Protein-polysaccharide-polyphenol ternary complex as well as preparation method and application thereof

Abstract

The invention discloses a protein-polysaccharide-polyphenol ternary complex as well as a preparation method and application thereof. The protein-polysaccharide-polyphenol ternary complex is prepared by mixing rice bran protein, sodium alginate and procyanidine serving as raw materials. The invention relates to a Pickering emulsion which is characterized in that the Pickering emulsion is used as a stabilizer and is delivered with an oil-phase substance or the oil-phase substance and quercetin (Que). A multispectral technology and microstructure analysis show that a long-chain fiber structure is formed through interaction of hydrogen bonds and static electricity under the condition that the pH is adjusted to be 9 in the preparation process, and the long-chain fiber structure of the ternary compound is adsorbed on an oil-water interface to form a compact net-shaped structure. The structures help to improve the stability of the Pickering emulsion. The stable Pickering emulsion prepared from the ternary complex forms a compact net structure when the pH value is 9, and pepsin hydrolysis can be resisted. According to the invention, the effective delivery of the Que is realized in the intestinal digestion process, and the biological accessibility of the Que is improved.

Description

Technical Field

[0001] The present invention relates to a protein-polysaccharide-polyphenol ternary complex and a preparation method and application thereof, in particular to a rice bran protein-sodium alginate-proanthocyanidin ternary complex and a preparation method and application thereof. Background Art

[0002] Quercetin (Que) is a naturally occurring polyphenolic compound with diverse biological functions, including anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. It has shown great potential in the food industry. However, limitations of Que, such as low water solubility, poor stability, and low bioaccessibility, restrict its application in the food industry. Therefore, designing suitable drug delivery systems to enhance the bioactivity of Que is crucial. Currently, Pickering emulsions are widely used as carriers to effectively encapsulate bioactive substances and improve their bioaccessibility. Solid particles play a crucial role in stabilizing Pickering emulsions by irreversibly attaching to the oil-water interface, forming an interfacial layer that significantly enhances stability. Unlike traditional emulsions, Pickering emulsions exhibit resistance to aggregation and Ostwald ripening and are environmentally friendly. Proteins, polysaccharides, polyphenols, and their complexes are commonly used to stabilize Pickering emulsions. These complexes exhibit greater applicability than the individual substances. Proteins are often used as stabilizers due to their amphiphilic nature. However, proteins alone have poor emulsifying properties. Polysaccharides can improve these properties by forming noncovalent interactions with proteins. Furthermore, polyphenols enhance the stability and antioxidant capacity of Pickering emulsions through noncovalent interactions with protein-polysaccharide complexes. Rice bran protein (RBP) is extracted from rice bran and consists of albumin (24%-43%), globulin (13%-36%), glutenin (22%-45%), and prolamin (1%-5%). It is a valuable plant protein containing 18 amino acids and 8 essential amino acids, which closely matches the FAO / WHO recommended profile. Due to its unique nutritional advantages and low allergenicity, RBP is widely used in functional foods as a health-promoting ingredient. However, due to the presence of a large number of sulfhydryl groups and disulfide bonds, RBP has poor water solubility, limiting its effectiveness as a sole stabilizer for emulsions. Sodium alginate (SA) is a linear anionic polysaccharide extracted from seaweed, composed of β-D-mannuronic acid and α-L-guluronic acid linked by (1→4) glycosidic bonds. The molecular chain contains abundant carboxyl groups. Proanthocyanidins (PC), also known as condensed tannins, are widely found in fruits, berries, nuts and seeds. Their polyphenolic properties give them significant biological activities, including antioxidant, anti-inflammatory, anti-diabetic and anti-cancer properties. Currently, most research focuses on protein-polysaccharide and protein-polyphenol stabilized emulsion systems. However, research on protein-polysaccharide-polyphenol ternary complexes as Pickering emulsion stabilizers is relatively limited. As amphiphilic molecules, the charge state and conformation of proteins change significantly with pH, ​​and these changes directly affect their emulsifying activity under different pH conditions. Therefore, it is of great significance to study the stability of protein-polysaccharide-polyphenol ternary complexes on Pickering emulsions under different pH conditions. Summary of the Invention

[0003] Objectives of the invention: One objective of the present invention is to provide a protein-polysaccharide-polyphenol ternary complex. Another objective of the present invention is to provide a method for preparing the protein-polysaccharide-polyphenol ternary complex. A third objective of the present invention is to provide a Pickering emulsion prepared using the protein-polysaccharide-polyphenol ternary complex.

[0004] Technical solution: The protein-polysaccharide-polyphenol ternary complex described in the present invention is obtained by mixing rice bran protein, sodium alginate and proanthocyanidins as raw materials.

[0005] Furthermore, the mass ratio of rice bran protein, sodium alginate and proanthocyanidins is 1:2:(0.05-0.5).

[0006] The method for preparing the protein-polysaccharide-polyphenol ternary complex of the present invention comprises the following steps:

[0007] The rice bran protein is dispersed in water, the pH is adjusted, the mixture is centrifuged, the supernatant is collected, and the mixture is mixed with a sodium alginate solution. The pH is adjusted again, proanthocyanidins are added, and the mixture is stirred.

[0008] Further, the pH is adjusted to be alkaline, preferably above 12, and then the pH is adjusted again to be 5-9.

[0009] The present invention also includes a Pickering emulsion comprising the protein-polysaccharide-polyphenol ternary complex of the present invention as a stabilizer.

[0010] Furthermore, peanut oil is included, and the mass ratio of the protein-polysaccharide-polyphenol ternary complex to the peanut oil is (1-2):(1-2). The protein-polysaccharide-polyphenol ternary complex and the peanut oil are mixed and sheared.

[0011] Furthermore, quercetin is added to peanut oil, which is then mixed with a protein-polysaccharide-polyphenol ternary complex and sheared. The ratio of quercetin to peanut oil and the protein-polysaccharide-polyphenol ternary complex is 0.1:(1-2):(1-2). The shearing is performed at 10,000 rpm for 2 minutes.

[0012] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the protein-polysaccharide-polyphenol ternary complex of the present invention forms different fiber structures under different pH conditions: a short-chain fiber structure is formed by hydrogen bonding and hydrophobic interactions under pH 5, a medium-chain fiber structure is formed by hydrogen bonding, hydrophobic and electrostatic interactions under pH 7, and a long-chain fiber structure is formed by hydrogen bonding and electrostatic interactions under pH 9. At a pH of 9, the long-chain fiber structure of the protein-polysaccharide-polyphenol ternary complex is adsorbed on the oil-water interface to form a dense network structure. These structures help to improve the stability of the Pickering emulsion. In particular, the Pickering emulsion stabilized by the protein-polysaccharide-polyphenol ternary complex forms a dense network structure at a pH of 9, which can better resist pepsin hydrolysis. In addition, they achieve effective delivery of Que during intestinal digestion and improve the bioaccessibility of Que. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The particle size, zeta potential, fluorescence spectrum, UV spectrum, FTIR spectrum and XRD patterns of PC, SA, RBP, RBP-SA and RBP-SA-PC under different pH conditions in Example 1 are shown;

[0014] Figure 2 TEM images, scanning electron microscope images, thermogravimetric curves, and contact angle graphs of PC, SA, RBP, RBP-SA, and RBP-SA-PC under different pH conditions in Example 1;

[0015] Figure 3 Optical microscope images, droplet sizes, and zeta potential diagrams of RBP PEs, RBP-SA PEs, and RBP-SA-PC PEs under different pH conditions in Example 2;

[0016] Figure 4 CLSM images of RBP-SA PEs and RBP-SA-PC PEs under different pH conditions in Example 2;

[0017] Figure 5 The SEM images, rheological images and low-field NMR images of RBP PEs, RBP-SA PEs and RBP-SA-PC PEs in Example 3 are shown;

[0018] Figure 6 is a schematic diagram of the stabilization mechanism and DPPH and ABTS radical scavenging activities of RBP PEs, RBP-SA PEs, and RBP-SA-PC PEs in Example 4;

[0019] Figure 7are CLSM images of the in vitro digestion of Que-loaded RBP-SA PEs and Que-loaded RBP-SA-PC PEs in Example 5;

[0020] Figure 8 Schematic diagram of the bioaccessibility and in vitro digestion mechanism of Que in Example 5. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1 Preparation method of RBP-SA-PC

[0023] RBP was dissolved in distilled water with stirring and stored at 4°C overnight to obtain a 2% (w / w) dispersion. Subsequently, the pH of the RBP dispersion was adjusted to 12.0 using 1 mol / L NaOH solution with continuous stirring. The mixture was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected as the RBP solution. SA was dissolved in distilled water with continuous stirring to prepare a 2% (w / w) SA solution. The RBP solution and SA solution were mixed in a 1:2 (w / w) ratio, and the pH was adjusted to 5, 7, and 9, respectively. The mixture was stirred continuously for 2 hours to form an RBP-SA solution. PC powder of varying weights was dissolved in RBP-SA and stirred continuously for 7 hours to obtain RBP-SA-PC. The resulting RBP-SA-PC solutions consisted of RBP-SA and various concentrations of PC, designated RBP-SA-0.05% PC, RBP-SA-0.1% PC, RBP-SA-0.3% PC, and RBP-SA-0.5% PC. RBP and RBP-SA were used as control groups. The above groups included three conditions of pH 5, 7, and 9.

[0024] The RBP, RBP-SA, RBP-SA-PC, SA, and PC prepared in this example were tested for particle size, potential, fluorescence, UV, IR, XRD, TEM, SEM, thermogravimetric, and contact angle. Figure 1-2 shown.

[0025] Figure 1 Figure 1 shows the particle size, zeta potential, fluorescence spectrum, UV spectrum, FTIR spectrum, and XRD patterns of PC, SA, RBP, RBP-SA, and RBP-SA-PC at different pH conditions in Example 1. (A represents particle size, B represents zeta potential, C represents fluorescence spectrum, D represents UV spectrum, E represents FTIR spectrum, and F represents XRD pattern.) Samples with different capital letters or samples with the same pH value have significantly different values ​​(p < 0.05).

[0026] Depend on Figure 1As shown in Figures A and B, the particle size of RBP reaches its maximum at pH 5, while the absolute value of its zeta potential is lowest at this pH. This pH is close to the isoelectric point (pI) of RBP. At this point, intermolecular electrostatic repulsion is minimized, favoring aggregation. At pH 7 and 9, as the pH is far from the pI, strong electrostatic repulsion exists between RBP molecules, inhibiting RBP aggregation. This results in a decrease in RBP particle size and an increase in the absolute value of its zeta potential. Compared with RBP, RBP-SA exhibits a larger particle size and higher absolute value of its zeta potential, indicating interactions between RBP and SA. These interactions may involve hydrogen bonding, electrostatic and hydrophobic interactions, and steric hindrance. With increasing pH, the particle size of RBP-SA decreases and the absolute value of its zeta potential increases. As the pH moves away from the pI, the negative charge density of RBP increases, and SA deprotonates, acquiring a negative charge. This strengthens the electrostatic repulsion between RBP and SA, promoting the formation of smaller, soluble complexes. Notably, the addition of PC significantly reduces the particle size of the ternary complex. As PC concentration increases, the particle size of RBP-SA-PC decreases and the absolute value of its zeta potential increases. This is likely due to the interaction of the phenolic hydroxyl groups of PC with RBP and SA, forming a more compact structure. At pH 9, the particle size of RBP-SA-PC is smallest and the absolute value of its zeta potential is largest. This is likely due to the denser structure at this pH, resulting in a smaller particle size. Under these conditions, PC undergoes maximum deprotonation, and both PC and RBP-SA carry more negative charges, thereby increasing the absolute value of the zeta potential of RBP-SA-PC.

[0027] The intrinsic fluorescence of proteins is mainly due to the fluorescence emitted by tryptophan and tyrosine residues when excited at 280nm. Intrinsic fluorescence spectra usually indicate structural changes of proteins by revealing changes in the microenvironment of hydrophobic amino acids. Figure 1 As shown in Figure C, the addition of SA significantly reduced the fluorescence intensity of RBP-SA, indicating that SA quenched the fluorescence signal of RBP. This suggests a change in the polarity of the microenvironment surrounding the aromatic amino acids in RBP. The fluorescence intensity of RBP-SA-PC decreased further, demonstrating a more pronounced quenching effect. A significant shift in the maximum fluorescence emission peak was observed compared to RBP-SA, indicating that the fluorophore shifted to a more hydrophobic environment after successful complexation of RBP-SA-PC. The fluorescence intensity of RBP-SA-PC decreased with increasing PC concentration, demonstrating a concentration-dependent quenching effect of PC on tryptophan and tyrosine residues in RBP. Interestingly, the fluorescence intensity of RBP-SA-PC also decreased with increasing pH. This pH-dependent decrease in fluorescence intensity may be due to the interaction between PC and RBP-SA, resulting in greater protein structural unfolding and stronger fluorescence quenching when the pH is far from the pI.

[0028] Ultraviolet spectroscopy is a method for studying the interactions between proteins, polysaccharides, and polyphenols. Figure 1 As shown in Figure D, RBP exhibits an absorption peak near 280 nm due to the absorption of tyrosine and tryptophan. After the addition of SA, the absorption peak position of RBP-SA did not shift significantly, but the peak intensity increased slightly. Compared with RBP-SA, the absorption peak position of RBP-SA-PC shifted and the intensity increased, indicating the interaction between PC, RBP, and SA. The formation of RBP-SA-PC increases the π→π* transition energy, changes the microenvironment of some amino acid residues in RBP, and causes structural changes in RBP. In addition, the absorption peak intensity of RBP-SA-PC increases with increasing PC concentration, and the absorption peak position continues to shift. This indicates that higher PC concentrations cause more obvious conformational changes in RBP, exposing more tryptophan and tyrosine residues. At the same time, the absorption peak position of RBP-SA-PC does not change significantly with increasing pH.

[0029] Depend on Figure 1 E can be seen in the PC spectrum at pH 5, 3416 cm -1 The peak at 2935 cm is the OH bond stretching vibration. -1 The peak at 1612cm is the CH bond stretching vibration. -1 The peak at 3445 cm corresponds to the stretching vibration of the carbonyl group. -1 The peak at 2929cm is the stretching vibration of the OH bond. -1 The peak at 1616cm is the stretching vibration of the C-H bond. -1 and 1417cm -1 The peaks at the corresponding COO - The asymmetric and symmetric stretching vibrations of PC and SA vary with the pH value, resulting in the shift of their characteristic peaks under different pH conditions. In the RBP spectrum at pH 5, 3403 cm -1 The peak at 2926 cm is the stretching vibration of the OH bond. -1 The peak at 1656cm is the stretching vibration of the C-H bond. -1 The peak is the stretching vibration of C=O. 1600-1700cm -1 Peaks within this range are usually associated with the secondary structure of proteins. Under different pH conditions, the characteristic peaks of RBP shifted, indicating that pH can change its secondary structure.

[0030] Compared with RBP, the OH bond stretching vibration peak of RBP-SA is significantly shifted, indicating hydrogen bonding between RBP and SA. Due to the presence of phenolic hydroxyl groups in PC, the OH bond stretching vibration peak of RBP-SA-PC is significantly shifted compared with RBP-SA, indicating hydrogen bonding between PC and RBP-SA. Compared with RBP, the C-H bond stretching vibration peak of RBP-SA is shifted at pH 5 and 7, indicating hydrophobic interactions between RBP and SA under these pH conditions. Similarly, the C-H bond stretching vibration peak of RBP-SA-PC is shifted compared with RBP-SA, indicating hydrophobic interactions between PC and RBP-SA at pH 5 and 7. Compared with RBP, the amide I band of RBP-SA is significantly shifted, indicating electrostatic interactions between RBP and SA. Similarly, the amide I band of RBP-SA-PC is shifted at pH 7 and 9 compared with RBP-SA, indicating electrostatic interactions between PC and RBP-SA.

[0031] In summary, the formation of RBP-SA-PC is highly dependent on pH conditions. At pH 5, RBP-SA-PC is primarily driven by hydrogen bonding and hydrophobic interactions between RBP-SA and PC. At pH 7, the formation of RBP-SA-PC involves hydrogen bonding, hydrophobic interactions, and electrostatic interactions. At pH 9, RBP-SA-PC is primarily formed through hydrogen bonding and electrostatic interactions.

[0032] XRD analysis is a method used to identify whether a molecular structure is crystalline or amorphous. Figure 1 As can be seen in Figure F, the broad peak of PC with a diffraction angle of about 22° indicates its amorphous nature, and pH has no significant effect on this amorphous structure. Similarly, SA shows two broad peaks near 13° and 25°, and pH has no significant effect on these diffraction peaks. RBP has five diffraction peaks near 20°, 27°, 31°, 45° and 56°, indicating its crystalline structure. The pH value affects the intensity of the RBP diffraction peak, but has no significant effect on its position. RBP-SA and RBP-SA-PC show amorphous diffusion peaks near 13° and 25°, indicating that they have an amorphous structure. Neither pH nor PC concentration has a significant effect on the amorphous structure of RBP-SA-PC.

[0033] Figure 2TEM images, scanning electron microscope images, thermogravimetric images and contact angle (θo / w) diagrams of PC, SA, RBP, RBP-SA, and RBP-SA-PC under different pH conditions in Example 1, wherein A is the TEM image of RBP, RBP-SA, RBP-SA-0.1% PC and RBP-SA-0.5% PC (all scale bars are 100 nm), B is the scanning electron microscope image of PC, SA, PC, RBP, RBP-SA and RBP-SA-PC (all scale bars are 100 μm) under different pH conditions, C is the thermogravimetric image of PC, SA, RBP, RBP-SA and RBP-SA-PC under different pH conditions, D is the θo / w of RBP and RBP-SA at pH 9; and the θo / w of RBP-SA-0.5% PC at pH 5, 7 and 9.

[0034] Depend on Figure 2 As shown in Figure A, pH significantly affects RBP morphology: at pH 5, RBP-SA forms nearly spherical particles, at pH 7, it forms irregular particles, and at pH 9, it forms irregular clusters. Upon addition of SA, RBP-SA forms distinct fibrous structures. Interestingly, the addition of PC further densifies these structures, forming short-chain fibers at pH 5, medium-chain fibers at pH 7, and long-chain fibers at pH 9. This pH-dependent behavior is likely due to changes in the noncovalent interaction between RBP-SA and PC, which regulates the length and density of the fiber network.

[0035] Depend on Figure 2 As shown in Figure B, the surface morphologies of PC, SA, RBP, RBP-SA, and RBP-SA-PC under different pH conditions are as follows: Figure 2 As shown in Figure B, RBP exhibits a lamellar structure at pH 5 and a dendritic, tangled network at pH 7 and 9. Meanwhile, PC exhibits irregular fragments at different pH values, while SA exhibits a network-like structure. Compared to RBP, RBP-SA exhibits an irregular lamellar structure. RBP-SA-PC exhibits both lamellar and dendritic structures, likely due to the abundant phenolic hydroxyl groups in PC forming non-covalent bonds with RBP-SA, resulting in a denser structure.

[0036] Depend on Figure 2As can be seen in Figure C, the thermal decomposition of RBP occurs in three stages. The first stage, with a temperature range of 30-100°C, is mainly attributed to water evaporation. The second stage occurs between 200-280°C and involves the denaturation of RBP and the beginning of the decomposition of its primary structure. The third stage occurs between 300-400°C and is characterized by the decomposition of the protein backbone and the volatilization of protein fragments, resulting in a sharp loss of mass of RBP. For SA, water evaporation occurs between 30-100°C, after which the polysaccharide structure begins to degrade in the range of 200-300°C. The thermal decomposition of PC is also divided into three stages: the first stage (30-100°C) is caused by water evaporation, the second stage (150-225°C) is caused by the breaking of weak chemical bonds, and the third stage (250-350°C) is caused by the degradation of the polyphenol backbone, resulting in a large mass loss of PC.

[0037] The thermal decomposition of RBP-SA is divided into two stages. The first stage occurs between 30-100°C and is due to mass loss caused by water evaporation. The second stage occurs between 200-300°C and involves the degradation of most of the RBP-SA structure. The initial decomposition temperature of RBP-SA is higher than that of RBP, indicating that SA enhances the thermal stability of RBP-SA. This improvement may be due to the non-covalent bonds between RBP and SA, which makes the internal structure of RBP-SA denser and further enhances the thermal stability of RBP-SA. The mass loss of RBP-SA-PC is divided into two stages: water evaporation at 30-100°C and extensive structural degradation at 200-300°C. The maximum peak on the DTG curve corresponds to the maximum weight loss rate (T max ) temperature. At pH 7 and 9, the T max The T values ​​of RBP-SA-PC were higher than those of RBP-SA, and increased with the increase of PC concentration. This indicates that the interaction between PC and RBP-SA improves the thermal stability of RBP-SA-PC. The higher the PC concentration, the better the stability at pH 7 and 9. Interestingly, the T values ​​of RBP-SA-PC max It also increases with the increase of pH value, indicating that higher pH value further enhances the thermal stability of RBP-SA-PC.

[0038] Depend on Figure 2As shown in Figure D, at pH 9, the θo / w of RBP is 33.04±2.49°, indicating that RBP is hydrophilic. The θo / w of RBP-SA is 78.48±2.39°, indicating that the addition of SA increases the oil-phase wettability of RBP. This is likely due to the ability of SA to unfold the RBP structure, exposing hydrophobic groups and improving interfacial wettability. The θo / w of RBP-SA-0.5% PC is 87.30±1.29°, indicating that the addition of PC can enhance the oil-phase wettability of RBP-SA. This is likely due to the large number of hydroxyl groups in PC, which form hydrogen bonds with RBP-SA and reduce the number of free hydroxyl groups in RBP-SA that can absorb water. The θo / w of RBP-SA-0.5% PC at pH 5 and 7 are 100.32±0.74° and 97.70±1.02°, respectively. At pH = 9, the θo / w of RBP-SA-0.5% PC is close to 90°, indicating that RBP-SA-0.5% PC is most conducive to the stability of Pickering emulsion at pH = 9.

[0039] Example 2

[0040] (1) Preparation of Pickering emulsion:

[0041] Peanut oil was mixed with each group of RBP-SA-PC prepared in Example 1 at a 1:1 (w / w) ratio and sheared at 10,000 rpm for 2 minutes to prepare RBP-SA-PC PEs. These were designated RBP-SA-0.05% PC PEs, RBP-SA-0.1% PC PEs, RBP-SA-0.3% PC PEs, and RBP-SA-0.5% PC PEs, respectively. As controls, RBP-stabilized Pickering emulsions (RBP PEs) and RBP-SA-stabilized Pickering emulsions (RBP-SA PEs) were prepared using the same process, using RBP and RBP-SA in place of RBP-SA-PC, respectively.

[0042] (2) Preparation of Que-loaded RBP-SA-PC PEs:

[0043] Que (0.1%, w / w) was added to peanut oil and then mixed with each of the RBP-SA-PC, RBP, or RBP-SA prepared in Example 1 at a 1:1 (w / w) ratio. The mixtures were sheared at 10,000 rpm for 2 minutes to produce Que-loaded RBP-SA-PC PEs. These Pickering emulsions were designated Que-loaded RBP-SA-0.05% PC PEs, Que-loaded RBP-SA-0.1% PC PEs, Que-loaded RBP-SA-0.3% PC PEs, and Que-loaded RBP-SA-0.5% PC PEs, respectively. Que-loaded RBP PEs and Que-loaded RBP-SA PEs served as controls.

[0044] The optical microscope images, droplet size and zeta potential of each group of RBP-SA-PC PEs, RBP PEs and RBP-SA PEs prepared in this example were analyzed. Figure 3 shown.

[0045] Figure 3 Figure 2 shows optical microscopy images, droplet size, and zeta potential of RBP PEs, RBP-SA PEs, and RBP-SA-PC PEs under different pH conditions in Example 2. (A) Optical microscopy image (all scales 25 μm), B droplet size, and C zeta potential. Different capital letters within the same sample and different lowercase letters under the same pH condition indicate significant differences (p < 0.05).

[0046] Depend on Figure 3 As can be seen in Figure A, the droplet size of RBP-SA-PC PEs is smaller than that of RBP PEs and RBP-SA PEs, and the droplet size further decreases with the increase of PC concentration. Figure 3Figure B shows the droplet size of the Pickering emulsion. Under specific pH conditions, the droplet size is in the following order: RBP-PEs>RBP-SA PEs>RBP-SA-PC PEs. In addition, as the PC concentration increases, the droplet size of RBP-SA-PC PEs decreases, indicating that the droplet size is affected by the number of solid particles at the oil-water interface. As the PC concentration increases, more solid particles are adsorbed onto the interface, thereby expanding the interfacial area and enhancing the stability of the Pickering emulsion. In addition, as the pH increases, the droplet size of RBP-SA-PC PEs decreases, indicating that the pH of the preparation of RBP-SA-PC affects the droplet size of the resulting Pickering emulsion. At a pH of 9, RBP-SA-PC may exhibit better interfacial wettability, allowing solid particles to adsorb on the oil-water interface, forming a spatial barrier, inhibiting oil droplet aggregation, and resulting in smaller oil droplet size. Therefore, the Pickering emulsion formed by RBP-SA-PC prepared at pH 9 was more stable than those prepared at pH 5 and 7.

[0047] Figure 3 Center (C) shows the zeta potential of the Pickering emulsions. The absolute value of the zeta potential of RBP-SA PEs is higher than that of RBP PEs. This is likely due to the non-covalent binding of SA, an anionic polysaccharide, which increases the interfacial charge of the Pickering emulsion. At high PC concentrations, the absolute value of the zeta potential of RBP-SA-PC PEs is greater than that of RBP-SA PEs. This is likely due to PC being a polyphenolic compound that can ionize and form negative charges in solution. The interaction between high-concentration PC and RBP-SA forms a dense interfacial layer on the oil droplet surface, increasing the surface charge, reducing droplet aggregation, and enhancing the stability of the Pickering emulsion. Furthermore, at high PC concentrations, the absolute value of the zeta potential of RBP-SA-PC PEs increases with increasing pH. This is likely due to the increased negative charge density of RBP and SA at high pH and the enhanced dissociation of PC in alkaline solutions. The higher absolute value of the zeta potential generates stronger electrostatic repulsion, enhancing the stability of RBP-SA-PC PEs.

[0048] CLSM image analysis was performed on each group of RBP-SA-PC PEs, RBP PEs and RBP-SA PEs prepared in this example. The results are as follows: Figure 4 shown.

[0049] Figure 4CLSM images of RBP-SA PEs and RBP-SA-PC PEs under different pH conditions in Example 1 (all scale bars are 25 μm). Figure 4 It can be seen that the emulsification performance of RBP PEs is poor, and the fluorescent dye further affects its stability. Since the water-in-oil structure of RBP PEs cannot be captured, its CLSM image is not presented. The microstructure of RBP-SA PEs and RBP-SA-PC PEs was observed by CLSM, as shown in Figure 2. Figure 4 As shown in Figure 2 , both RBP-SA PEs and RBP-SA-PC PEs exhibit an oil-in-water structure. Under certain pH conditions, the droplet size of RBP-SA-PC PEs is smaller than that of RBP-SA PEs, indicating that RBP-SA-PC is more easily adsorbed at the oil-water interface. With increasing PC concentration, the droplet size of RBP-SA-PC PEs decreases, likely due to the formation of a thicker interfacial layer at the oil-water interface, which inhibits droplet flocculation and agglomeration and enhances the stability of the RBP-SA-PC PEs. CLSM analysis also revealed that the particle size of the Pickering emulsions further decreases with increasing pH, and the structure of the Pickering emulsions becomes denser at higher pH values. This may be due to the varying adsorption capacity of RBP-SA-PC at the oil-water interface, influenced by different non-covalent interactions under different pH conditions. With increasing pH, the adsorption capacity of RBP-SA-PC at the oil-water interface increases, resulting in a more uniform distribution and improved stability of the RBP-SA-PC PEs at higher pH levels. These findings are consistent with the optical microscopy and droplet size results of Pickering emulsions.

[0050] Example 3

[0051] To further investigate the interfacial structure of Pickering emulsions, dodecane was used instead of peanut oil to prepare Pickering emulsions. Because peanut oil is difficult to freeze-dry, dodecane was used instead. The preparation process was the same as in Example 2. After mixing, the water and dodecane were removed by freeze-drying to produce RBP PEs, RBP-SA PEs, and RBP-SA-PC PEs, respectively.

[0052] The interface micromorphology of each group of Pickering emulsions prepared in this example was observed by scanning electron microscopy, and the rheological properties of the Pickering emulsions were evaluated by measuring their viscoelasticity and the transverse relaxation time was measured by LF-NMR. The results are shown in Figure 2. Figure 5 shown.

[0053] Figure 5SEM images, rheological and low-field NMR images of RBP PEs, RBP-SA PEs and RBP-SA-PC PEs in Example 3; where, A is the SEM image (the scale bar is 100 μm), B is the rheological image, and C is the low-field NMR image.

[0054] As Figure 5 shown in A. Under certain pH conditions, RBP-SA PEs exhibit a network structure. With the increase in PC concentration, RBP-SA-PC PEs form a denser network structure at the oil-water interface, while the pore size decreases. These results indicate that the network structure is crucial for stabilizing RBP-SA-PC PEs, and the higher the PC concentration, the smaller the pore size. High concentrations of PC may enhance the adsorption of RBP-SA-PC at the oil-water interface, forming a dense network on the surface of the oil droplets. This structure inhibits droplet aggregation, thereby enhancing the stability of the Pickering emulsion. For RBP-SA-0.5% PC PEs, obvious structural collapse occurs at pH 5, and the structural collapse decreases at pH 7. At pH 9, a dense network structure is observed without collapse. This pH-dependent behavior may be due to the change in non-covalent interactions between 0.5% PC and RBP-SA, which alters the interfacial adsorption capacity and network gel strength.

[0055] The rheological properties of the Pickering emulsion are evaluated by measuring its viscoelasticity. The variations of G' and G" of the Pickering emulsion with the angular frequency are as Figure 5 shown in B. At low frequencies, G">G' for RBP PEs, indicating poor emulsifying performance of RBP PEs. RBP PEs mainly exhibit a viscous liquid state and are easy to flow and deform. In contrast, G'>G" for RBP-SA PEs and RBP-SA-PC PEs, indicating that these Pickering emulsions have gel properties dominated by elasticity, and the dependencies of G' and G" on frequency are very small. Under specific pH conditions, the order of G' and G" in the Pickering emulsion is RBP PEs < RBP-SA PEs < RBP-SA-PC PEs. This indicates that the addition of SA and PC enhances the gel-like structure of the Pickering emulsion. This improvement may be due to the decrease in droplet size in RBP-SA-PC PEs after adding SA and PC, which increases the oil-water interface contact area and forms a denser gel network structure, thereby improving the viscoelasticity of the Pickering emulsion. In addition, G' and G" of RBP-SA-PC PEs increase with the increase in PC concentration, indicating that the increase in PC concentration contributes to the formation of a denser network structure. This structure can prevent oil droplet flocculation and aggregation, thereby maintaining the stability of RBP-SA-PC PEs.

[0056] LF-NMR is a powerful tool for understanding the distribution and mobility of water in Pickering emulsions, as water plays a key role in the formation and rheological properties of Pickering emulsions. To further evaluate the binding capacity of water molecules in Pickering emulsions, the transverse relaxation time (T2) was measured using LF-NMR. Figure 5 Figure C shows the T2 distribution spectrum of Pickering emulsions. All Pickering emulsions have three different proton signals in their relaxation time, including bound water (T 21 )、Fixed Water(T 22 ) and free water (T 23 ). A longer T2 indicates better water mobility in the system, while a shorter T2 indicates stronger binding ability of water to the Pickering emulsion. Under certain pH conditions, the T2 of RBP-SA-PC PEs is shorter than that of RBP PEs and RBP-SA PEs, indicating that the addition of SA and PC limits the water migration of the Pickering emulsion. In addition, the T2 of RBP-SA-PC PEs is 22 and T 23 The value of the water-binding capacity of the Pickering emulsion decreases with increasing PC concentration, indicating that increasing PC concentration enhances the water-binding capacity of the Pickering emulsion. This phenomenon may be due to the formation of a denser gel network structure in the RBP-SA-PC PEs as the PC concentration increases. This dense network effectively restricts water mobility and contributes to the stability of the Pickering emulsion.

[0057] Example 4

[0058] Based on the above studies, the mechanism of RBP-SA-PC stabilizing Pickering emulsions under different pH conditions was proposed, such as Figure 6 As shown in A. Figure 6 The diagrams are the stabilization mechanism diagrams and DPPH and ABTS free radical scavenging activity diagrams of RBP PEs, RBP-SA PEs and RBP-SA-PC PE in Example 4, wherein A is the stabilization mechanism diagram, B is the DPPH free radical scavenging activity, and C is the ABTS free radical scavenging activity. Figure 6As shown in Figure A, pH primarily affects the noncovalent interactions between proteins, polysaccharides, and polyphenols, leading to the formation of ternary complexes with varying shapes. At pH 5, RBP-SA and PC form short-chain, fibrous ternary complexes through hydrogen bonding and hydrophobic interactions. These complexes adsorb at the oil-water interface, forming a partially collapsed network. At pH 7, RBP-SA and PC form medium-chain, fibrous ternary complexes through hydrogen bonding, hydrophobic bonding, and electrostatic interactions. These complexes adsorb at the interface, forming a network-like, viscoelastic interfacial layer with minimal collapse, forming a spatial barrier on the oil droplet surface. At pH 9, RBP-SA and PC form long-chain, fibrous ternary complexes through hydrogen bonding and electrostatic interactions. These complexes adsorb more rapidly at the interface, forming a denser, network-like, viscoelastic interfacial layer that effectively captures and restricts the displacement of oil droplets, prevents aggregation, and enhances the stability of the Pickering emulsion. Secondly, the droplet size of RBP-SA-PCPEs decreases with increasing pH. Smaller droplets adsorb faster at the interface, forming a dense network structure with smaller pores, which further stabilizes the Pickering emulsion. In summary, the Pickering emulsion stabilized by the long-chain fiber ternary complex has good stability.

[0059] The antioxidant activity of Pickering emulsion was studied using DPPH and ABTS free radical scavenging activities. 0.1 mL of the Pickering emulsion sample prepared in step (1) of Example 2 was mixed with 3.9 mL of 0.1 mmol / L DPPH ethanol solution and incubated in the dark for 30 min. The absorbance was then recorded at 517 nm using a UV-visible spectrophotometer. Equal volumes of 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate solution were mixed and reacted in the dark for 16 hours. The resulting ABTS stock solution was then diluted to an absorbance of 0.700 ± 0.02 at 734 nm. 0.3 mL of the Pickering emulsion sample was mixed with 3.5 mL of the diluted ABTS solution and incubated in the dark for 10 minutes. The absorbance was measured at 734 nm using a UV-visible spectrophotometer.

[0060] The antioxidant capacity of the samples was evaluated by measuring the change in absorbance after the Pickering emulsion scavenged free radicals. Both DPPH and ABTS free radical scavenging activities reflect the hydrogen donating capacity of the samples. Figure 6 As shown in B and C. Figure 6As shown in Figures B and C, RBP has been shown to have antioxidant activity according to the literature, and therefore, RBP PEs exhibit antioxidant properties under certain pH conditions. The addition of SA enhanced the antioxidant activity of RBP-SA PEs compared to RBP PEs, likely due to a synergistic effect between RBP and SA. Because PC contains phenolic hydroxyl groups, its antioxidant activity originates from these groups. Compared to RBP-SA PEs, the antioxidant activity of RBP-SA-PC PEs increased significantly in a concentration-dependent manner. This improvement may be related to the increased availability of phenolic hydroxyl groups at higher PC concentrations, thereby enhancing hydrogen supply capacity. Furthermore, the antioxidant activity of RBP-SA-PC PEs increased with increasing pH, as Pickering emulsions are more stable at high pH, ​​providing a more stable system for enhancing antioxidant properties.

[0061] Example 5

[0062] The Pickering emulsion prepared in step (2) of Example 2 was mixed with simulated gastric fluid (pH 1.2, containing 3.2 mg / mL pepsin) at a ratio of 1:14 and incubated at 37°C at 100 rpm for 2 h. The gastric digestion product was then mixed with simulated intestinal fluid (pH 7.5 PBS buffer, containing 4 mg / mL pancreatic lipase, 4.3 mg / mL sodium cholate, and 0.6 mmol / L calcium chloride) at a ratio of 1:1. The pH was adjusted to 7.5 with 1 mol / L NaOH, and the mixture was incubated at 37°C at 100 rpm for 2 h. The samples were stained with Nile blue and Nile red and observed under CLSM.

[0063] CLSM was used to monitor the microstructure of Pickering emulsions during in vitro digestion. Figure 7 shown. Figure 7 is the CLSM image of the in vitro digestion of Que-loaded RBP-SA PEs and Que-loaded RBP-SA-PC PEs in Example 5; Figure 7It can be seen that Que-loaded RBP PEs do not exhibit an oil-in-water structure. Therefore, CLSM images of their in vitro digestion are not provided. In the initial stage, both Que-loaded RBP-SA PEs and Que-loaded RBP-SA-PC PEs formed an oil-in-water structure. With the increase of PC concentration and pH value, the droplet size of RBP-SA-PC PEs decreased. During the gastric digestion stage, at pH 5, the interfacial structure of Que-loaded RBP-SA PEs disappeared and a flocculated structure was presented. Similarly, no interfacial structure was observed in the Que-loaded RBP-SA-0.05% PC PEs and Que-loaded RBP-SA-0.1% PC PEs emulsions, indicating that at PC concentrations of 0.05% and 0.1%, the droplet surface of these two Pickering emulsions was hydrolyzed by pepsin, resulting in the destruction of the oil-in-water structure. Surprisingly, an oil-in-water structure was observed in Que-loaded RBP-SA-0.3% PC PEs and Que-loaded RBP-SA-0.5% PC PEs, indicating that a portion of these Pickering emulsions resisted hydrolysis by pepsin at PC concentrations of 0.3% and 0.5%, maintaining their structural integrity. This resistance may be due to the increased PC concentration at pH 5, which forms a denser gel network at the oil-water interface, thereby mitigating the effects of pepsin hydrolysis. Disruption of the oil-in-water structure was observed in most Que-loaded RBP-SA PEs at pH 7 compared to pH 5. While the interfacial structure remained visible in Que-loaded RBP-SA-PC PEs, the interfacial layer was weakened by pepsin hydrolysis. This weakening is attributed to dilution of the Pickering emulsion during gastric digestion, disrupting the RBP-SA-PC network at the oil-water interface. However, a portion of the RBP-SA-PC remained adsorbed at the interface, maintaining the stability of the Pickering emulsion. Notably, Que-loaded RBP-SA-PC PEs exhibited resistance to pepsin hydrolysis at all PC concentrations at pH 9. This resistance may be due to the formation of a denser network structure at the oil-water interface by RBP-SA-PC at pH 9, which effectively resists pepsin hydrolysis. During the intestinal digestion stage, the oil-in-water structure of all Pickering emulsions undergoes significant changes as the oil droplets are broken down into free fatty acids (FFA) by pancreatic lipase and bile salts. The mixture of FFA, protein hydrolysates, and oil droplets forms the orange and red regions observed in the CLSM images.

[0064] After simulated intestinal fluid digestion, a part of the digested sample was centrifuged at 12,000 rpm for 30 min, the middle-layer micelles were collected, the Que content in the digestion and micelles was quantified, and the bioaccessibility of Que was calculated. The bioaccessibility of bioactive substances is a key index for evaluating the administration effect of Pickering emulsions. Figure 8 is a schematic diagram of the bioaccessibility and in vitro digestion mechanism of Que in Example 5, where A is the bioaccessibility and B is the schematic diagram of the in vitro digestion mechanism. As Figure 8 shown in A. Peanut oil containing Que showed the lowest bioaccessibility, probably due to limited digestion and reduced release of Que in the small intestine. Under certain pH conditions, the bioaccessibility of Que was in the order of: Que-loaded RBP PEs < Que-loaded RBP-SA PEs < Que-loaded RBP-SA-0.05% PC PEs < Que-loaded RBP-SA-0.1% PC PEs < Que-loaded RBP-SA-0.3% PC PEs < Que-loaded RBP-SA-0.5% PC PEs. This phenomenon may be because RBP-SA-PC formed a denser gel network at higher PC concentrations, thus protecting Que from degradation by pepsin. In addition, in Pickering emulsions with higher PC concentrations, smaller droplet sizes increased the contact area with lipase, promoted the transfer of Que to mixed micelles, and improved the bioaccessibility. The pH-dependence of RBP-SA-PC further affected the bioaccessibility of Que. At different pH values, RBP-SA and PC formed RBP-SA-PC with different chain lengths through non-covalent interactions, resulting in different pore network structures at the oil-water interface. SEM and CLSM observations found that higher pH values promoted the formation of the gel network, thus inhibiting the hydrolysis of pepsin and reducing oil droplet aggregation. This structural enhancement improved the bioaccessibility of Que.

[0065] Under different pH conditions, the digestion mechanism of Que-load RBP-SA-PC PEs is as Figure 8As shown in Figure 2 (B). pH affects the noncovalent interactions between proteins, polysaccharides, and polyphenols, leading to the formation of ternary complexes of varying chain lengths. During gastric digestion, the density of the interfacial layer formed at the oil-water interface by RBP-SA-PC prepared under different pH conditions can limit the hydrolysis of the Pickering emulsion by pepsin. At pH 5, at higher PC concentrations, Que-loaded RBP-SA-PC PEs exhibited resistance to pepsin hydrolysis, with some Pickering emulsions maintaining an oil-in-water structure. At pH 7, while the structural integrity of the Pickering emulsion was maintained, Que-loaded RBP-SA-PC PEs showed a weakened interfacial layer during gastric digestion. At pH 9, Que-loaded RBP-SA-PC PEs exhibited strong resistance to pepsin hydrolysis. This resistance may be due to the long-chain fibrous structure of RBP-SA-PC at pH 9, which forms a dense network at the oil-water interface, effectively resisting pepsin hydrolysis and maintaining the stability of the Pickering emulsion during gastric digestion. During intestinal digestion, the droplet size of the Pickering emulsion decreases with increasing pH. Smaller droplet sizes increase the contact area between pancreatic lipase, bile salts, and the Pickering emulsion, forming more mixed micelles that solubilize Que and improve its bioaccessibility. In summary, at pH 9, long-chain, fibrous ternary complexes adsorb at the oil-water interface, forming a dense network structure that maintains the stability of the Pickering emulsion during gastric digestion. Furthermore, these ternary complexes facilitate the formation of more mixed micelles during intestinal digestion, significantly improving the bioaccessibility of Que.

Claims

1. A protein-polysaccharide-polyphenol ternary complex, characterized in that: The invention is obtained by mixing rice bran protein, sodium alginate and proanthocyanidins as raw materials.

2. The protein-polysaccharide-polyphenol ternary complex according to claim 1, characterized in that: The mass ratio of rice bran protein, sodium alginate and proanthocyanidins is 1:2:(0.05-0.5).

3. The method for preparing the protein-polysaccharide-polyphenol ternary complex according to claim 1 or 2, characterized in that: The following steps are involved: The rice bran protein is dispersed in water, the pH is adjusted, the mixture is centrifuged, the supernatant is collected, and the mixture is mixed with a sodium alginate solution. The pH is adjusted again, proanthocyanidins are added, and the mixture is stirred.

4. The preparation method according to claim 3, characterized in that Adjust the pH to alkaline and then adjust the pH to 5-9.

5. A Pickering emulsion, characterized in that The invention comprises the protein-polysaccharide-polyphenol ternary complex according to claim 1 or 2.

6. The Pickering emulsion according to claim 5, characterized in that It also includes peanut oil, and the mass ratio of the protein-polysaccharide-polyphenol ternary complex to the peanut oil is (1-2):(1-2).

7. The Pickering emulsion according to claim 5, characterized in that Mix the protein-polysaccharide-polyphenol ternary complex with peanut oil and shear.

8. The Pickering emulsion according to any one of claims 5 to 7, characterized in that Also includes quercetin.

9. The Pickering emulsion according to claim 8, characterized in that Add quercetin to peanut oil, then mix the protein-polysaccharide-polyphenol ternary complex with the peanut oil and shear.

10. The use according to claim 9, characterized in that The mass ratio of quercetin, peanut oil and protein-polysaccharide-polyphenol ternary complex is 0.1:(1-2):(1-2).

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