SPI-CS-EGCG ternary compound and preparation method and application thereof

Abstract

The invention provides an SPI-CS-EGCG ternary complex as well as a preparation method and application thereof. The preparation method of the ternary complex comprises the following steps: respectively preparing an SPI solution, a CS solution and an EGCG solution; adjusting the pH value of the SPI solution, and heating to obtain SPI microgel; the SPI microgel and a CS solution are mixed and stirred, and an SPI-CS mixed solution is obtained; and mixing and stirring the SPI-CS mixed solution and the EGCG solution, and centrifuging to obtain the SPI-CS-EGCG ternary complex. According to the SPI-CS-EGCG ternary complex, the functional characteristics of SPI are remarkably improved through non-covalent interaction (hydrogen bonds, hydrophobic interaction and Van der Waals' force), and the SPI-CS-EGCG ternary complex has good thermal stability, rheology and tribology characteristics, so that the SPI-CS-EGCG ternary complex has wide application prospects in the food industry.

Description

Technical Field

[0001] The present invention belongs to the field of protein / polysaccharide, and in particular relates to a SPI-CS-EGCG ternary complex and a preparation method and application thereof. Background Art

[0002] Proteins, polysaccharides, and polyphenols are three important functional components in food systems, each with unique chemical structures, physical properties, and functional roles. Proteins have excellent emulsifying, gelling, foaming, and structural functions, polysaccharides are known for their thickening, gelling, water-retention, and stabilizing abilities, and polyphenols possess antioxidant, antimicrobial, and nutritional health benefits. In recent years, research has gradually expanded from binary interactions between proteins and polysaccharides to ternary interactions between proteins, polysaccharides, and polyphenols to combine the beneficial properties of each component. However, existing research has primarily focused on the preparation and application of binary complexes, while the preparation and functional properties of ternary complexes have not been fully explored. For example, Zhihua Pang et al. used pure whey protein isolate (WPI) particles to form WPI-chitosan composite particles under heating conditions through electrostatic interaction. These WPI-chitosan composite particles exhibited improved lubricity and have broad application prospects in the food industry.

[0003] Chitosan (CS) is a linear polysaccharide composed of β-(1-4)-2-amino-D-glucose units and some β-(1-4)-N-acetyl-D-glucosamine units. It carries a positive charge, which allows it to interact with anionic substances and exhibits excellent mucosal adhesion properties. Epigallocatechin-3-gallate (EGCG), a flavan-3-ol phenolic compound found in green tea, readily forms complexes with natural macromolecules due to its high solubility in water. Structurally, EGCG acts as an excellent hydrogen donor, forming hydrogen bonds with hydrogen acceptors in proteins. Its benzene ring also facilitates interactions with hydrophobic regions of proteins, enhancing its binding potential in complex systems. However, existing soy protein isolate (SPI)-based particles have a high surface hydrophobicity and are prone to aggregation, resulting in poor water dispersibility, limiting their application in food applications. Furthermore, SPI-based components tend to aggregate when subjected to changes in solution or environmental conditions (such as temperature, pH, and ionic strength), compromising their stability and functionality. Although the prior art discloses a technical solution for preparing nanoparticles by modifying some proteins with chitosan and EGCG, it does not disclose a technical solution for preparing a ternary complex using soy protein as a raw material. For example, the Chinese invention patent with publication number CN116746657A discloses a sophorolipid complex edible emulsion and its preparation method and application. With zein and chitosan (CS) as the base, epigallocatechin gallate (EGCG), perilla extract and sophorolipid as fillers are added to the zein and chitosan (CS) mixture to improve the antibacterial effect. The Chinese invention patent with publication number CN118985910A discloses a method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles. The rice bran protein-epigallocatechin gallate-carboxymethyl chitosan (REC) ternary nanoparticles are prepared by pH cycling method to prolong the antioxidant effect of epigallocatechin gallate. The above preparation method is not applicable to soy protein and cannot solve the problems of soy protein isolate in terms of taste and stability. Therefore, how to overcome the defects of soy protein's easy aggregation and poor stability by preparing soy protein-based ternary complexes and improve its taste has become a hot topic in current research. Summary of the Invention

[0004] In view of this, the present invention aims to propose a SPI-CS-EGCG ternary complex and its preparation method and application to improve the functional properties of SPI and enhance thermal stability, rheological and tribological properties.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] In a first aspect, the present invention provides a method for preparing a SPI-CS binary complex, the method comprising the following steps:

[0007] (1) Prepare SPI solution and CS solution respectively;

[0008] (2) adjusting the pH of the SPI solution to 5.6 and heating it at 95°C for more than 30 min to obtain SPI microgels;

[0009] (3) mixing the SPI microgel with the CS solution to obtain an SPI-CS mixed solution;

[0010] (4) Centrifuging the SPI-CS mixed solution to obtain the SPI-CS binary complex.

[0011] Furthermore, the preparation method of the SPI solution comprises the following steps: mixing SPI powder with distilled water, heating and stirring until completely dissolved, and then high-pressure homogenization to obtain an SPI solution; preferably, the heating temperature is 50-60°C, the stirring speed is 500-600 rpm, the stirring time is 2-3 hours, and the homogenization pressure is 4000-6000 psi; preferably, the concentration of the SPI solution is 0.9%.

[0012] Furthermore, the preparation method of the CS solution comprises the following steps: mixing CS powder with distilled water and stirring until fully hydrated; preferably, the stirring speed is 500-600 rpm and the stirring time is 1-2 hours; preferably, the concentration of the CS solution is 0.1%.

[0013] Furthermore, the mass ratio of SPI to CS in the SPI-CS mixed solution is 8 to 10:1; preferably, the mass ratio of SPI to CS is 9:1; preferably, the stirring rate in step (3) is 8000 to 12000 rpm, and the stirring time is 1 to 4 minutes; preferably, the stirring rate is 10000 rpm, and the stirring time is 2 minutes.

[0014] Furthermore, the step (3) includes the following steps: mixing the SPI microgel with the CS solution, adjusting the pH to 5.6, and stirring to obtain a SPI-CS mixed solution.

[0015] Furthermore, in step (4), the centrifugal force is 9000 g and the centrifugation time is 30 min.

[0016] In a second aspect, the present invention provides a SPI-CS binary complex prepared by the preparation method described in the first aspect.

[0017] In a third aspect, the present invention provides a method for preparing a ternary complex of SPI-CS-EGCG, the method for preparing the ternary complex comprising the following steps:

[0018] (1) Prepare SPI solution, CS solution and EGCG solution respectively;

[0019] (2) adjusting the pH value of the SPI solution and heating it to obtain SPI microgels;

[0020] (3) mixing the SPI microgel with the CS solution to obtain an SPI-CS mixed solution;

[0021] (4) The SPI-CS mixed solution and the EGCG solution were mixed and stirred, and centrifuged to obtain the SPI-CS-EGCG ternary complex.

[0022] Furthermore, in step (2), the pH value is 5.6, the heating temperature is 95° C., and the heating time is more than 30 minutes.

[0023] Furthermore, the preparation method of the SPI solution comprises the following steps: mixing SPI powder with distilled water, heating and stirring until completely dissolved, and then high-pressure homogenization to obtain an SPI solution; preferably, the heating temperature is 50-60°C, the stirring speed is 500-600 rpm, the stirring time is 2-3 hours, and the homogenization pressure is 4000-6000 psi; preferably, the concentration of the SPI solution is 0.9%.

[0024] Furthermore, the preparation method of the CS solution comprises the following steps: mixing CS powder with distilled water and stirring until fully hydrated; preferably, the stirring speed is 500-600 rpm and the stirring time is 1-2 hours; preferably, the concentration of the CS solution is 0.1%.

[0025] Furthermore, the preparation method of the EGCG solution comprises the following steps: mixing EGCG powder with distilled water and stirring until fully hydrated; preferably, the stirring speed is 500-600 rpm and the stirring time is 1-2 h; preferably, the concentration of the EGCG solution is 50 mg / mL.

[0026] Furthermore, the mass ratio of SPI to CS in the SPI-CS mixed solution is 8 to 10:1; preferably, the mass ratio of SPI to CS is 9:1; preferably, the stirring rate in step (3) is 8000 to 12000 rpm, and the stirring time is 1 to 4 minutes; preferably, the stirring rate is 10000 rpm, and the stirring time is 2 minutes.

[0027] Furthermore, the step (3) includes the following steps: mixing the SPI microgel with the CS solution, adjusting the pH to 5.6, and stirring to obtain a SPI-CS mixed solution.

[0028] Furthermore, the step (4) includes the following steps: mixing the SPI-CS mixed solution and the EGCG solution, adjusting the pH to 5.6, and centrifuging to obtain the SPI-CS-EGCG ternary complex.

[0029] Furthermore, the reagents used to adjust pH were 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH.

[0030] Furthermore, in step (4), the stirring speed is 400-600 rpm, the stirring time is 40-80 min, the centrifugal force is 8000-10000 g, and the centrifugal time is 20-40 min; preferably, the stirring speed is 500 rpm, the stirring time is 60 min, the centrifugal force is 9000 g, and the centrifugal time is 30 min.

[0031] Furthermore, in the mixed solution of the SPI-CS mixed solution and the EGCG solution in step (4), the mass percentage of SPI is 0.405% to 0.45%, the mass percentage of CS is 0.045% to 0.05%, and the mass concentration of EGCG is 0.10 mg / mL to 0.50 mg / mL; preferably, the total biopolymer concentration in the mixed solution is 0.5%.

[0032] In a fourth aspect, the present invention provides a SPI-CS-EGCG ternary complex prepared according to the preparation method described in the first aspect.

[0033] Furthermore, the ternary complex is a composite particle with SPI as the core, CS as the shell, and EGCG as the middle layer, wherein SPI-CS has the same core-shell structure as the WPI-chitosan composite particles formed by the electrostatic interaction of whey protein isolate and chitosan. This core-shell structure can provide stable microgel particles; the addition of EGCG as a functional factor not only retains the core-shell structure of SPI-CS, but also effectively solves the problems of poor solubility and low thermal stability of SPI, while giving the complex antioxidant and antibacterial functions, expanding its application potential in health foods.

[0034] In a fifth aspect, the present invention provides use of the SPI-CS binary complex described in the first aspect or the SPI-CS-EGCG ternary complex described in the third aspect in food or medicine.

[0035] Preferably, the application includes one of the following applications:

[0036] (1) Applications for improving the resilience of food or medicine;

[0037] (2) Applications to reduce friction of food or medicine;

[0038] (3) Applications to enhance the lubricity of food or medicine

[0039] Furthermore, the application is the use of the SPI-CS-EGCG ternary complex as a food additive in food; preferably, the food additive is a functional food additive.

[0040] Compared with the prior art, the SPI-CS-EGCG ternary complex of the present invention and its preparation method and application have the following advantages:

[0041] (1) The present invention successfully prepared a soy protein-based SPI-CS-EGCG ternary complex by first preparing SPI into a microgel, then mixing it with CS to prepare SPI-CS particles with a core-shell structure, and finally adding EGCG for reinforcement. The SPI-CS-EGCG ternary complex of the present invention significantly improved the functional properties of SPI through non-covalent interactions (hydrogen bonds, hydrophobic interactions and van der Waals forces), and had good thermal stability, rheological and tribological properties, making it have broad application prospects in the food industry.

[0042] (2) The present invention optimizes the mixing step of SPI and CS, adjusts the pH of the SPI solution to 6.5, and heats the SPI solution at 95°C for 30 min to microgel the SPI solution before mixing it with CS, thereby preparing an SPI-CS binary complex with a core-shell structure and improving the stability of the SPI-CS binary complex.

[0043] (3) The present invention mixes EGCG with SPI-CS and adjusts the pH to 5.6 to prepare the SPI-CS-EGCG ternary complex. EGCG promotes the stability of the particles through hydrogen bonds, hydrophobic interactions, and electrostatic forces, and increases the denaturation temperature of the protein. At the same time, the SPI-CS-EGCG ternary complex has good viscoelasticity and friction properties, and has broad application prospects in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0045] Figure 1 The figure shows the preparation process and principle of the SPI-CS-EGCG ternary complex of the present invention.

[0046] Figure 2Schematic diagram of the water holding capacity of particles of the SPI-CS binary complex prepared in Comparative Examples 1-3;

[0047] Figure 3 Schematic diagram of the test results of the composite particles prepared in Examples 1-3 and Comparative Examples 1-5.

[0048] Figure 4 Schematic diagram of the test results of fluorescence intensity, ellipticity and binding efficiency of the complexes prepared in Examples 1-3 and Comparative Examples 1, 4 and 5.

[0049] Figure 5 Schematic diagram of surface property test results of the composites prepared in Examples 1-3 and Comparative Example 1.

[0050] Figure 6 Schematic diagram of the test results of rheological properties and friction properties of the composites prepared in Examples 1-3 and Comparative Example 1.

[0051] Figure 7 AFM and SEM images of the composites prepared in Examples 1-3 and Comparative Example 1.

[0052] Figure 8 Schematic diagram of the molecular docking of SPI-CS and EGCG in the present invention. DETAILED DESCRIPTION

[0053] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0054] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0055] Example 1

[0056] like Figure 1 As shown, the preparation method of the SPI-CS-EGCG ternary complex of this embodiment includes the following steps:

[0057] Step 1: Mechanically stir at 500 rpm for 2 hours at 50°C to completely dissolve the soy protein isolate in the aqueous solution, then homogenize once at a homogenizing pressure of 5000 psi, and hydrate at 4°C for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stir at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrate at 4°C for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%; stir at 500 rpm for 1 hour to completely dissolve EGCG in the aqueous solution, and hydrate at 4°C for 12 hours to obtain an EGCG aqueous solution with a mass concentration of 50 mg / mL;

[0058] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps are used to prepare the SPI-CS binary mixed solution, and the specific method is as follows:

[0059] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain a SPI-CS mixed solution.

[0060] Then, the SPI-CS mixed solution prepared in the above steps and the EGCG aqueous solution were used to prepare the SPI-CS-EGCG ternary complex, and the specific method was as follows:

[0061] In step 3, the EGCG aqueous solution was then added to the SPI-CS mixed solution to a final concentration of 0.1 mg / mL and a total biopolymer concentration of 0.5%. The mixture was stirred at 500 rpm for 60 minutes at room temperature. The pH of the dispersion was adjusted to 5.6 using 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH. The solution was then centrifuged at 9000 g for 30 minutes, and the precipitate was collected to obtain the SPI-CS-EGCG ternary complex.

[0062] Example 2

[0063] The preparation method of the SPI-CS-EGCG ternary complex of this embodiment comprises the following steps:

[0064] Step 1: Mechanically stir at 500 rpm for 2 hours at 50°C to completely dissolve the soy protein isolate in the aqueous solution, then homogenize once at a homogenizing pressure of 5000 psi, and hydrate at 4°C for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stir at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrate at 4°C for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%; stir at 500 rpm for 1 hour to completely dissolve EGCG in the aqueous solution, and hydrate at 4°C for 12 hours to obtain an EGCG aqueous solution with a mass concentration of 50 mg / mL;

[0065] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps are used to prepare the SPI-CS binary mixed solution, and the specific method is as follows:

[0066] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain a SPI-CS mixed solution.

[0067] Then, the SPI-CS mixed solution prepared in the above steps and the EGCG aqueous solution were used to prepare the SPI-CS-EGCG ternary complex, and the specific method was as follows:

[0068] In step 3, the EGCG aqueous solution was then added to the SPI-CS mixed solution to a final concentration of 0.25 mg / mL and a total biopolymer concentration of 0.5%. The mixture was stirred at 500 rpm for 60 minutes at room temperature. The pH of the dispersion was adjusted to 5.6 using 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH. The solution was then centrifuged at 9000 g for 30 minutes, and the precipitate was collected to obtain the SPI-CS-EGCG ternary complex.

[0069] Example 3

[0070] The preparation method of the SPI-CS-EGCG ternary complex of this embodiment comprises the following steps:

[0071] Step 1: Mechanically stir at 500 rpm for 2 hours at 50°C to completely dissolve the soy protein isolate in the aqueous solution, then homogenize once at a homogenizing pressure of 5000 psi, and hydrate at 4°C for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stir at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrate at 4°C for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%; stir at 500 rpm for 1 hour to completely dissolve EGCG in the aqueous solution, and hydrate at 4°C for 12 hours to obtain an EGCG aqueous solution with a mass concentration of 50 mg / mL;

[0072] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps are used to prepare the SPI-CS binary mixed solution, and the specific method is as follows:

[0073] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain a SPI-CS mixed solution.

[0074] Then, the SPI-CS mixed solution prepared in the above steps and the EGCG aqueous solution were used to prepare the SPI-CS-EGCG ternary complex, and the specific method was as follows:

[0075] In step 3, the EGCG aqueous solution was then added to the SPI-CS mixed solution to a final concentration of 0.5 mg / mL and a total biopolymer concentration of 0.5%. The mixture was stirred at 500 rpm for 60 minutes at room temperature. The pH of the dispersion was adjusted to 5.6 using 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH. The solution was then centrifuged at 9000 g for 30 minutes, and the precipitate was collected to obtain the SPI-CS-EGCG ternary complex.

[0076] Comparative Example 1

[0077] The preparation method of the SPI-CS binary complex of this comparative example is different from that of Example 1 except that EGCG is not added. Specifically, the following steps are included:

[0078] Step 1: Mechanically stirring at 500 rpm for 2 hours at 50° C. to completely dissolve the soy protein isolate in the aqueous solution, then homogenizing once at a homogenizing pressure of 5000 psi, and hydrating at 4° C. for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stirring at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrating at 4° C. for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%;

[0079] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps were used to prepare the SPI-CS binary complex, and the specific method was as follows:

[0080] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain an SPI-CS mixed solution. Finally, the solution was centrifuged at 9,000g for 30 minutes, and the precipitate was collected to obtain the SPI-CS binary complex.

[0081] Comparative Example 2

[0082] The preparation method of the SPI-CS binary complex of this comparative example differs from that of comparative example 1 only in that the pH of the soy protein isolate aqueous solution is adjusted to 6.0 instead of 5.6 in step 2. Specifically, the method comprises the following steps:

[0083] Step 1: Mechanically stirring at 500 rpm for 2 hours at 50° C. to completely dissolve the soy protein isolate in the aqueous solution, then homogenizing once at a homogenizing pressure of 5000 psi, and hydrating at 4° C. for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stirring at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrating at 4° C. for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%;

[0084] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps were used to prepare the SPI-CS binary complex, and the specific method was as follows:

[0085] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 6.0 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain an SPI-CS mixed solution. Finally, the solution was centrifuged at 9,000g for 30 minutes, and the precipitate was collected to obtain the SPI-CS binary complex.

[0086] Comparative Example 3

[0087] The preparation method of the SPI-CS binary complex of this comparative example differs from that of comparative example 1 only in that the pH of the soy protein isolate aqueous solution is not adjusted. Specifically, the method comprises the following steps:

[0088] Step 1: Mechanically stirring at 500 rpm for 2 hours at 50° C. to completely dissolve the soy protein isolate in the aqueous solution, then homogenizing once at a homogenizing pressure of 5000 psi, and hydrating at 4° C. for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stirring at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrating at 4° C. for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%;

[0089] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps were used to prepare the SPI-CS binary complex, and the specific method was as follows:

[0090] In step 2, the soy protein isolate aqueous solution was heated at 95°C for 30 minutes and rapidly cooled to room temperature (approximately 24°C) with ice water. Subsequently, a chitosan aqueous solution was added at a 1:1 ratio of protein to polysaccharide (SPI:CS) to a weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain an SPI-CS mixed solution. Finally, the solution was centrifuged at 9,000g for 30 minutes, and the precipitate was collected to obtain the SPI-CS binary complex.

[0091] Comparative Example 4

[0092] The preparation method of the SPI-CS-EGCG ternary complex of this comparative example is different from that of Example 1, except that the pH adjustment in step 3 is changed from 5.6 to 7.0. Specifically, the following steps are included:

[0093] Step 1: Mechanically stir at 500 rpm for 2 hours at 50°C to completely dissolve the soy protein isolate in the aqueous solution, then homogenize once at a homogenizing pressure of 5000 psi, and hydrate at 4°C for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stir at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrate at 4°C for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%; stir at 500 rpm for 1 hour to completely dissolve EGCG in the aqueous solution, and hydrate at 4°C for 12 hours to obtain an EGCG aqueous solution with a mass concentration of 50 mg / mL;

[0094] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps are used to prepare the SPI-CS binary mixed solution, and the specific method is as follows:

[0095] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain a SPI-CS mixed solution.

[0096] Then, the SPI-CS mixed solution prepared in the above steps and the EGCG aqueous solution were used to prepare the SPI-CS-EGCG ternary complex, and the specific method was as follows:

[0097] In step 3, the EGCG aqueous solution was then added to the SPI-CS mixed solution to a final concentration of 0.25 mg / mL and a total biopolymer concentration of 0.5%. The mixture was stirred at 500 rpm for 60 minutes at room temperature. The pH of the dispersion was adjusted to 7.0 using 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH. The solution was then centrifuged at 9000 g for 30 minutes, and the precipitate was collected to obtain the SPI-CS-EGCG ternary complex.

[0098] Comparative Example 5

[0099] The preparation method of the SPI-CS-EGCG ternary complex of this comparative example is different from that of Example 1, except that NaCl is added in step 3 to adjust the ion concentration. Specifically, the following steps are included:

[0100] Step 1: Mechanically stir at 500 rpm for 2 hours at 50°C to completely dissolve the soy protein isolate in the aqueous solution, then homogenize once at a homogenizing pressure of 5000 psi, and hydrate at 4°C for 12 hours to obtain a soy protein isolate aqueous solution with a mass concentration of 0.9%; stir at 500 rpm for 1 hour to completely dissolve chitosan in the aqueous solution, and hydrate at 4°C for 12 hours to obtain a chitosan aqueous solution with a mass concentration of 0.1%; stir at 500 rpm for 1 hour to completely dissolve EGCG in the aqueous solution, and hydrate at 4°C for 12 hours to obtain an EGCG aqueous solution with a mass concentration of 50 mg / mL;

[0101] Then, the soy protein isolate aqueous solution and chitosan aqueous solution prepared in the above steps are used to prepare the SPI-CS binary mixed solution, and the specific method is as follows:

[0102] In step 2, the pH of the soy protein isolate aqueous solution was adjusted to 5.6 using 1.0M and 0.1M HCl solutions, then heated at 95°C for 30 minutes and quickly cooled to room temperature (about 24°C) with ice water. Subsequently, a chitosan aqueous solution was added in a 1:1 ratio to a protein to polysaccharide (SPI:CS) weight ratio of 9:1 and a total biopolymer concentration of 0.5%. The pH of the mixed solution was then readjusted to 5.6 using 0.1M HCl. Finally, the mixed solution was sheared at 10,000 rpm for 2 minutes using a high-speed homogenizer to obtain a SPI-CS mixed solution.

[0103] Then, the SPI-CS mixed solution prepared in the above steps and the EGCG aqueous solution were used to prepare the SPI-CS-EGCG ternary complex, and the specific method was as follows:

[0104] In step 3, the EGCG aqueous solution was then added to the SPI-CS mixed solution to a final concentration of 0.25 mg / mL and a total biopolymer concentration of 0.5%. The solution was stirred at 500 rpm for 60 minutes at room temperature. The pH of the dispersion was adjusted to 5.6 using 1.0 M HCl, 0.1 M HCl, and 0.1 M NaOH. NaCl was added to a final ion concentration of 400 mM. The solution was then centrifuged at 9000 g for 30 minutes, and the precipitate was collected to obtain the SPI-CS-EGCG ternary complex.

[0105] Performance test case

[0106] The particle characteristics and rheological properties of the composites prepared in the examples and comparative examples were measured, including particle size, potential, turbidity, surface hydrophobicity, storage modulus and loss modulus as a function of frequency, oral lubrication behavior, and microstructural changes, and were compared.

[0107] Performance Test Example 1 Water Holding Capacity of SPI-CS Binary Composite

[0108] 10 g of each sample prepared in Comparative Examples 1-3 was placed in a glass beaker and allowed to stand for 30 minutes before taking a visual image.

[0109] The results are as follows Figure 2 As shown, the SPI-CS binary complex of Comparative Example 1 has a smoother, more cohesive structure, indicating that microgelling SPI prior to combining it with CS results in the strongest SPI-CS interaction, favoring gel formation. However, significant water separation was observed in Comparative Example 2, indicating that changing the pH of SPI microgelation weakens the SPI-CS gel structure and reduces water retention. The surface of Comparative Example 3 is rough, demonstrating that the reduction in the positive charge of chitosan after combining unmicrogelled SPI with CS weakens the interaction with SPI, resulting in a rougher microgel structure.

[0110] Performance Test Example 2 Particle Characteristics of SPI-CS-EGCG Ternary Complex

[0111] Zeta potential is the electrostatic potential near the surface of particles suspended in a liquid, especially caused by particle-particle interactions in colloidal systems. It can be used as an indicator of particle aggregation stability and interaction effects. It is generally believed that the higher the absolute value of the Zeta potential (positive or negative), the more stable the system. Figure 3 Figure A shows the zeta potential of the SPI-CS-EGCG ternary complex at different EGCG concentrations. As can be seen from the figure, the zeta potential of all samples is positive, and according to the test results of Examples 1-3, the zeta potential does not change significantly with increasing EGCG concentration, indicating that EGCG does not significantly contribute to the electrical properties. The potential of Comparative Examples 1-2 is significantly higher than that of Comparative Example 3, indicating that microgel treatment can promote the aggregation stability of the SPI-CS binary complex. The low potential values ​​of Comparative Examples 4-5 also indicate that neutral pH and the presence of salt ions are detrimental to the stability of the SPI-CS-EGCG ternary complex.

[0112] Figure 3 Figure B shows the average particle size of the SPI-CS-EGCG ternary complex at different EGCG concentrations. Comparative Example 1 exhibited an average diameter of approximately 665 nm, higher than that of Comparative Examples 2-3, while Examples 1-3 exhibited significantly larger average diameters than Comparative Example 1, demonstrating that the addition of EGCG to the dispersion resulted in a significant increase in particle size. This increase in particle size is consistent with the formation of the SPI-CS-EGCG complex through non-covalent interactions. The hydroxyl groups in the polyphenols act as excellent hydrogen donors, promoting hydrogen bonding with carbonyl groups in proteins and ionic interactions with amino groups. The hydrophobic amino acids in proteins and the non-polar aromatic rings of the polyphenols approach and aggregate, generating mutual attractive forces between the hydrophobic moieties. These interactions generate attractive forces that are much stronger than the electrostatic repulsion between the particles, allowing the polyphenols to act as bridges. This results in the formation of molecular aggregates resembling supramolecular structures, thereby increasing the particle size of the SPI-CS-EGCG complex. Comparative Example 4, which incorporates EGCG but maintains a neutral pH, exhibited a smaller particle size than Comparative Example 1, as the pH, far from the isoelectric point of SPI, inhibits SPI aggregation. The high particle size of Comparative Example 5 suggests that salt ions contribute to excessive aggregation of the complex.

[0113] Turbidity tests help identify complex formation because they typically result in increased light scattering. In the present invention, turbidity measurements were used to confirm the interaction between EGCG and SPI-CS composite microgels. The observed increase in turbidity may be due to an increase in the number and size of complex aggregates. Figure 3C shows the turbidity of the SPI-CS-EGCG ternary complex at different EGCG concentrations. The turbidity of the dispersions of Examples 1-3 is significantly higher than that of Comparative Example 1, indicating that there is a non-covalent interaction between SPI-CS and EGCG after the addition of EGCG. In addition, the presence of polyphenols exposes more hydrophobic groups on the protein, reduces its solubility, and thus increases the turbidity of the system. The turbidity of Comparative Example 4, in which EGCG is added but the system pH is 7.0, is less than that of Comparative Example 1, which once again proves that the environmental pH of 5.6 plays an important role in the formation of the SPI-CS-EGCG ternary complex. The high turbidity of Comparative Example 5 corresponds to the high particle size value, combined with the low potential value, indicating that the presence of salt ions will promote significant aggregation between SPIs or between SPI-CS, which is not conducive to the binding of EGCG.

[0114] Performance Test Example 3 Spectrum of SPI-CS-EGCG Ternary Complex

[0115] The emission spectrum of proteins at 280 nm can provide information about protein conformation, dynamic properties, and intermolecular interactions, mainly from tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Figure 4 Figure A shows the fluorescence spectrum of the SPI-CS complex after interaction with EGCG. The results of Examples 1-3 and Comparative Example 1 indicate that the addition of EGCG reduces the fluorescence intensity of the protein. This fluorescence quenching is due to the interaction between the phenolic ring of EGCG and the hydrophobic groups of the tryptophan (Trp) residues in SPI, indicating that EGCG binds to the SPI-CS complex. This interaction is similar to the binding of the aromatic groups of polyphenols to the hydrophobic pockets of human serum albumin and lysozyme through hydrophobic interactions.

[0116] Generally, if the maximum fluorescence emission (λmax) of tryptophan (Trp) is below 330 nm, it is considered to be in a hydrophobic environment, while a λmax above 330 nm indicates that Trp is in a hydrophilic environment. Figure 4 As shown in Figure A, Comparative Example 1 shows a strong fluorescence peak at 319 nm. After EGCG is added and combined in the embodiment, the fluorescence intensity is significantly quenched, and the λmax of SPI-CS-EGCG red-shifts to 322 nm, 328 nm and 332 nm, corresponding to Example 1, Example 2 and Example 3, respectively. This red shift of λmax and the reduction in fluorescence intensity indicate that the tertiary structure of soy protein isolate has changed, exposing the residues to a more hydrophilic environment. This shows that the addition of EGCG causes the tryptophan residues to be exposed, resulting in protein unfolding, reduced fluorescence intensity, and red shift of the emission peak. In addition, compared with Example 2, Comparative Example 5 shows that the addition of NaCl in Comparative Example 5 causes the fluorescence intensity to gradually increase, which may be due to the electrostatic shielding effect of salt ions, which weakens the molecular interactions within the SPI-CS-EGCG complex.

[0117] The change in molar ellipticity caused by the binding of EGCG to the SPI-CS complex was measured using CD spectroscopy in the far-UV range (190-250 nm). Figure 4 B). The data were analyzed to determine the proportions of α-helix, β-sheet, β-turn and random coil conformations (as shown in Table 1). The addition of different concentrations of EGCG in Comparative Example 1 and Examples 1-3 resulted in significant changes in the SPI peak intensity, indicating that its secondary structure was affected, which may be due to the fact that hydrophobic interactions changed the solvent environment and destroyed the intramolecular and intermolecular hydrogen bonds. Figure 4 As shown in Figure B, Examples 1-3 showed broad positive and negative peaks near 195nm and 210nm, corresponding to α-helix and β-sheet content, respectively. Further analysis of the results of Comparative Example 1 and Examples 1-3 confirmed that with the increase of EGCG concentration, the β-sheet content decreased significantly from 47.1% to 24.6%, while the α-helix content increased from 17.5% to 55.3% (as shown in Table 1). These structural changes indicate that the interaction between SPI-CS and EGCG after the addition of EGCG in the examples causes the spatial conformation of the protein to change, which is driven by hydrophobic interactions and hydrogen bonds. The increase in α-helical structure generally indicates that the protein is partially unfolded, which may be related to the increased exposure of the hydrophobic group. The combination of EGCG and SPI-CS to form ternary complex particles induced conformational changes in soy protein, stabilizing soy protein through additional hydrogen bonds and electronic interactions. A reduction in random coil structure was also observed, which is consistent with the findings in other ternary complexes (such as soy protein hydrolysate / β-glucan / ferulic acid), in which stronger intermolecular interactions, especially hydrogen bonds, increased the α-helix content and reduced the disordered structure. Comparing Comparative Example 4 with Example 1, it can be seen that after the EGCG aqueous solution was added to the SPI-CS mixed solution, when the pH changed from 5.6 in Example 1 to 7.0 in Comparative Example 4, the α-helix content of the protein in Comparative Example 4 was significantly reduced compared to Example 1, while the β-sheet content increased, indicating a transition from α-helix to β-sheet, forming more compact particles, which may lead to a decrease in the solubility of the protein and affect its emulsification and foaming ability.

[0118] Table 1 Secondary structure content of SPI-CS-EGCG ternary complex at different EGCG concentrations, pH and ionic strength

[0119]

[0120] Different letters represent different significance levels (p < 0.05).

[0121] In order to clarify the ability and efficiency of EGCG binding to SPI-CS, the binding efficiency of EGCG in the SPI-CS-EGCG ternary complex was evaluated (e.g. Figure 4 C). According to the results of Examples 1-3, as the concentration of EGCG increases, its binding to SPI-CS significantly improves, confirming that EGCG is successfully bound to the SPI-CS complex. However, the binding efficiency of Example 3 is similar to that of Example 2 (65.17% and 64.38%, respectively). This may be due to the fact that the EGCG concentration reaches or exceeds the number of SPI side chain binding sites in the reaction system and the limited amino acid residues available for binding in SPI. In addition, the binding efficiency of Comparative Example 4 is similar to that of Example 2 (62.63% and 64.38%, respectively). This may be due to the high dispersion of SPI under these conditions, the increase in binding sites with EGCG, but the poor binding stability (lower potential value). In contrast, the binding efficiency of Comparative Example 5 is lower than that of Example 2 and lower than that of Example 1, indicating that the intermolecular attraction between SPI-CS and EGCG is weakened in the presence of salt ions.

[0122] The prior art generally believes that salt ions promote complex aggregation. However, a comparison between Comparative Example 5 and Example 2 demonstrates that salt ions negatively impact the formation of a stable SPI-CS-EGCG ternary complex, and neutral pH is also detrimental to the formation of a stable ternary complex. Therefore, to further determine the optimal EGCG concentration for forming a high-performance ternary complex, subsequent experiments were conducted using Comparative Example 5 and Examples 1-3.

[0123] Performance Test Example 4 Surface Properties and Formation Mechanism of SPI-CS-EGCG Ternary Complex

[0124] To further confirm the involvement of hydrophobic interactions in the SPI-CS-EGCG ternary complex, samples were analyzed by surface hydrophobicity. Surface hydrophobicity reflects the number of hydrophobic groups on the protein surface and depends on the number of nonpolar amino acid residues exposed to the surrounding aqueous phase, thus exhibiting group additivity. Figure 5 Figure A shows the surface hydrophobicity of the SPI-CS-EGCG ternary complex. Comparison between Comparative Example 1 and Examples 1-3 shows that the surface hydrophobicity of SPI decreases significantly with increasing EGCG concentration, indicating a significant reduction in the number of hydrophobic groups on the protein surface. The aromatic rings of EGCG may bind to the hydrophobic groups of SPI through hydrophobic interactions, thereby reducing the number of ANS binding sites. This reduction in surface hydrophobicity may be attributed to protein aggregation induced by polyphenol cross-linking or the introduction of new hydrophilic groups.

[0125] Figure 5B shows the FTIR spectra of the SPI-CS complex with different concentrations of EGCG. The introduction of EGCG did not lead to the appearance of new absorption bands, indicating that the interaction involved was non-covalent and no covalent bond was formed. However, the key peaks of Comparative Example 1 changed significantly, including the key peaks at 3272.6 cm -1 (NH and OH stretching vibration), 2929.3cm -1 (CH stretching vibration of -CH2 and -CH3 groups), 1630.3 cm -1 (amide I vibration, C=O stretching) and 1514.3 cm -1 (Amide II vibration, CN and NH bending). These changes indicate that EGCG affects the interaction mechanism within the SPI-CS complex. The NH stretching peaks of Example 1, Example 2, and Example 3 red-shift to 3278.3, 3283.8, and 3277.5 cm, respectively. -1 , indicating spectral changes and the formation of hydrogen bonds between the amino and carboxyl groups of SPI and the hydroxyl groups of EGCG. The strength of these hydrogen bonds depends on the EGCG concentration, as higher concentrations of phenolic hydroxyl groups promote the formation of hydrogen bonds between SPI-CS and EGCG. In addition, the amide I peaks of Examples 1, 2, and 3 shifted to 1627.4 cm -1 、1627.4cm -1 and 1625.6cm -1 , compared with 1630.0cm of Comparative Example 1 -1 The peaks were blue-shifted. The amide II peaks (1517.7 cm -1 、1520.1cm -1 and 1521.5cm -1 ) and 1514.3cm of Comparative Example 1 -1 The peaks were red-shifted compared to those of Comparative Example 1. These changes indicate that hydrophobic and electrostatic interactions are involved between bioactive molecules and that EGCG induces changes in the secondary and tertiary structures of proteins. The CH stretching vibrations of Examples 1, 2, and 3 also shifted from 2929.3 cm-1 in Comparative Example 1 to 3929.3 cm-2 in Comparative Example 1. -1 shifted to 2929.8 cm in the SPI-CS-EGCG complex -1 、2932.8cm -1 and 2930cm -1 , indicating that EGCG may be involved in hydrophobic interactions. Taken together, these FTIR results further confirmed the formation of the SPI-CS-EGCG ternary complex, and EGCG promoted the stability of the particles through hydrogen bonding, hydrophobic interactions, and electrostatic forces.

[0126] The thermal stability of the SPI-CS-EGCG ternary complex was analyzed using DSC thermograms. Figure 5 As shown in Figure C, after EGCG was attached to SPI-CS in Examples 1-3, the denaturation temperatures of the SPI-CS-EGCG ternary complex increased to 125°C, 132°C, and 133°C (Examples 1, 2, and 3), respectively, significantly higher than those in Comparative Example 1, indicating improved thermal properties of the protein. Therefore, the increase in denaturation temperature indicates that the thermal stability of the SPI-CS-EGCG ternary complex is superior to that of the SPI-CS binary complex particles prepared in Comparative Example 1.

[0127] Performance Test Example 5 Rheological and Friction Properties of SPI-CS-EGCG Ternary Composite

[0128] Figure 6 A shows the rheological properties of the SPI-CS-EGCG ternary complex dispersions at different EGCG concentrations, including apparent viscosity, storage modulus (G′), and loss modulus (G″). Figure 6 Aa shows the relationship between the apparent shear viscosity and shear rate of these dispersions. -1 Increase to 100s -1 , the apparent viscosity of the dispersions of Comparative Example 1 and Examples 1-3 gradually decreased, indicating that the dispersions exhibited shear-thinning behavior, which is a characteristic of pseudoplastic fluids. Comparing Comparative Example 1 with Examples 1-3, it can be seen that the apparent viscosity of the SPI-CS-EGCG ternary complex increases with the increase in EGCG concentration. This is because non-covalent interactions promote the formation of soluble aggregates, which contribute to the formation of a network structure, thereby increasing the flow resistance, and can give products such as puddings and yogurt a delicate touch and a full chewing feel, avoiding a granular or loose structure. The Herschel-Bulkley model is used to describe the flow behavior of the SPI-CS-EGCG ternary complex. As shown in Table 2, the ternary complexes obtained in Examples 1-3 exhibit higher yield stress and consistency coefficient (K) values ​​than the SPI-CS binary complex obtained in Comparative Example 1. The flow index values ​​of all samples are less than 1, confirming the shear-thinning behavior.

[0129] The viscoelastic behavior of SPI-CS-EGCG dispersion was analyzed by measuring the changes of storage modulus (G′) and loss modulus (G″) with frequency. Figure 6As shown in Figures 6Ab and 6Ac, for SPI-CS-EGCG dispersions with different EGCG concentrations, G′ and G″ increase with increasing frequency in the frequency range of 0.1-10 Hz, indicating that the modulus is frequency-dependent. Throughout the frequency range of the study, the G′ value is always higher than G″, indicating that the dispersion is mainly elastic. In addition, according to the comparison between Comparative Example 1 and Examples 1-3, the G′ and G″ values ​​further increase with increasing EGCG concentration, indicating that non-covalent interactions enhance molecular cross-linking, thereby improving viscoelastic properties. Compared with Examples 1-3, the tangent angle tanδ value of the dispersion of Comparative Example 1 is higher (results not shown), indicating that the dispersions of Examples 1-3 are more elastic, while the dispersion of Comparative Example 1 is more viscous. The modulus at 1 Hz shown in Table 2 shows that the addition of EGCG significantly increases G′ and G″. Highly elastic additives can help form a fibrous network, simulate the bite and resilience of animal meat, and improve the problem of fragile and powdery taste of vegetarian meat.

[0130] Table 2 Flow curves and rheological parameters of the ternary complex SPI-CS-EGCG

[0131]

[0132] Different letters represent different significance levels (p < 0.05).

[0133] Tribology is an effective tool for characterizing the properties of foods during oral processing, such as smoothness and creaminess. A polydimethylsiloxane (PDMS) membrane with a surface roughness of less than 50 nm and a contact angle of 95.33° was prepared and used as a lower contact surface to simulate the tongue. A 1 ml test sample was spread onto the lower contact surface to form a layer approximately 2 mm thick. The coefficient of friction was measured at 37°C and a constant normal force of 2 N. Figure 6 B shows the friction curves of SPI-CS-EGCG ternary composites with different EGCG contents. All samples showed similar trends. In the initial sliding stage, the friction curve shows an increase in friction, indicating high friction in the boundary friction region. In this region, the gap size is small, allowing only a limited number of particles to enter between the contact surfaces and be trapped in the surface grooves, and these particles carry a relatively high load. Therefore, the friction coefficient is high because there is direct physical contact between the two contacting surfaces (or only a very small amount of lubricant is present). As the speed increases, the friction coefficient decreases, similar to the mixed friction region in the Stribeck curve. In this region, more particles enter the contact zone, promoting the separation of the friction surfaces. The larger gap enables the particles to roll freely, producing a ball bearing lubrication effect.

[0134] Regarding the effect of EGCG on lubrication behavior, Examples 1 and 2 reduced friction in the entire sliding speed range compared to Comparative Example 1 (e.g. Figure 6 Ba). This reduction in friction may be attributed to the non-covalent interactions between EGCG and SPI-CS, which may have changed the surface properties of the complex and improved its lubricity. The reduction in friction is consistent with the observations of foods rich in phenolic compounds, partly due to the non-covalent interactions between the hydroxyl groups in the phenolic compounds and salivary proteins, which lead to the formation of a precipitated complex, thereby reducing oral lubricity. The non-covalent interactions between SPI-CS and hydroxyl groups compete for the binding sites of phenolic compounds, which can limit their interaction with salivary proteins. In this case, the experimental results confirmed that the non-covalent interactions between SPI-CS and EGCG played an important role in hindering the ability of EGCG to reduce friction, thereby improving the lubricity of the ternary complex. Therefore, the increase in the lubricity of the SPI-CS-EGCG ternary complex after EGCG addition may be related to the increase in viscosity. The higher EGCG concentration in Example 3 compared to Comparative Example 1 will lead to increased friction, which may be due to the saturation of binding sites on the SPI-CS complex, resulting in poor surface modification effect and the possible formation of larger, more aggregated structures, increasing surface roughness and friction.

[0135] In order to better simulate the oral processing conditions, the effect of saliva on lubrication was also considered. The PDMS membrane was soaked in artificial saliva for at least 2 hours and then rinsed with water to form a saliva-coated surface. The remaining conditions were consistent with the tribological test. On the surface treated with saliva, the friction of Examples 1-3 over the entire speed range was significantly reduced (e.g., Figure 6 Bb and 6Bc). This reduction in the friction coefficient is likely attributed to the positively charged surface of the composite particles, as shown by the zeta potential results. This positive charge likely facilitates the adsorption of the particles onto the PDMS surface, which retains the mucin film after immersion in artificial saliva. These adsorbed particles may act as "ball bearings," filling friction gaps and providing a smoother sliding experience. These results demonstrate that the non-covalent interaction between EGCG and SPI-CS effectively weakens the interaction between EGCG and human saliva, thereby enhancing the lubricity of the ternary composite.

[0136] Performance Test Example 6 Microstructure of SPI-CS-EGCG Ternary Complex

[0137] The microstructure morphology of SPI-CS-EGCG dispersions at different EGCG concentrations was analyzed using AFM and SEM. Figure 7A shows typical 2D and 3D views of AFM images. The particles of the composites of Examples 1-3 exhibited heterogeneous particle structures, and the particle size was larger than that of Comparative Example 1, which may be due to the interaction between EGCG and SPI-CS. According to the comparison between Comparative Example 1 and Examples 1-3, it can be seen that the morphology of the ternary composite was significantly affected by the EGCG concentration. The composite of Example 3, which had the highest EGCG concentration, exhibited a more uniform elliptical rod-like structure and a smaller particle size than that of Comparative Example 1 and Examples 1-2. Figure 3 The particle size results in B are consistent.

[0138] The surface morphology of the SPI-CS-EGCG complex was observed using SEM. Figure 7 As shown in Figure B, the ternary complex exhibits a blocky structure with particles bound together. This binding is likely due to the formation of a molecular interaction network between adjacent particles. The particle surfaces of Examples 1 and 2 are relatively smooth, while those of Example 3 are roughened. These differences are attributed to the protective effect of CS on the particle outer layer at the lower EGCG concentrations in Examples 1 and 2.

[0139] Performance Test Example 7 Molecular Docking Conformation and Interaction Analysis of SPI-CS-EGCG Ternary Complex

[0140] We also investigated molecular docking between SPI-CS and EGCG to identify the optimal binding site and interaction mode. First, the main components of SPI are 7S and 11S. Therefore, CS was docked to the 7S and 11S proteins, respectively. The SPI-CS complexes with the best docking scores (7S-CS: -5.7 kcal / mol and 11S-CS: -8.0 kcal / mol) were then selected for secondary docking with EGCG. Finally, the binding site with the lowest binding energy was selected. Therefore, the 7S-CS-EGCG (-7.2 kcal / mol) and 11S-CS-EGCG (-7.4 kcal / mol) complexes were chosen as representatives for detailed analysis of high-affinity binding interactions. Specifically, the EGCG structure was downloaded from the PubChem database and geometry optimized using the mmff94 force field to correct for any unreasonable bond lengths and angles. The protein, chitosan, and EGCG structures were processed using Autodock Tools (Vision: 1.5.6), followed by the addition of hydrogens, Gasteiger charges, and nonpolar hydrogens. During the docking process, blind docking was performed using a docking box, with the entire protein as the search space. Semi-flexible molecular docking was performed using the Vina program. After docking, the optimal complex conformation was selected for further analysis based on the binding posture and binding energy. The docking results showed that the binding affinity of the 11S-CS-EGCG complex was slightly higher than that of the 7S-CS-EGCG complex, indicating a stronger interaction. Figure 8Figure 2 shows the interactions of CS and EGCG with 7S and 11S proteins. (A) 7S-CS-EGCG complex; (B) EGCG in the hydrophobic pocket of 7S protein; (C) 3D interaction diagram of 7S-CS-EGCG; (D) 2D interaction diagram of 7S-CS-EGCG; (E) 11S-CS-EGCG complex; (F) EGCG in the hydrophobic pocket of 11S protein; (G) 3D interaction diagram of 11S-CS-EGCG; (H) 2D interaction diagram of 11S-CS-EGCG (the redder the hydrophobic pocket, the more hydrophobic the region). For 7S protein, EGCG interacts with Ala 392, Leu 350, Leu 262, Val 327, Pro 324, Tyr 323, Leu 285, and Gln 305 through van der Waals interactions. EGCG also has a hydrogen bond with Asp 391. Furthermore, hydrophobic interactions were observed between EGCG and Leu 263, Ile 283, and Phe 325. Notably, three hydrogen bonds were observed between CS and EGCG, confirming the presence of a ternary complex. Regarding the 11S protein, EGCG interacted with Glu 252, Pro 298, Trp 250, Pro 302, His 297, Gln 92, Glu 254, Lys 90, and Ile 246 via van der Waals interactions, and with Val 245, Ser 244, Gln 300, Gln 251, and Gln 253 via hydrogen bonds. A single pi-alkyl interaction was observed between EGCG and Pro 91. Furthermore, a single hydrogen bond was observed between CS and EGCG, confirming the formation of a ternary complex.

[0141] The results indicate that the binding of EGCG to the SPI-CS complex is facilitated by multiple types of interactions, including van der Waals forces, hydrogen bonds, and hydrophobic interactions. These diverse interactions contribute to the stability and specificity of the ternary complex. Hydrogen bonds exist between CS and EGCG in both the 7S and 11S complexes, demonstrating the importance of these non-covalent interactions in maintaining the structural integrity of the SPI-CS-EGCG complex.

[0142] In summary, the SPI-CS-EGCG ternary complex prepared by the method of the present invention has good thermal stability, rheological and tribological properties, and can be widely used in the food industry as a functional food additive to improve the taste, stability and nutritional properties of food.

[0143] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a SPI-CS binary complex, characterized in that: The preparation method of the binary complex comprises the following steps: (1) Prepare SPI solution and CS solution respectively; (2) adjusting the pH of the SPI solution to 5.6 and heating it at 95°C for more than 30 min to obtain SPI microgels; (3) mixing the SPI microgel with the CS solution to obtain an SPI-CS mixed solution; (4) Centrifuging the SPI-CS mixed solution to obtain the SPI-CS binary complex.

2. The SPI-CS binary complex obtained by the preparation method according to claim 1.

3. A method for preparing a ternary complex of SPI-CS-EGCG, characterized in that: The preparation method of the ternary complex comprises the following steps: (1) Prepare SPI solution, CS solution and EGCG solution respectively; (2) adjusting the pH of the SPI solution to 5.6 and heating it at 95°C for more than 30 min to obtain SPI microgels; (3) mixing the SPI microgel with the CS solution to obtain an SPI-CS mixed solution; (4) The SPI-CS mixed solution and the EGCG solution were mixed and stirred, and centrifuged to obtain the SPI-CS-EGCG ternary complex.

4. The preparation method according to claim 3, characterized in that The SPI solution preparation method comprises the following steps: mixing SPI powder with water, heating and stirring until completely dissolved, and then performing high-pressure homogenization to obtain the SPI solution; preferably, the heating temperature is 50-60° C., the stirring speed is 500-600 rpm, the stirring time is 2-3 hours, and the homogenization pressure is 4000-6000 psi; preferably, the concentration of the SPI solution is 0.9%.

5. The preparation method according to claim 3, characterized in that The preparation method of the CS solution comprises the following steps: mixing CS powder with water and stirring until fully hydrated; preferably, the stirring speed is 500-600 rpm and the stirring time is 1-2 hours; preferably, the concentration of the CS solution is 0.1%.

6. The preparation method according to claim 3, characterized in that The preparation method of the EGCG solution comprises the following steps: mixing EGCG powder with water and stirring until fully hydrated; preferably, the stirring speed is 500-600 rpm and the stirring time is 1-2 hours; preferably, the concentration of the EGCG solution is 50 mg / mL.

7. The preparation method according to claim 3, characterized in that: The mass ratio of SPI to CS in the SPI-CS mixed solution is 8 to 10:1; preferably, the mass ratio of SPI to CS is 9:1; preferably, in step (3), the stirring rate is 8000 to 12000 rpm, and the stirring time is 1 to 4 minutes; preferably, the stirring rate is 10000 rpm, and the stirring time is 2 minutes.

8. The preparation method according to claim 3, wherein: In step (4), the mass percentage of SPI in the mixed solution of the SPI-CS mixed solution and the EGCG solution is 0.405% to 0.45%, the mass percentage of CS is 0.045% to 0.05%, and the mass concentration of EGCG is 0.10 mg / mL to 0.50 mg / mL; preferably, in step (4), the stirring speed is 400 to 600 rpm, the stirring time is 40 to 80 min, the centrifugal force is 8000 to 10000 g, and the centrifugation time is 20 to 40 min; further preferably, the stirring speed is 500 rpm, the stirring time is 60 min, the centrifugal force is 9000 g, and the centrifugation time is 30 min.

9. The SPI-CS-EGCG ternary complex prepared according to the preparation method according to any one of claims 3 to 8.

10. Use of the SPI-CS binary complex according to claim 2 or the SPI-CS-EGCG ternary complex according to claim 9 in food or medicine; preferably, the use comprises one of the following applications: (1) Applications for improving the resilience of food or medicine; (2) Applications to reduce friction of food or medicine; (3) Applications to enhance the lubricity of food or medicine.

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