protein) solution, stirred rapidly for 30 min under a stirrer, and the pH was adjusted to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-zein binary functional protein complex, recorded as WPI-Z. The stirrer parameters were as follows: stirring speed was 800 r / min, and the ambient temperature was 25±2°C.

[0073] Comparative Example 2

[0074] Using deionized water as solvent, add 0.045g water-soluble protein (whey protein) to 50 mL deionized water, hydrate overnight at 4°C, adjust the pH to 12 with 2 M NaOH, and stir well at room temperature (25±2°C) for 2 h. Add 0.01g sodium alginate to the water-soluble protein (whey protein) solution and stir rapidly for 30 min under a stirrer. Adjust the pH to 7 with 1 M HCl to obtain a water-soluble protein (whey protein)-sodium alginate binary functional protein complex, recorded as WPI-SA. The stirrer parameters are as follows: stirring speed is 800 r / min, and the ambient temperature is 25±2°C.

[0075] Test Example 1 Scanning electron microscope test:

[0076] After the protein complex prepared above is freeze-dried, it is ground into fine powder, which is then sprayed with gold and placed in a scanning electron microscope to observe the microscopic morphology. The purpose of spraying gold is to enhance the conductivity of the sample in order to obtain a clearer microscopic morphology.

[0077] The results are as follows Figure 1 The results show that the complex with sodium alginate added has obvious particles and a smoother surface. Compared with the binary functional protein complex WPI-Z, the ternary functional protein complex has a clearer edge and a firmer surface. 10：5：5 Best.

[0078] Test Example 2 Determination of Fourier Transform Infrared Spectroscopy:

[0079] Fourier transform infrared spectroscopy is one of the most commonly used methods to study the structure, chemical properties and physical properties of substances. The changes in the absorption peaks and the direction of the shift shown in the spectrum can prove the changes in the internal structure of the substance. The protein complex prepared above was freeze-dried and ground into fine powder. The sample powder was mixed with KBr at a ratio of 1:100, and then the sample and KBr were ground into a finer powder in a mortar to make them evenly mixed. After the film was formed for 1 minute, it was placed in the instrument for measurement. The scanning range was 4000~400 cm -1 , with a resolution of 4 cm -1 , scanned 32 times. Potassium bromide tablets were used as blank controls during the measurement, and the infrared spectra obtained were analyzed using Origin software.

[0080] The results are as follows Figure 2 and Figure 3 As shown in the figure, it can be seen that the main absorption peak of natural WPI appears in the amide A band (3290 cm -1 OH stretching vibration), amide I band (1654 cm -1 , C=O stretching vibration), amide II band (1539cm -1 NC bending vibration and CN stretching vibration). Amide I band and amide II band are sensitive areas of protein secondary structure. The red shift and blue shift of the absorption peak in this area prove that the protein secondary structure has changed. Figure 2 and Figure 3 It is known that the absorption peak of the amide I band of the ternary functional protein complex undergoes a red shift, and the absorption peak of the amide II band undergoes a blue shift, indicating that non-covalent interactions occur during the formation of the complex. The movement of the amide I band is related to the interaction of the NH, C=O or COO- groups of the protein backbone, and the movement of the amide II band is related to the group vibration of the protein backbone.

[0081] Test Example 3 Determination of thermal stability:

[0082] Differential scanning calorimeter is an instrument for measuring the thermal stability of substances. Take 3-5 mg of freeze-dried powder sample and place it in an aluminum crucible. After pressing the cover, place the crucible with the sample and the empty crucible inside the instrument. The empty crucible is kept in the instrument as a control until it is taken out after the measurement is completed. The test conditions are: starting temperature 20℃, heating rate 10℃ / min, reaction termination temperature 200℃, nitrogen flow rate 50 mL / min. After the test, use the analysis software matched with the instrument to analyze the thermal characteristics of the sample.

[0083] The results are as follows Figure 4As shown, the thermal denaturation temperature (Td) of the water-soluble protein (whey protein)-zein binary functional protein complex is 129℃; the thermal denaturation temperature (Td) of the water-soluble protein (whey protein)-sodium alginate binary functional protein complex is 113℃. The Td value of the ternary functional protein complex has a significant change compared with the binary functional protein complex. The thermal stability of the water-soluble protein (whey protein)-zein binary functional protein complex has a more obvious downward trend after the addition of sodium alginate. It can be explained that in this process, due to the addition of sodium alginate, zein has a non-covalent interaction with it, which leads to a change in the thermal denaturation temperature of the complex. When the volume of zein and sodium alginate is 5:5, the Td value is 123℃, and the thermal denaturation temperature of the ternary functional protein complex is the highest. However, as the total amount of zein continued to increase, the Td value gradually decreased. The change of zein would lead to the change of thermal stability of protein. The appropriate amount of zein would improve the thermal stability of soy protein isolate. 10：5：5 Best thermal stability.

[0084] Test Example 4 Determination of particle size potential:

[0085] The prepared samples were diluted to 1 mg / mL with ultrapure water, and the zeta potential and particle size of the ternary functional protein complexes with different mass ratios were measured using a Nano-ZS laser particle size analyzer. The particle size and zeta potential of the samples were calculated using the Stokes-Einstein equation and the Smoluchowski model, respectively. All samples were measured three times in parallel and the average value was taken.

[0086] Figure 5 and Figure 6 The displayed result is the particle size potential of the test sample. Figure 5 As shown in the figure, with the addition of water-soluble protein (whey protein) and sodium alginate, more water-soluble protein (whey protein) and sodium alginate molecules will be adsorbed on the surface of zein molecules, resulting in strong electrostatic repulsion and spatial repulsion between the ternary functional protein complexes. The increase in repulsion between the ternary functional protein complexes further prevents the aggregation of zein. Among all the prepared samples, the particle size of the basic water-soluble protein (whey protein)-sodium alginate binary functional protein complex is the largest. However, when there is a small amount of zein in the system, the particle size increases slightly, but gradually decreases as the mass ratio of zein increases. This may be because the hydrophobicity of zein is enhanced during the pH-driven method, which promotes its interaction with water-soluble protein (whey protein) and sodium alginate, thereby forming a more compact structure and reducing the particle size.

[0087] like Figure 6The zeta potential of the ternary functional protein complexes with different mass ratios is shown in the figure. It can be seen that the zeta potential of all samples is negative, indicating that the surface of the protein is mostly negatively charged amino acids. The zeta potential value of the water-soluble protein (whey protein)-sodium alginate binary functional protein complex is -33.19 mV; the zeta potential value of the water-soluble protein (whey protein)-zein binary functional protein complex is -33.19 mV and the absolute value of the zeta potential is the smallest. As the mass ratio of zein increases, the absolute value of the zeta potential shows a trend of first decreasing and then increasing. When the mass ratio of zein to sodium alginate is 5:5, the absolute value of the zeta potential is the smallest. This shows that the better compounding of zein with water-soluble protein (whey protein) and sodium alginate makes the structure more compact, exposes fewer negatively charged amino acid groups, and reduces the charge on the molecular surface, which is consistent with the particle size experiment.

[0088] Test Example 5 Determination of molecular dynamics simulation:

[0089] Model preparation: The crystal structure of zein is not included in the protein database. Therefore, different software were used for homology modeling to construct the structure of zein. The sequence used was the amino acid sequence (22-265) of Zein (ID: Q41844) in the Uniprot database. The software used was the I-TASSER software web version and the Modeller software web version. The two models obtained were subjected to 50 ns molecular dynamics simulation. The structure modeled by Modeller was unstable and unfolded to a certain extent. The structure modeled by I-TASSER was relatively stable, so the model built by I-TASSER was retained for further docking. WPI, Zein and SA were docked using the Glide module of the Schrödinger (Schrödinger, LLC: New York, NY, 2015) software suite. The protein structures used in the docking were all structures after molecular dynamics simulation equilibrium of protein monomers. For each model, the potential protonation state of each model residue was corrected at pH 7.0 using the Epik module of Schrödinger's software. Then, hydrogen atoms and a 12nm cube of 80% ethanol mixed solvent box were added to each model using the "Tleap" module in Gromacs 2022.02. Counterions were added to the box to neutralize the charge of the system. All systems were parameterized using the Gromacs 2022.02 force field. The RESP charges of Zein, WPI and SA were calculated using Gaussian09 at the HF / 6-31G* level, and the other force field parameters were treated with the general Gromacs 2022.02 force field (GAFF). Finally, periodic boundary conditions (PBC) were applied to all models to eliminate boundary effects.

[0090] Molecular dynamics simulation: Molecular dynamics simulation was performed using the GPU-accelerated PMEMD program in the Gromacs 2022.02 software package. First, each model was optimized in three steps. In the first step, all protein and ligand molecules were constrained, only solvent molecules were released, and solvent molecules were optimized separately; in the second step, protein side chain molecules were released, the side chains of protein residues were optimized, and water molecules were further optimized; in the third step, the entire system was released and the entire system was optimized. After that, a 50 ps heating process was applied under the canonical ensemble (NVT) to maintain the system temperature at room temperature. Next, a 200 ps simulation was applied under the isothermal and isobaric ensemble (NPT) to pull the system density back to the normal solution density (density of 80% ethanol solution at 1 atm). Finally, a 50 ns molecular dynamics simulation was performed for each model under the NVT ensemble. In all simulations, the temperature and pressure were controlled using a Langevin thermostat and a Berendsen barometer. All bonds involving hydrogen atoms were constrained using the SHAKE method. Afterwards, the free energy was calculated using the Molecular Mechanics / Generalized Born Surface Area (MM / GBSA) method (using the MMPBSA.py script).

[0091] To investigate the stability of molecular dynamics simulations, the RMSD of water-soluble protein (whey protein), zein, and sodium alginate before and after binding were analyzed.

[0092] The results are as follows Figure 7 When the molecular dynamics simulation was performed for 25ns, the system had reached equilibrium, and the RMSD value of zein (holo-Zein-SA) after compounding with sodium alginate was lower than that of zein (apo-Zein) without sodium alginate, indicating that the structure of zein (holo-Zein-SA) after compounding with sodium alginate was more stable. For water-soluble protein (whey protein), the RMSD values ​​before compounding with sodium alginate (apo-WPI) and after compounding with sodium alginate (holo-WPI-SA) did not change much.

[0093] In addition, in order to understand the changes in the flexibility of each region of the protein before and after the combination of water-soluble protein (whey protein), zein and sodium alginate, the equilibrium trajectory (25ns-50ns) was intercepted and RMSF analysis was performed.

[0094] The results are as follows Figure 8As shown in the figure, compared with the zein (apo-Zein) not bound to sodium alginate, the flexibility of the zein (holo-Zein-SA) bound to sodium alginate was significantly enhanced, among which the five regions of 0-20, 25-40, 50-75, 130-150 and 175-200 decreased significantly, and its fluctuation range became narrower, indicating that sodium alginate promoted the stability of the zein structure. For WPI, compared with the water-soluble protein (apo-WPI) not bound to sodium alginate, the overall fluctuation of the water-soluble protein (holo-WPI-SA) bound to sodium alginate was almost unaffected. This is also consistent with the results obtained by RMSD.

[0095] The amino acid residues of zein and water-soluble protein (whey protein) (res-Zein-WPI) in molecular dynamics simulation results are shown in Fig. 9 As shown in the figure, the amino acid residues 30-60 of zein and water-soluble protein (whey protein) came into contact after 10 ns of the molecular dynamics simulation, with a contact distance of 0.08 nm; while the amino acid residues 120-150 of zein and water-soluble protein (whey protein) came into contact at the beginning of the simulation and continued to the end, with a contact area of ​​0.08-0.15 nm.

[0096] The amino acid residues of zein and sodium alginate (res-Zein-SA) in molecular dynamics simulation results are shown in Fig.10 As shown in the figure, the amino acid residues 0-10, 30-60, and 130-160 of zein and sodium alginate came into contact at 15-40 ns of the molecular dynamics simulation, with a contact area of ​​0.08 nm; while the amino acid residues 10-30 and 180-190 of zein and sodium alginate came into contact after 40 ns of the molecular dynamics simulation, with a contact area of ​​0.08 nm.

[0097] Based on the above experimental results, the following conclusions are drawn:

[0098] (1) Scanning electron microscopy results showed that the composite particles with sodium alginate added were obvious and had a smoother surface. Compared with the pure protein composite, the ternary composite had a clearer overall structure with clear edges and a firmer surface.

[0099] (2) Fourier transform infrared spectroscopy showed that during the formation of the ternary functional protein complex, covalent bonding occurred between the three components, resulting in changes in the amino acid microenvironment and a certain degree of red shift or blue shift of each group at different wavelengths;

[0100] (3) By analyzing the thermal properties of the ternary functional protein complex, it was found that the thermal stability of the ternary complex with the addition of zein was higher than that of the binary complex. When the volume ratio of zein and sodium alginate was 5:5, the Td value was 123°C, at which point the thermal denaturation temperature of the ternary complex was the highest;

[0101] (4) Through the particle size potential test, it was found that among all the prepared samples, the particle size of the basic water-soluble protein (whey protein)-sodium alginate binary complex was the largest. However, when a small amount of zein was present in the system, the particle size increased slightly, but gradually decreased as the mass ratio of zein increased. This may be because the hydrophobicity of zein increased during the pH-driven method, which promoted its interaction with water-soluble protein (whey protein) and sodium alginate, thereby forming a more compact structure and resulting in a decrease in particle size. The zeta potential value of the water-soluble protein (whey protein)-sodium alginate binary complex was -33.19 mV; the zeta potential value of the water-soluble protein (whey protein)-zein binary complex was -33.19 mV and the absolute value of the zeta potential was the smallest. As the mass ratio of zein increased, the absolute value of the zeta potential showed a trend of first decreasing and then increasing. The absolute value of the zeta potential was the smallest when the mass ratio of zein to sodium alginate was 5:5. This indicates that the better complexation of zein with water-soluble protein (whey protein) and sodium alginate makes the structure more compact, exposing fewer negatively charged amino acid groups and reducing the charge on the molecular surface, which is consistent with the particle size experiment.

[0102] (5) Through molecular dynamics simulation tests, water-soluble protein (whey protein) and sodium alginate promoted the stability of the zein structure. As for the water-soluble protein (whey protein), its overall fluctuation was almost unaffected. Different amino acid contacts occurred between zein-sodium alginate and zein and water-soluble protein (whey protein).

[0103] In summary, the ternary functional protein complex of the present invention exhibits more excellent characteristics in terms of micromorphology, microstructure, thermal stability, etc., among which WPI-Z-SA 10：5：5 At the same time, the raw materials are widely available and cheap, the preparation process is green and pollution-free, and the safety is guaranteed, so its development prospects are even broader.

[0104] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a ternary functional protein complex, characterized in that: A ternary composite system is obtained by using a pH-driven method with a water-soluble protein solution, zein and sodium alginate, wherein the water-soluble protein is whey protein; The preparation method consists of the following steps: S1: Using deionized water as solvent, add 0.045 g of water-soluble protein into 50 mL of deionized water and incubate overnight to obtain a water-soluble protein solution; S2: Using deionized water as solvent, prepare 2 M NaOH solution and 1 M HCl solution; S3: The pH of the overnight water-soluble protein solution was adjusted to 12 with 2M NaOH solution, and stirred for 2 hours under a stirrer. 0.005g of zein was added to the water-soluble protein solution, and stirred for 30 minutes under a stirrer. 0.005g of sodium alginate was added to the water-soluble protein-zein composite solution, and stirred for 30 minutes under a stirrer. The composite solution was adjusted to 7 with 1M HCl solution to obtain a ternary functional protein complex. The parameters of the stirrer were set as follows: a stirring speed of 800r / min and an ambient temperature of 25°C.

2. A ternary functional protein complex, characterized in that: The method according to claim 1 is used to make the 3. Use of the ternary functional protein complex according to claim 2 in the preparation of edible nutritional factor delivery products.

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