Camellia oleifera cake protein-polysaccharide and polyphenol covalent or non-covalent compound and preparation thereof

Abstract

The invention discloses an oil tea cake protein-polysaccharide and polyphenol covalent or non-covalent compound as well as preparation and application thereof, and belongs to the technical field of food processing. The preparation method comprises the following steps: adding polyphenol into an oil-tea cake protein-polysaccharide compound solution, adjusting the pH value to 9.0 + / -0.05, and stirring under an aerobic condition to carry out covalent cross-linking reaction, so as to prepare oil-tea cake protein-polysaccharide compound-polyphenol covalent composite nanoparticles; or adjusting the pH value to 7.0 + / -0.05, and stirring and self-assembling under the anaerobic condition to obtain the oil tea cake protein-polysaccharide compound-polyphenol non-covalent composite nanoparticles. The particles are endowed with differentiated functions through covalent or non-covalent modification, and the application adaptability is expanded. The camellia oleifera cake is used as a raw material, high-value utilization of agricultural by-products is achieved, and application of the camellia oleifera cake in a functional component delivery system is further expanded.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a covalent or non-covalent complex of camellia seed cake protein-polysaccharide and polyphenol and its preparation. Background Technology

[0002] Nanoparticles, as highly efficient delivery carriers, have broad prospects in the food, pharmaceutical, and cosmetic fields. Among them, nanoparticles constructed from natural polymer materials such as proteins and polysaccharides are particularly favored due to their excellent biocompatibility and biodegradability. Proteins and polysaccharides can form composite carriers through covalent or non-covalent interactions, effectively encapsulating hydrophobic active ingredients. For example, patent document CN118236347A discloses the use of a tangerine peel pectin-zein nanoparticle delivery system to load curcumin, thereby improving the encapsulation stability and bioavailability of curcumin while also enhancing its stability and antioxidant activity in vivo.

[0003] However, existing technologies face a dual challenge: on the one hand, single protein-polysaccharide complex systems often lack stability and are prone to dissociation at specific pH, ionic strength, or high temperatures, leading to premature leakage of the encapsulated components. On the other hand, many current delivery systems rely on inorganic or synthetic polymer materials with poor biocompatibility. In contrast, widely available and inexpensive plant proteins are ideal alternatives, but their generally poor water solubility and dispersibility severely limit the efficient preparation and application of functional nanoparticles. Although methods such as antisolvent methods can improve the emulsification properties of plant proteins, the resulting nanoparticles often have low yields, excessive rigidity, and poor dispersibility. Existing improvement technologies, such as patent document CN 110129395 A, disclose the use of restrictive enzymatic modification, but this may introduce new problems such as difficulties in recovering free enzymes, increased production costs, and the influence of salt ions on nanoparticle formation and stability during hydrolysis.

[0004] Polyphenols, such as tannic acid and epigallocatechin gallate (EGCG), are often used as functional agents due to their excellent antioxidant and anti-inflammatory activities. Furthermore, the abundant phenolic hydroxyl groups in their molecular structure enable them to interact with proteins through covalent bonding or non-covalent forces such as hydrophobic interactions and hydrogen bonds. This interaction provides a new pathway for modifying protein and polysaccharide complexes.

[0005] Camellia oil cake, a major byproduct of camellia oil extraction, is rich in protein and polysaccharides, making it a valuable resource for preparing functional components. However, current utilization of camellia oil cake is mostly concentrated in primary processing or animal feed, with limited applications at higher value levels. In particular, research on developing its proteins and polysaccharides into functional nanodelivery systems is still insufficient.

[0006] Therefore, developing a new method based on camellia oil cake, an agricultural byproduct, to efficiently combine protein-polysaccharide complexes with polyphenols through synergistic covalent or non-covalent interactions, thereby preparing structurally stable and functionally enhanced nanoparticles, can not only enhance the added value of camellia oil cake but also provide a new solution for the efficient delivery of active ingredients, which has important theoretical significance and application value. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing structurally stable and functionally enhanced nanoparticles by efficiently combining camellia oil cake protein-polysaccharide complexes with polyphenols through covalent or non-covalent interactions, thereby realizing the high-value application of camellia oil cake.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing a covalent complex of protein-polysaccharide and polyphenol from camellia seed cake. The method includes: adding polyphenols to a solution of protein-polysaccharide complex from camellia seed cake, adjusting the pH to 9.0 ± 0.05, stirring under aerobic conditions to induce a covalent cross-linking reaction, obtaining a dispersion of covalently composite nanoparticles of protein-polysaccharide complex and polyphenol from camellia seed cake, dialyzing the product with a molecular weight cutoff ≥3500 Da, and freeze-drying to obtain the covalent complex of protein-polysaccharide and polyphenol from camellia seed cake.

[0009] This invention introduces polyphenols into a protein-polysaccharide complex system of camellia seed cake. By adjusting the system to alkaline and maintaining aerobic conditions, it aims to promote the oxidation of polyphenols to generate electrophilic quinone intermediates, while enhancing the reactivity of the nucleophilic groups of the protein. This drives the quinone intermediates to undergo an efficient covalent cross-linking reaction with the nucleophilic groups (such as amino and thiol groups) on the protein of the camellia seed cake protein-polysaccharide complex, forming structurally stable covalently composite nanoparticles of camellia seed cake protein-polysaccharide and polyphenols.

[0010] Specifically, an alkaline environment (pH = 9.0 ± 0.05) facilitates the deprotonation of the ε-amino group (-NH2) of lysine residues in protein molecules, significantly enhancing their nucleophilicity (electron-rich ability) and making them more susceptible to attacking electrophilic reagents. Secondly, alkaline conditions promote the oxidation of polyphenols (especially catechol structures). Dissolved oxygen in the solution acts as an oxidant in the oxidation of polyphenols, and under alkaline conditions, it can more efficiently oxidize polyphenols (such as EGCG and the catechol structure in tannins) to highly reactive quinone intermediates. These quinone intermediates then undergo efficient Michael addition or Schiff base reactions with nucleophilic groups on proteins, forming stable covalent bonds.

[0011] The present invention also provides a method for preparing a protein-polysaccharide and / or non-covalent complex of camellia seed cake meal. The preparation method includes: adding polyphenols to a protein-polysaccharide complex solution of camellia seed cake meal, adjusting the pH value to 7.0 ± 0.05, stirring under anaerobic conditions until the liquid is clear or semi-transparent to obtain a non-covalent composite nanoparticle dispersion of protein-polysaccharide complex-polyphenol from camellia seed cake meal, dialyzing the product with a molecular weight cutoff ≥3500 Da, and freeze-drying to obtain a non-covalent composite of protein-polysaccharide and polyphenol from camellia seed cake meal.

[0012] This invention introduces polyphenols into a protein-polysaccharide composite system of camellia seed cake. By controlling the system in a neutral state (pH=7.0 ± 0.05) and creating an anaerobic environment, the aim is to inhibit the oxidation and covalent reaction of polyphenols, so that the polyphenols and protein-polysaccharide complexes mainly rely on non-covalent forces such as hydrogen bonds and hydrophobic interactions to self-assemble, forming composite nanoparticles with mild conditions and good activity retention.

[0013] Specifically, in a neutral environment, the charged state and conformation of proteins are closer to their native physiological state. Most of the phenolic hydroxyl groups in polyphenols remain undissociated, which facilitates the formation of intermolecular hydrogen bonds. Simultaneously, the well-defined hydrophobic regions on the protein surface facilitate binding with the aromatic rings of polyphenols through hydrophobic interactions. Oxygen exclusion is to actively inhibit the oxidation of polyphenols and subsequent covalent reactions, ensuring that interactions are limited to non-covalent forces. An anaerobic environment is the key control condition for achieving "pure" non-covalent assembly in this step. This condition maximizes the preservation of the native active structure of polyphenols.

[0014] In this invention, the protein-polysaccharide complex from camellia oil cake meal is extracted from camellia oil cake meal. The extraction method includes: crushing the camellia oil cake meal and washing it sequentially with anhydrous ethanol, acetone, and anhydrous ethanol to obtain alcohol-insoluble substances; then adding water for ultrasonic dissolution and collecting the supernatant; then adding anhydrous ethanol and using alcohol precipitation to form a precipitate of the protein and polysaccharide substances in the supernatant for further separation and purification; finally, using dialysis to retain the extract with a molecular weight cutoff greater than 14 kDa, and freeze-drying to obtain the protein-polysaccharide complex from camellia oil cake meal.

[0015] In this invention, the camellia oil cake protein-polysaccharide complex solution is prepared by dissolving camellia oil cake protein-polysaccharide complex powder in deionized water. Preferably, the mass-volume percentage concentration of the camellia oil cake protein-polysaccharide complex solution is 2-6%. This concentration range ensures sufficient dissolution of the complex and maintains system stability, providing a basis for subsequent effective compounding with polyphenols. Studies have found that when the concentration is below 2%, the density of effective active sites of the protein-polysaccharide complex in the solution is insufficient, leading to a significant decrease in the probability of its interaction with polyphenols (e.g., 2 mg / mL). When the concentration exceeds 6%, the solution viscosity increases sharply. Excessively high viscosity severely hinders the uniform mixing of the reaction system, affects the accuracy of pH adjustment, and limits the diffusion of polyphenol molecules to protein active sites (mass transfer limitation).

[0016] More preferably, the mass-volume percentage concentration of the camellia oil cake protein-polysaccharide complex solution is 4%.

[0017] As a preferred method, the preparation method of the camellia seed cake protein-polysaccharide complex solution is as follows: the camellia seed cake protein-polysaccharide complex powder is added to deionized water, stirred continuously for 2 h at room temperature and stirring speed of 500-800 rpm, and then placed at 4℃ for 12 h.

[0018] Preferably, the polyphenol is epigallocatechin gallate or tannic acid. The polyphenol is added to a camellia seed cake protein-polysaccharide complex solution and stirred until the polyphenol is completely dispersed in the solution.

[0019] Preferably, the final concentration of polyphenols in the solution system is 1.0-3.0 mg / mL. In a system with a protein-polysaccharide complex concentration of 4% (w / v) from camellia seed cake, when the polyphenol concentration is below 1.0 mg / mL, it is insufficient to fully interact with the protein, resulting in low composite degree and insufficient loading; when it is above 3.0 mg / mL, polyphenol self-aggregation or supersaturation occurs, affecting the uniformity and stability of the nanoparticles.

[0020] More preferably, the final concentration of polyphenols in the solution system is 2 mg / mL. Studies have found that in a system with a 4% (w / v) concentration of the protein-polysaccharide complex from camellia oil cake, the polyphenol concentration within this range exhibits the best binding efficiency and particle properties (such as particle size distribution and stability), achieving efficient loading while ensuring the structural integrity and dispersibility of the nanoparticles.

[0021] Preferably, the pH value is adjusted using 1 mol / L NaOH and 1 mol / L HCl.

[0022] In this invention, camellia seed cake protein-polysaccharide complex-polyphenol covalently composite nanoparticles are prepared under aerobic conditions. Preferably, when preparing camellia seed cake protein-polysaccharide complex-polyphenol covalently composite nanoparticles, the reaction system is kept open with air circulation, and the reaction is carried out continuously for 24 h at a constant stirring speed of 500-800 rpm at 25±1℃.

[0023] In this invention, non-covalent composite nanoparticles of camellia seed cake protein-polysaccharide complex-polyphenol are prepared under anaerobic conditions. Preferably, when preparing the non-covalent composite nanoparticles of camellia seed cake protein-polysaccharide complex-polyphenol, the mixed solution after pH adjustment is placed in a closed reactor, nitrogen is introduced to remove oxygen, and the mixture is continuously stirred for 2 hours at 25±1℃ and 500-800 rpm.

[0024] After the reaction, the reaction product was dialyzed under the following conditions: temperature 4℃, time 48-72 h, and a molecular weight cutoff of 3500 Daltons. This molecular weight cutoff was designed to allow the removal of small molecule impurities (such as unreacted polyphenols and salt ions) while retaining larger protein-polysaccharide-polyphenol composite nanoparticles, avoiding loss of the target product, thereby purifying the product and maintaining its structural integrity. These conditions are a crucial step in ensuring product purity and achieving effective separation and purification of nanoparticles.

[0025] This invention also provides covalent or non-covalent complexes of camellia seed cake protein-polysaccharide and polyphenols prepared by the above-described method. Particle performance verification results show that the covalent or non-covalent composite nanoparticles prepared by the above method exhibit differentiated characteristics in structure and function, making them suitable for different application scenarios. For example, covalent composite nanoparticles have a more stable structure and are suitable for delivery systems with high stability requirements; non-covalent composite nanoparticles have milder preparation conditions and better thermal stability, making them suitable for systems with high requirements for rapid release of active ingredients and structural integrity.

[0026] This invention endows composite nanoparticles with differentiated functions through covalent or non-covalent modification, thereby expanding their application adaptability: Covalent composite particles form a stable structure through quinone-protein crosslinking, which maintains its integrity under high temperature conditions. They also have a uniform particle size distribution and high surface charge. These characteristics make the particles particularly suitable as carriers for functional foods or oral drugs that need to withstand processing conditions (such as heat sterilization and specific pH environments).

[0027] Non-covalent composite particles achieve rapid binding through hydrogen bonds and hydrophobic interactions. They have higher thermal denaturation temperatures and looser structures, which facilitates the rapid release of encapsulated active ingredients (such as polyphenols) under mild conditions, making them suitable for liquid beverages, dairy products, or instant nutritional supplements.

[0028] This invention significantly enhances the performance of composite nanoparticles through both covalent and non-covalent modifications, facilitating the efficient delivery of active ingredients: both covalently and non-covalently modified complexes exhibit enhanced UV absorption and fluorescence quenching behavior, indicating that polyphenols are effectively encapsulated and interact with protein-polysaccharide complexes; changes in surface hydrophobicity and interfacial tension suggest that the complexes have potential application value in emulsification and dispersion systems, and can be used as natural emulsifiers or stabilizers.

[0029] Therefore, this invention provides the application of the aforementioned camellia seed cake protein-polysaccharide-polyphenol covalent complex in the preparation of functional foods or oral medications as a carrier, emulsifier, or stabilizer. Based on performance test data from the embodiments of this invention, the camellia seed cake protein-polysaccharide-polyphenol covalent complex, due to its structural stability, uniform particle size, and high surface charge under high temperature conditions, can be specifically applied in the following product fields: as a heat-resistant carrier in the preparation of functional solid beverages, nutritional powders, or oral sustained-release formulations, encapsulating active ingredients such as vitamins and probiotics; as an emulsifying stabilizer in the preparation of plant-based dairy products, sauces, and baking fillings requiring high-temperature processing; and also as a natural emulsifier in cosmetic systems such as face creams and sunscreens to meet their requirements for long-term storage stability.

[0030] This invention provides the application of the aforementioned non-covalent complex of camellia seed cake protein-polysaccharide and polyphenol in the preparation of nutritional supplements, emulsifiers or stabilizers.

[0031] The beneficial effects of this invention are as follows: (1) Green and safe, simple process: This invention does not require the introduction of exogenous chemical components, avoiding potential impurity pollution and safety risks, and conforms to the green and environmentally friendly preparation concept. At the same time, the method has simple process steps and mild operating conditions, and can achieve effective compounding of protein-polysaccharide complex and polyphenol and construction of nanoparticles in one-step reaction, which significantly shortens the production cycle and reduces equipment and energy costs.

[0032] (2) Comprehensive utilization of resources and high added value: This invention uses camellia oil cake as raw material to realize the high-value utilization of agricultural by-products. It not only solves the problem of waste cake disposal, but also expands its application in functional component delivery systems, which has significant economic and environmental benefits.

[0033] (3) Broad application prospects and strong adaptability: The nanoparticles prepared by this invention have good dispersibility, stability and controllable release characteristics based on covalent / non-covalent modification. They can be widely used in the fields of nutritional fortification and active ingredient encapsulation according to the needs of the target application scenario (such as long-term sustained release or rapid release), and have good industrialization potential and market prospects. Attached Figure Description

[0034] Figure 1 The thermal stability of the composite nanoparticles prepared in Examples 1-4.

[0035] Figure 2 Fourier transform infrared spectroscopy analysis (A) and secondary structure diagram (B) of the composite nanoparticles prepared in Examples 1-4.

[0036] Figure 3 UV-Vis spectroscopy analysis (A), fluorescence analysis (B), and hydrophobicity diagram (C) of the composite nanoparticles prepared in Examples 1-4.

[0037] Figure 4 The images show the three-dimensional fluorescence patterns of the composite nanoparticles prepared in Examples 1-4.

[0038] Figure 5 The contact angle diagrams are for the composite nanoparticles prepared in Examples 1-4.

[0039] Figure 6 Interfacial tension diagrams of the composite nanoparticles prepared in Examples 1-4 Figure 7 Turbidity (A), potential (B), and particle size distribution (C) of the composite nanoparticles prepared in Examples 1-4.

[0040] Figure 8 Scanning electron microscope images of the composite nanoparticles prepared in Examples 1-4. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0043] The protein-polysaccharide complex protein powder used in the following examples was extracted from camellia oil cake, and the extraction method was based on patent document CN 119366579 A. The specific steps are as follows: (1) Raw material pretreatment: Camellia oil cake was first crushed and then passed through an 80-mesh sieve to obtain camellia oil cake powder; (2) Preparation of alcohol-insoluble substances: Anhydrous ethanol was added to the camellia oil cake powder at a material-liquid ratio of 1:3, and the mixture was magnetically stirred at 25°C and 1000 rpm for 30 min, and then centrifuged at 4°C, 10000 rpm, and 10 min. The supernatant was discarded, and the above operation was repeated until the supernatant became clear. Acetone was added to the precipitate at a ratio of 1:3, and the mixture was magnetically stirred at 25°C and 1000 rpm for 30 minutes. Then, the mixture was centrifuged at 4°C, 10000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was dried at room temperature to obtain a dry alcohol-insoluble substance. (3) Ultrasound: The alcohol-insoluble substance was added to ultrapure water at a ratio of 1:3 and ultrasonicated (power 450W) for 30 minutes at 25°C. Centrifuge for 15 min to obtain the supernatant of camellia seed cake; repeat the above operation and combine the supernatants from the two times; (4) add anhydrous ethanol to the supernatant at a volume ratio of 1:3 and stir, then let it stand at 4℃ for 12 h to separate the layers; then centrifuge at 4℃ and 3500 rpm for 15 min, and the lower precipitate obtained is the water-soluble camellia seed cake substance; (5) dialyze the lower precipitate, the dialysis bag is 14 kDa, dialyze at 4℃ for 3 days, and change the ultrapure water every 8 h. Collect the liquid in the dialysis bag and freeze dry it to finally obtain the camellia seed cake protein-polysaccharide complex. Camellia seed cake comes from Zhejiang Jiusheng Camellia Technology Co., Ltd., and is the residue cake after camellia seeds are pressed for oil.

[0044] Epigallocatechin gallate powder (CAS No.: 989-51-5) and tannic acid powder (CAS No.: 1401-55-4) were both purchased from Aladdin Reagent Company.

[0045] Example 1 This embodiment prepared a non-covalent composite nanoparticle of camellia seed cake protein-polysaccharide complex and epigallocatechin gallate, and the preparation method is as follows: 1) Accurately weigh 2.00 g of camellia seed cake protein-polysaccharide complex powder into a beaker, add 50 mL of purified water, and stir continuously for 2 h at room temperature and a stirring speed of 500-800 rpm to ensure that the protein is fully dispersed and hydrated. Then, place the protein solution in a refrigerator at 4℃ and let it stand for 12 h to allow it to fully hydrate, finally obtaining a 4.0% (w / v) concentration of camellia seed cake protein-polysaccharide complex protein stock solution for later use.

[0046] 2) Accurately weigh 100 mg of epigallocatechin gallate powder and add it to 50 mL of the camellia seed cake protein-polysaccharide complex stock solution prepared in step 1). This step brings the final polyphenol concentration in the system to 2.0 mg / mL. Stir until the polyphenols are completely dispersed in the protein solution.

[0047] 3) Using 1 M HCl or NaOH solution, precisely adjust the pH of the series of polyphenol-camellia cake protein-polysaccharide complex mixed solutions prepared in step 2) to 7.0 ± 0.05 to create a suitable solution environment for non-covalent complexation.

[0048] 4) The pH-adjusted mixed solution from step 3) was placed in a sealed reactor, and nitrogen gas was introduced to remove oxygen and create an anaerobic environment. The reaction was then carried out under conditions of 25 ± 1℃ and a stirring speed of 500 rpm for 2 hours. During this process, polyphenols and camellia seed cake protein-polysaccharide complex proteins spontaneously self-assembled through intermolecular hydrophobic interactions and non-covalent forces such as hydrogen bonds, ultimately forming a clear or semi-transparent non-covalent composite nanoparticle dispersion of camellia seed cake protein-polysaccharide complex-polyphenol (CME-EGCG-N).

[0049] 5) The dispersion of camellia seed cake protein-polysaccharide complex-polyphenol non-covalent composite nanoparticles obtained in step 4) was dialyzed. The dialysis bag was 3500 Daltons. The dialysis conditions were: temperature 4℃, time 72 h, with ultrapure water replaced every 8 h. The liquid in the dialysis bag was collected and dispensed into lyophilization bottles. After pre-freezing, it was freeze-dried in a freeze dryer to obtain a solid powder of camellia seed cake protein-polysaccharide complex-polyphenol non-covalent composite nanoparticles, which is convenient for long-term storage and subsequent application.

[0050] Example 2 This embodiment prepares a non-covalent composite nanoparticle of camellia seed cake protein-polysaccharide complex and tannic acid, and the preparation method is as follows: 1) Accurately weigh 2.00 g of camellia seed cake protein-polysaccharide complex powder into a beaker, add 50 mL of purified water, and stir continuously for 2 h at room temperature and a stirring speed of 500-800 rpm to ensure that the protein is fully dispersed and hydrated. Then, place the protein solution in a refrigerator at 4℃ and let it stand for 12 h to allow it to fully hydrate, finally obtaining a 4.0% (w / v) concentration of camellia seed cake protein-polysaccharide complex protein stock solution for later use.

[0051] 2) Accurately weigh 100 mg of tannic acid powder and add it to 50 mL of the camellia seed cake protein-polysaccharide complex stock solution prepared in step 1). This step brings the final polyphenol concentration in the system to 2.0 mg / mL. Stir until the polyphenols are completely dispersed in the protein solution.

[0052] 3) Using a 1 M HCl or NaOH solution, precisely adjust the pH of the tannic acid-camellia cake protein-polysaccharide complex mixed solution prepared in step 2) to 7.0 ± 0.05 to create a suitable solution environment for non-covalent complexation.

[0053] 4) The pH-adjusted mixed solution from step 3) was placed in a sealed reactor, and nitrogen gas was introduced to remove oxygen and create an anaerobic environment. The reaction was then carried out under conditions of 25 ± 1℃ and a stirring speed of 500 rpm for 2 hours. During this process, polyphenols and camellia seed cake protein-polysaccharide complex proteins spontaneously self-assembled through intermolecular hydrophobic interactions and non-covalent forces such as hydrogen bonds, ultimately forming a clear or semi-transparent non-covalent composite nanoparticle dispersion of camellia seed cake protein-polysaccharide complex-tannic acid (CME-TA-N).

[0054] 5) The non-covalent composite nanoparticle dispersion of camellia seed cake protein-polysaccharide complex-tannic acid obtained in step 4) was dialyzed. The dialysis bag was 3500 Daltons. The dialysis conditions were: temperature 4℃, time 72 h, with ultrapure water changed every 8 h. The liquid in the dialysis bag was collected and dispensed into lyophilization bottles. After pre-freezing, it was freeze-dried in a freeze dryer to obtain a solid powder of non-covalent composite nanoparticles of camellia seed cake protein-polysaccharide complex-polyphenol, which is convenient for long-term storage and subsequent application.

[0055] Example 3 This embodiment prepared a covalently composite nanoparticle of camellia seed cake protein-polysaccharide complex and epigallocatechin gallate, and the preparation method is as follows: 1) Accurately weigh 2.00 g of camellia seed cake protein-polysaccharide complex powder into a beaker, add 50 mL of purified water, and stir continuously for 2 h at room temperature and a stirring speed of 500-800 rpm to ensure that the protein is fully dispersed and hydrated. Then, place the protein solution in a refrigerator at 4℃ and let it stand for 12 h to allow it to fully hydrate, finally obtaining a 4.0% (w / v) concentration of camellia seed cake protein-polysaccharide complex protein stock solution for later use.

[0056] 2) Accurately weigh 100 mg of epigallocatechin gallate powder and add it to 50 mL of the camellia seed cake protein-polysaccharide complex stock solution prepared in step 1). This step brings the final polyphenol concentration in the system to 2.0 mg / mL. Stir until the polyphenols are completely dispersed in the protein solution.

[0057] 3) Using a 1 M NaOH solution, precisely adjust the pH of the series of polyphenol-camellia seed cake protein-polysaccharide complex mixed solutions prepared in step 2) to 9.0 ± 0.05. Then, keep the reaction system open or maintain contact with air, and continue the reaction for 24 h under aerobic conditions at a constant stirring speed of 500 rpm at 25 ± 1℃. During this process, dissolved oxygen oxidizes the polyphenols to quinone intermediates, which undergo a highly efficient covalent cross-linking reaction with nucleophilic groups (such as amino and thiol groups) on the camellia seed cake protein-polysaccharide complex protein to form a camellia seed cake protein-polysaccharide complex-epigallocatechin gallate covalent complex (CME-EGCG-C).

[0058] 4) The dispersion of camellia seed cake protein-polysaccharide complex-polyphenol covalent composite nanoparticles obtained in step 3) was dialyzed. The dialysis bag was 3500 Daltons. The dialysis conditions were: temperature 4℃, time 72 h, with ultrapure water replaced every 8 h. The liquid in the dialysis bag was collected and dispensed into lyophilization bottles. After pre-freezing, it was freeze-dried in a freeze dryer to obtain a solid powder of camellia seed cake protein-polysaccharide complex-polyphenol non-covalent composite nanoparticles, which is convenient for long-term storage and subsequent application.

[0059] Example 4 This embodiment prepares a covalently composite nanoparticle of camellia seed cake protein-polysaccharide complex and tannic acid, and the preparation method is as follows: 1) Accurately weigh 2.00 g of camellia seed cake protein-polysaccharide complex powder into a beaker, add 50 mL of purified water, and stir continuously for 2 h at room temperature and a stirring speed of 500-800 rpm to ensure that the protein is fully dispersed and hydrated. Then, place the protein solution in a refrigerator at 4℃ and let it stand for 12 h to allow it to fully hydrate, finally obtaining a 4.0% (w / v) concentration of camellia seed cake protein-polysaccharide complex protein stock solution for later use.

[0060] 2) Accurately weigh 100 mg of tannic acid powder and add it to 50 mL of the camellia seed cake protein-polysaccharide complex stock solution prepared in step 1). This step brings the final polyphenol concentration in the system to 2.0 mg / mL. Stir until the polyphenols are completely dispersed in the protein solution.

[0061] 3) Using a 1 M NaOH solution, precisely adjust the pH of the series of polyphenol-camellia seed cake protein-polysaccharide complex mixed solutions prepared in step 2) to 9.0 ± 0.05. Then, keep the reaction system open or maintain contact with air, and continue the reaction for 24 h under aerobic conditions at a constant stirring speed of 500 rpm at 25 ± 1℃. During this process, dissolved oxygen oxidizes the polyphenols to quinone intermediates, which undergo a highly efficient covalent cross-linking reaction with nucleophilic groups (such as amino and thiol groups) on the camellia seed cake protein-polysaccharide complex protein to form a camellia seed cake protein-polysaccharide complex-tannic acid covalent complex (CME-TA-C).

[0062] 4) The dispersion of camellia seed cake protein-polysaccharide complex-polyphenol covalent composite nanoparticles obtained in step 3) was dialyzed. The dialysis bag was 3500 Daltons. The dialysis conditions were: temperature 4℃, time 72 h, with ultrapure water replaced every 8 h. The liquid in the dialysis bag was collected and dispensed into lyophilization bottles. After pre-freezing, it was freeze-dried in a freeze dryer to obtain a solid powder of camellia seed cake protein-polysaccharide complex-polyphenol non-covalent composite nanoparticles, which is convenient for long-term storage and subsequent application. Test Example 1: Thermal Stability of Particles The camellia seed cake protein-polysaccharide complexes prepared in Examples 1-4 and polyphenols were covalently or non-covalently prepared into composite nanoparticles, and camellia seed cake protein-polysaccharide complex samples (3 mg-10 mg) were placed in sealed aluminum crucibles and heated from 20 °C to 200 °C at a rate of 10 °C / min under nitrogen purging at 20 mL / min. A blank aluminum crucible was used as a control.

[0063] like Figure 1 It was found that both covalent and non-covalent binding of the protein-polysaccharide complex from camellia oil cake meal to polyphenols increased the heat denaturation temperature. The non-covalent binding exhibited a higher heat denaturation temperature than the covalent binding, possibly because non-covalent binding is a reversible process. During heating, these reversible forces can continuously reform and break, allowing the protein structure to maintain a certain degree of stability at high temperatures.

[0064] Test Example 2: Fourier Transform Infrared Analysis of Particles According to the general rules of infrared spectroscopy analysis method in national standard GB / T 6040-2019, the composite nanoparticles prepared by covalently or non-covalently with polyphenols from camellia seed cake protein-polysaccharide complexes obtained in Examples 1-4, and nanoparticles containing only camellia seed cake protein-polysaccharide complexes, were analyzed at 400 cm⁻¹. -1 ~4000cm -1 Fourier transform infrared spectra were measured within the specified wavelength range. All nanoparticles were mixed with KBr powder at a mass ratio of 1:100, and the resolution was 4 cm⁻¹.-1 The total number of scans was 64.

[0065] like Figure 2 The A band indicates a red shift in the amide A band after the addition of polyphenols, suggesting the formation of hydrogen bonds. The red shift is more pronounced for covalently bonded polyphenols than for non-covalently bonded polyphenols. Because tannic acid contains more phenolic hydroxyl groups, its change is more significant than that of catechin epigallocatechin ester. Changes in the absorption peaks in the amide I and amide II bands indicate that the addition of polyphenols altered the original secondary structure of the camellia seed cake protein-polysaccharide complex. After the addition of polyphenols, the absorption peak at 500 cm⁻¹... -1 ~1300cm -1 The presence of characteristic peaks for polyphenols indicates that the protein-polysaccharide complex and polyphenols in the camellia oil cake meal reacted.

[0066] like Figure 2 B indicates that the secondary structure of the sample changed after the addition of polyphenols, with a higher proportion of random coils (red), indicating that the internal structure became looser, leading to more protein unfolding. Specifically, the β-sheet of the protein secondary structure decreased, while random coils increased, indicating a shift towards disorder in the protein.

[0067] Test Example 3: Ultraviolet-Vis Spectroscopic Analysis of Particles According to industry standard JY / T 0570-2020, the camellia seed cake protein-polysaccharide complex prepared in Examples 1-4 and the composite nanoparticles prepared by covalent or non-covalent means with polyphenols, and the camellia seed cake protein-polysaccharide complex alone were dissolved in ultrapure water at a concentration of 1 mg / mL, and ultraviolet-visible absorption spectroscopy analysis was performed, with ultrapure water as a blank.

[0068] like Figure 3 As shown in Figure A, a characteristic absorption peak exists near 280 nm. After binding with polyphenols, the absorption peak at 280 nm is significantly enhanced and undergoes a slight red shift. This is likely because the addition of polyphenols introduces hydroxyl groups, which alter the absorption characteristics of the camellia seed cake protein-polysaccharide complex, resulting in a significant increase in its absorption capacity at 280 nm. The absorption peak intensity increases with the degree of binding. The covalent binding of both polyphenols to the protein has a higher absorption peak intensity than the non-covalent binding, indicating that the covalent binding of protein and polyphenols is superior to the non-covalent binding.

[0069] Test Example 4: Fluorescence Spectroscopy Analysis of Particles The camellia seed cake protein-polysaccharide complexes prepared in Examples 1-4 were covalently or non-covalently prepared with polyphenols to form composite nanoparticles, and camellia seed cake protein-polysaccharide complexes alone were diluted with distilled water to 0.2 mg / mL. Fluorescence spectra were measured using a fluorescence spectrophotometer according to this method. The excitation wavelength was set to 295 nm (to stimulate tryptophan fluorescence), and the emission wavelength range was set to 300–500 nm. The scan rate was 600 nm / min, and the slit width was 5 nm. Fluorescence emission spectra were measured at 25 °C, using ultrapure water as a blank.

[0070] like Figure 3 B indicates that, compared to the camellia seed cake protein-polysaccharide complex, the camellia seed cake protein-polysaccharide complex-polyphenol complex exhibited significant fluorescence quenching. A slight red shift (higher wavenumber) also occurred, suggesting that the addition of polyphenols may affect the surrounding polar environment, causing the environment of the chromophore to change from polar to nonpolar.

[0071] Test Example 5: Surface Hydrophobicity The camellia seed cake protein-polysaccharide complexes prepared in Examples 1-4 were covalently and non-covalently prepared with polyphenols to form composite nanoparticles, as well as the camellia seed cake protein-polysaccharide complexes alone. 8-phenylamino-1-naphthalenesulfonic acid (ANS) was used as a fluorescent probe to measure the samples. All samples were diluted with ultrapure water to obtain a final protein concentration of 0.05-0.25 mg / mL. Then, 1 mL of sample solution was mixed with 10 μL of 8 mM ANS solution. After incubation in the dark for 10 min, the fluorescence intensity was recorded at emission wavelengths of 390 nm and 470 nm using a fluorescence spectrophotometer. H0 was calculated by measuring the initial slope of the fluorescence intensity graph relative to the protein concentration.

[0072] like Figure 3 The "C" indicates that compared to the protein-polysaccharide complex of camellia seed cake, the addition of polyphenols increases surface hydrophobicity, possibly because polyphenols disrupt the protein structure, exposing hydrophobic groups. The addition of polyphenols provides hydrophobic groups to the protein, such as benzene rings. Covalent cross-linking (such as the reaction between phenolic hydroxyl groups oxidized to quinones and the -NH2 / -SH group of the protein) may induce conformational contraction or exposure of the hydrophobic core, leading to enhanced protein surface hydrophobicity.

[0073] Test Example 6: Three-dimensional fluorescence map In the spectral range of 200 to 500 nm, and in continuous scanning mode, 3D fluorescence spectra of the camellia seed cake protein-polysaccharide complex and polyphenols prepared by covalent and non-covalent methods in Examples 1-4, as well as the camellia seed cake protein-polysaccharide complex alone, were recorded in a quartz cell using a fluorophotometer. The excitation wavelength was initially set to 200 nm, and then incremented by 10 nm, for a total of 16 scans.

[0074] like Figure 4 As shown, the fluorescence at Ex=280 and Em=340 nm primarily reflects the fluorescence of tyrosine (Tyr) residues. The peak decreases after reaction with the polyphenol, indicating an interaction between Try and the polyphenol. The covalent complex exhibits a more pronounced peak decrease, suggesting a stronger covalent interaction. This may be due to the formation of quinones that can bind to Try through covalent oxidation.

[0075] Test Example 7: Contact Angle Diagram of Particles The granular powder obtained by freeze-drying was pressed into tablets with a thickness of 2 mm. The tablets were then immersed in optical glass cuvettes containing camellia oil. Deionized water (2 μL) was gently placed on the tablet surface using a high-precision syringe. The three-phase contact angle θ was obtained by simulating the profile of the imaging droplet using the LaPlace-Young equation. The measurements were averaged over at least three droplets. All measurements were repeated three times.

[0076] like Figure 5 The results show that at 0 min, the contact angles are all greater than 90°, indicating that the particles are lipophilic. After covalent binding with polyphenols, the contact angles decrease. At 5 min, the contact angles are all less than 90°, indicating that the particles tend to be hydrophilic, which is beneficial for preparing oil-in-water emulsions.

[0077] Test Example 8: Interfacial Tension Diagram of Particles 20 mL of 0.1% (w / v) Camellia oleifera cake protein-polysaccharide complex prepared in Examples 1-4 was prepared using ultrapure water. Composite nanoparticles were prepared covalently and non-covalently with polyphenols, along with Camellia oleifera cake protein-polysaccharide complex alone. The nanoparticles were magnetically stirred for 2 h until completely dissolved, and the static interfacial tension at the water / oil interface was evaluated. The Young-Laplace equation was used for data fitting the droplet shape curve. The 0.1% (w / v) extract was tested at 120 min and 25 ± 1 °C. The droplet volume was approximately 19–21 μL. The experiment was repeated at least three times.

[0078] like Figure 6 The results indicate that the interfacial tension increased after the addition of polyphenols compared to the protein-polysaccharide complex made solely from camellia seed cake residue. This suggests that polyphenols significantly interacted with the interfacial active components (such as proteins) in the system, forming a more hydrophilic complex that tends to remain in the aqueous phase. When the complex's hydrophilicity increases and it no longer tends to occupy the interface, it self-assembles within the aqueous phase to achieve an energy-stable state, encapsulating the hydrophobic portion and forming nanoparticles. Covalent bonding, by creating a strongly hydrophilic, rigid, and stable complex, significantly weakens the ability of the original interfacial active components to migrate and adsorb to the interface. This covalent bonding effect is far more thorough and persistent than non-covalent bonding, thus manifesting as higher interfacial tension.

[0079] Test Example 9: Turbidity, Potential, and Particle Size Diagram The particle size, PDI, and Zeta potential of the composite nanoparticles prepared by covalently or non-covalently with polyphenols from camellia seed cake meal prepared in Examples 1-4, and the protein-polysaccharide composite nanoparticles from camellia seed cake meal alone, were determined using a Malvern particle size analyzer. The specific operating method is as follows: Particle powder was prepared into a nanoparticle solution with a concentration of 1.0 mg / mL using deionized water. This solution was then diluted 50 times with deionized water. The refractive indices of the protein and water were set to 1.46 and 1.33, respectively, and the measurements were performed at 20°C.

[0080] like Figure 7 The results indicate that the covalent complex exhibits a less pronounced increase in turbidity compared to the non-covalent complex. This may be because the covalent interaction between the camellia oil cake protein-polysaccharide complex and the polyphenols after oxidation to quinone effectively inhibits further oxidative degradation of the protein and polyphenols, reducing the formation of suspended particles and thus resulting in lower turbidity. The increase in the absolute value of the potential upon addition of polyphenols may be due to the negative charge carried by epigallocatechin gallate and tannic acid, increasing the surface charge. Compared to the camellia oil cake protein-polysaccharide complex, the particle size of the complex decreased after the addition of polyphenols, possibly due to electrostatic interactions and steric mechanics leading to a more compact structure.

[0081] Test Example 10: Morphological observation of particles under a scanning electron microscope According to the national standard GB / T36422-2018, the camellia seed cake protein-polysaccharide complex prepared in Examples 1-4 and polyphenols were covalently and non-covalently prepared into composite nanoparticles, and the camellia seed cake protein-polysaccharide complex alone was ground and adhered onto conductive adhesive, and observed by scanning electron microscopy.

[0082] like Figure 8 The study, conducted using SEM at 2000x magnification, revealed that the surface of the camellia oil cake protein-polysaccharide complex was relatively smooth. Upon binding with polyphenols, particularly through non-covalent bonding, fine particles were observed on the surface, suggesting that polyphenols may adsorb onto the surface of the camellia oil cake protein-polysaccharide complex via electrostatic interactions and hydrogen bonds. Epigallocatechin gallate, with its lower molecular weight than tannins, exhibits higher reactivity and can more effectively covalently bind to amino acid residues (such as lysine or arginine) in proteins. The surface of its covalently bound protein is smoother than that of tannins.

[0083] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a covalent or non-covalent complex of camellia seed cake protein-polysaccharide and polyphenol, characterized in that, The preparation method includes: adding polyphenols to a camellia seed cake protein-polysaccharide complex solution, adjusting the pH value to 9.0 ± 0.05, stirring under aerobic conditions to undergo a covalent cross-linking reaction, and obtaining a camellia seed cake protein-polysaccharide complex-polyphenol covalent composite nanoparticle dispersion; dialyzing the product with a molecular weight cutoff ≥ 3500 Da; and freeze-drying to obtain a camellia seed cake protein-polysaccharide and polyphenol covalent complex. Alternatively, polyphenols are added to a solution of camellia seed cake protein-polysaccharide complex, the pH is adjusted to 7.0 ± 0.05, and the mixture is stirred under anaerobic conditions until the liquid is clear or translucent to obtain a non-covalent composite nanoparticle dispersion of camellia seed cake protein-polysaccharide complex-polyphenol. Products with a molecular weight cutoff of ≥3500 Da are dialyzed and then freeze-dried to obtain a non-covalent composite of camellia seed cake protein-polysaccharide and polyphenol.

2. The preparation method according to claim 1, characterized in that, The camellia oil cake protein-polysaccharide complex solution is prepared by dissolving camellia oil cake protein-polysaccharide complex powder in deionized water, with a mass-volume percentage concentration of 2-6%.

3. The preparation method according to claim 2, characterized in that, The protein-polysaccharide complex powder from camellia seed cake was added to deionized water and stirred continuously for 2 hours at room temperature and a stirring speed of 500-800 rpm, and then placed at 4℃ for 12 hours.

4. The preparation method according to claim 1, characterized in that, The polyphenols are epigallocatechin gallate or tannic acid.

5. The preparation method according to claim 1 or 4, characterized in that, The final concentration of polyphenols in the solution system is 1.0-3.0 mg / mL.

6. The preparation method according to claim 1, characterized in that, When preparing camellia seed cake protein-polysaccharide complex-polyphenol covalent composite nanoparticles, the reaction system was kept open with air circulation, and the reaction was carried out for 24 h under constant stirring at 25±1℃ and 500-800 rpm.

7. The preparation method according to claim 1, characterized in that, When preparing non-covalent composite nanoparticles of camellia seed cake protein-polysaccharide complex-polyphenol, the mixed solution after pH adjustment was placed in a closed reactor, nitrogen was introduced to remove oxygen, and the mixture was stirred continuously for 2 hours at 25±1℃ and 500-800 rpm.

8. A protein-polysaccharide-polyphenol covalent or non-covalent complex of camellia seed cake meal prepared by the preparation method according to any one of claims 1-7.

9. The application of the camellia seed cake protein-polysaccharide-polyphenol covalent complex as described in claim 8 in the preparation of functional foods or oral drugs as a carrier, emulsifier or stabilizer.

10. The use of the non-covalent complex of camellia seed cake protein-polysaccharide and polyphenol as described in claim 8 in the preparation of nutritional supplements, emulsifiers or stabilizers.

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