Casein-tea polysaccharide-polyphenol composite nanoparticles and preparation method thereof

Abstract

The invention relates to the technical field of food processing, and discloses casein-tea polysaccharide-polyphenol composite nanoparticles and a preparation method of the casein-tea polysaccharide-polyphenol composite nanoparticles, and the preparation method comprises the following steps: mixing large yellow tea polysaccharide with a casein solution to obtain a CS / TPS mixed solution; performing glycosylation modification on casein in the CS / TPS mixed solution by adopting a Maillard reaction to prepare a casein-polysaccharide copolymer solution; the preparation method comprises the following steps: mixing a plurality of catechins, dissolving the catechins in ultrapure water to obtain a mixture stock solution, mixing the mixture stock solution and a casein-polysaccharide copolymer solution according to a volume ratio of 1: 16, stirring for 1 hour for self-assembly, pre-freezing the stirred solution at-80 DEG C, and freeze-drying for 48 hours to obtain CS-TPS-MIX nano-composite powder. According to the invention, a polyphenol delivery system with a dual antioxidant synergistic mechanism is constructed through a Maillard reaction mediated nano-composite technology, and the system is stable in structure, so that the release of polyphenol is more controllable.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly relates to a casein-polysaccharide-polyphenol nanocomposite particle and a preparation method thereof. Background Art

[0002] Huangda tea is one of the characteristic tea categories in Anhui Province, and its unique quality characteristics originate from the special yellowing and over-firing processes during processing. Research shows that the over-firing process significantly increases the contents of gallic acid, pyrogallic acid, and isomerized catechins in the tea leaves. The changes in these components may affect the health effects of Huangda tea. Through chemical composition analysis and enzyme activity experiments, it is found that the changes in the components in Huangda tea are closely related to its hypoglycemic activity. There are various polyphenolic compounds in Huangda tea, with tea polyphenols represented by catechins as the main body, accounting for 18%-36% of the dry weight. These compounds can show significant blood sugar control effects in both cell and animal models by improving insulin resistance and inhibiting digestive enzyme activity, and are regarded as the core targets in the research of tea functional components. More and more research shows that tea polyphenols have anti-inflammatory effects and can prevent cancer, improve cognitive impairments caused by obesity, metabolic syndrome, and circadian rhythm disorders, etc.

[0003] However, the structure of polyphenolic compounds contains multiple phenolic hydroxyl groups, which causes most polyphenols to show high instability under light, heat, and alkaline conditions. Moreover, their oral absorption effect is poor and the bioavailability is low. These factors severely limit their applications. In recent years, various delivery systems have been developed to effectively improve the solubility, stability, and bioavailability of bioactive compounds, among which the encapsulation technology is the most widely used.

[0004] The core principle of the encapsulation technology is to encapsulate tea polyphenols in specific carriers (such as macromolecular materials like polysaccharides and proteins) to effectively block the interference of the external environment on tea polyphenols. However, some delivery systems have the following problems: 1. The active groups such as phenolic hydroxyl groups on polyphenols interact with carriers such as proteins (such as hydrogen bonds and hydrophobic interactions), resulting in a reduced chance of contact with free radicals and insufficient antioxidant activity of the entire delivery system; 2. Polyphenols are encapsulated inside the composite, and the rate of their release or diffusion into the reaction system is limited, reducing the reaction efficiency with free radicals and affecting the function of polyphenols.

[0005] Therefore, how to further improve the antioxidant activity of the delivery system has become an urgent problem to be solved. Summary of the Invention

[0006] The purpose of the present invention is to provide a casein-tea polysaccharide-polyphenol composite nanoparticle and a preparation method thereof to solve the technical problem of a significant decrease in the polyphenol oxidation activity in the existing polyphenol delivery system.

[0007] To solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides a method for preparing casein-tea polysaccharide-polyphenol composite nanoparticles, comprising the following steps:

[0009] Mix the Huangdacha polysaccharide with the casein solution to obtain a CS / TPS mixture, and subject the casein in the CS / TPS mixture to glycosylation modification by Maillard reaction to prepare a casein-polysaccharide copolymer solution. In the CS / TPS mixture, the mass ratio of Huangdacha polysaccharide to casein is 1:1.

[0010] Mix several catechins and dissolve them in ultrapure water to obtain a mixture stock solution. Mix the mixture stock solution with the casein-polysaccharide copolymer solution at a volume ratio of 1:16, stir for 1 h for self-assembly, and place the stirred solution at -80 °C for pre-freezing and freeze-dry for 48 h to obtain CS-TPS-MIX nanocomposite powder.

[0011] As a preferred embodiment of the present invention, the Maillard reaction comprises the following steps:

[0012] Adjust the pH of the CS / TPS mixture to 7.4, heat and react at 80 °C for 1.5 h. After the reaction, quickly cool it in an ice-water bath and store it at 4 °C for standby.

[0013] As a preferred embodiment of the present invention, the preparation steps of the casein solution are as follows:

[0014] Dissolve casein in ultrapure water, continuously stir until completely dissolved, adjust the pH to 7.4 to make the final concentration of casein 8 mg / mL to prepare the casein solution.

[0015] Store the prepared casein solution at 4 °C for static preservation until completely hydrated.

[0016] As a preferred embodiment of the present invention, four catechins are selected: (-)-epigallocatechin gallate (EGCG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG), gallic acid (GA). The above catechins can be selected mixedly, or individually or in any combination.

[0017] As a preferred embodiment of the present invention, the preparation method of the Huangdacha polysaccharide is as follows: Using Huangdacha as the raw material, extract and purify the Huangdacha polysaccharide through steps such as water extraction, alcohol precipitation, protein removal, and pigment removal.

[0018] The present invention also provides a casein-tea polysaccharide-polyphenol composite nanoparticle, and this casein-tea polysaccharide-polyphenol composite nanoparticle is a ternary complex in which polyphenols are loaded by casein-tea polysaccharide.

[0019] The present invention has the following beneficial effects compared with the prior art:

[0020] The present invention uses casein and the physiologically active Huangda tea polysaccharide as raw materials to prepare a copolymer with excellent functional properties. The encapsulation rate of this copolymer is as high as 90%, the average particle size is between 100-200 nm, the particle size distribution is more uniform, and it is easily soluble in water. It not only greatly improves the stability of tea polyphenols, but also has gastrointestinal sustained and controlled release capabilities, and has prospects for wide application in fields such as food processing, healthcare, and daily chemical industry;

[0021] The present invention constructs a polyphenol delivery system with a dual antioxidant synergistic mechanism. A protein-tea polysaccharide composite carrier is obtained by the Maillard reaction. This reaction can not only obtain the carrier, but also the intermediate products (such as reductones) and end products (such as melanoidins) generated by it can participate in free radical scavenging to a certain extent, making up for the loss of the antioxidant activity of polyphenols caused by encapsulation and enabling it to have sufficient antioxidant activity;

[0022] Compared with the conventional micro-nano carrier technology, the polyphenol loading amount of the CS-TPS-MIX nano-carrier system is further improved. At the same time, the CS-TPS-MIX nano-carrier system has a stable structure, the release of polyphenols is more controllable, more active sites are exposed, and problems caused by the encapsulation of polyphenols are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0024] Figure 1 It is a schematic structural diagram of the preparation method of the casein-tea polysaccharide-polyphenol composite nanoparticle provided by the present invention;

[0025] Figure 2 It is a PDI statistical chart of CS, CS / TPS, and CS-TPS provided by the present invention;

[0026] Figure 3 It is a potential value statistical chart of CS, CS / TPS, and CS-TPS provided by the present invention;

[0027] Figure 4The present invention provides Fourier transform infrared spectra of CS, CS / TPS, CS-TPS, and CS-TPS-MIX;

[0028] Figure 5 The present invention provides a statistical chart of the encapsulation efficiency and loading capacity of EGCG, GCG, ECG, and GA in the CS-TPS-MIX nanocomposite;

[0029] Figure 6 The present invention provides a statistical chart of the particle size and PDI analysis of CS, CS-MIX, and CS-TPS-MIX;

[0030] Figure 7 The present invention provides a statistical chart of the DSC curves of CS, MIX, CS-MIX, and CS-TPS-MIX;

[0031] Figure 8 The present invention provides a DPPH radical scavenging rate chart of CS, CS-TPS, CS-MIX, and CS-TPS-MIX;

[0032] Figure 9 The present invention provides an ABTS radical scavenging rate chart of CS, CS-TPS, CS-MIX, and CS-TPS-MIX;

[0033] Figure 10 The present invention provides a statistical chart of the gastrointestinal simulated digestion of MIX and CS-TPS-MIX nanocomposites. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] As Figure 1 shown, the present invention provides a preparation method of casein-tea polysaccharide-polyphenol composite nanoparticles, including the following steps:

[0036] Mix equal proportions of Huangdacha polysaccharide and casein solution to obtain a CS / TPS mixture, and perform Maillard reaction on the casein in the CS / TPS mixture for glycosylation modification to obtain a casein-polysaccharide copolymer solution, where the mass ratio of casein to Huangdacha polysaccharide in the CS / TPS mixture is 1:1;

[0037] Mix several kinds of catechins and dissolve them in ultrapure water to obtain a mixture stock solution (MIX solution). Mix the mixture stock solution with the casein-polysaccharide copolymer solution at a volume ratio of 1:16, and stir for 1 h for self-assembly. Place the stirred solution at -80 °C for pre-freezing and freeze-dry for 48 h to obtain CS-TPS-MIX nanocomposite powder.

[0038] In the above Maillard reaction, in addition to the casein-polysaccharide copolymer, there are other oxidation systems in the reaction. For example, reductones are generated in the middle stage of the Maillard reaction. Reductones are a class of compounds containing enediol structures (R—C(OH)=C(OH)—R). As intermediate products of the Maillard reaction, reductones scavenge free radicals through hydrogen donor, electron transfer, and metal chelation mechanisms, inhibiting further oxidative damage.

[0039] The brown to black polymeric product melanoidin formed at the final stage of the Maillard reaction. The main product, the casein-yellow big tea polysaccharide copolymer, is included in the generated melanoidin. Melanoidin is mainly composed of nitrogen-containing heterocyclic compounds, polycyclic aromatic hydrocarbons, and sugar-amino acid cross-linking products. Its structure and function are significantly affected by reaction conditions (such as temperature, pH, reactant type). Melanoidins with similar structures can scavenge free radicals and inhibit further oxidative damage.

[0040] Specifically, first, the synergistic antioxidant effect of tea polysaccharide. Tea polysaccharide itself has certain antioxidant ability (by donating hydrogen or chelating metal ions). After forming a complex with casein, the overall antioxidant performance may be enhanced through multi-component synergistic effects. Second, after the Maillard reaction between CS and TPS, the intermediate products (such as reductones) and final products (such as melanoidins) generated can directly participate in free radical scavenging, making up for the loss of decreased polyphenol activity, thereby further improving the activity of the polyphenol delivery system.

[0041] Furthermore, due to the long-chain structure of yellow big tea polysaccharide wrapping casein, a core-shell structure is theoretically formed. The core-shell structure contains voids for catechins (such as tea polyphenols, caffeine) to enter. The addition of polyphenols may fill the voids, making the structure denser. Polyphenols may also bind to polysaccharides to form a more stable complex. In this case, the closer cross-linking may lead to a decrease in particle size. At the same time, the addition of polyphenols may introduce more interactions, reducing the aggregation between particles, forming more uniform nanoparticles, thereby reducing PDI, increasing solubility, better releasing polyphenolic substances, and improving the stability of the polyphenol delivery system.

[0042] The present invention can form a complex with unique physical and chemical properties and functional characteristics through the covalent binding of proteins and polysaccharides. Only by heating, it can drive the covalent cross-linking of proteins and polysaccharides to generate a protein-polysaccharide covalent complex. This delivery system has good solubility, excellent emulsifying ability, strong antioxidant activity and high stability. When constructing such a delivery system, natural proteins and polysaccharides, with their outstanding advantages such as high safety, good biocompatibility and biodegradability, become ideal materials for developing drug delivery systems.

[0043] The drug delivery system constructed by the invention based on proteins or polysaccharides has multiple advantages. On the one hand, it can effectively mask the astringency of bioactive compounds such as polyphenols and improve the sensory quality of products; on the other hand, through encapsulation, protection and controlled release mechanisms, it greatly enhances the stability and biological activity of these compounds.

[0044] It can be seen that in the present invention, Maillard reaction is used to glycosylate casein in the CS / TPS mixture. After the Maillard reaction of the CS-TPS copolymer, not only the polysaccharide itself has antioxidant activity, but the substances generated by the Maillard reaction can directly participate in free radical scavenging, including reductones and melanoidins, which can double compensate for the loss of decreased polyphenol activity and at the same time have a better taste, etc. At the same time, tea polyphenols are encapsulated in the CS-TPS copolymer to effectively block the interference of the external environment on tea polyphenols, form a more stable nanostructure, ensure that bioactive substances or components are not damaged in the gastrointestinal environment, achieve the goals of stable preservation, slow release and enhanced biological activity, and have a better taste, better solubility, emulsibility, high stability and strong antioxidant activity, showing broad application prospects as a delivery carrier for bioactive substances in the food and pharmaceutical industries.

[0045] The following provides examples for specific description of the delivery system:

[0046] Example 1:

[0047] (1) Preparation of huangdacha polysaccharide (TPS): Using huangdacha as the raw material, huangdacha polysaccharide (TPS) was extracted and purified through steps such as water extraction, alcohol precipitation, deproteinization and decolorization;

[0048] (2) Preparation of casein (CS) solution: Dissolve a certain mass of casein (Casein, CS) in ultrapure water and continuously stir until completely dissolved; adjust the pH of the CS solution to 7.4 to make the final protein concentration 8 mg / mL, and place the prepared protein solution at 4 °C overnight to ensure complete hydration;

[0049] (3) Preparation of CS-TPS copolymer by Maillard reaction: Mix the casein (CS) solution and the yellow big tea polysaccharide (TPS) in a mass ratio of casein: yellow big tea polysaccharide of 1:1, adjust the pH to 7.4, and heat and react at 80 °C for 1.5 h. After the reaction, quickly cool it in an ice-water bath and store it at 4 °C for later use to obtain the CS-TPS copolymer solution;

[0050] (4) Preparation of casein-yellow big tea polysaccharide-polyphenol (CS-TPS-MIX) nanocomposite by self-assembly method: Mix four catechins (17.79 mg EGCG, 4.05 mg ECG, 3.72 mg GCG, 1 mg GA) and dissolve them in 15 mL of ultrapure water to prepare a catechin mixture stock solution (MIX); then mix the MIX stock solution with the CS-TPS solution in a volume ratio of 1:16, stir magnetically for 1 h to allow them to interact fully, and finally pre-freeze at -80 °C and freeze-dry for 48 h to obtain the CS-TPS-MIX nanocomposite powder.

[0051] EGCG is (-)-epigallocatechin gallate, GCG is (-)-gallocatechin gallate, ECG is (-)-epicatechin gallate, and GA is gallic acid.

[0052] The CS-TPS copolymer solution and the CS-TPS-MIX nanocomposite are characterized as follows.

[0053] 1. Successfully constructed CS-TPS copolymer solution

[0054] First, compare the particle sizes of the CS solution, the CS / TPS physical mixture solution with those of the CS-TPS copolymer solution prepared in Example 1. As Figure 2 can be seen, compared with CS and CS / TPS, the PDI value of the CS-TPS copolymer is significantly reduced, indicating that the CS-TPS copolymer has a compact structure, a significantly reduced particle size span, and a more uniform particle size distribution.

[0055] It can be seen that although two macromolecules are combined, their average particle size is reduced, and folding and binding occur in the structural space. It can be seen that after the Maillard reaction, CS and TPS undergo covalent interaction to form a glycosylation product with CS, reducing the average particle size of CS-TPS.

[0056] The glycosylation process usually increases the negative charge of the protein being measured. Zeta potential can explore the attractive or repulsive interactions between charged particles. The absolute value of the Zeta potential is proportional to the number of particles with the same charge, that is, the larger the absolute value, the more particles with the same charge, and the more stable the particles. The influence of the Maillard reaction on the Zeta potential of casein is as Figure 3As shown, the absolute value of the potential of the CS-TPS copolymer solution obtained in Example 1 is the largest. This result indicates that through the Maillard reaction, the glycosylation of CS is achieved. The polysaccharide changes the surface charge distribution of the protein through the glycosylation process and slows down the aggregation of the protein by increasing the electrostatic repulsion. This system is more stable than a simple mixture of protein and polysaccharide.

[0057] 2. Successfully constructed CS-TPS-MIX nanocomposites

[0058] See Figure 4 as shown Figure 4 Figure 1 is the Fourier transform infrared spectroscopy (FT-IR) of different nanoparticles. FT-IR is used to characterize the potential molecular interactions between proteins, polysaccharides, and polyphenols.

[0059] As can be seen from the figure, the absorption peaks of different samples in the range of 3200-3400 cm -1 are attributed to the stretching vibration of the -OH group. The absorption peak of CS-TPS is located at about 3416 cm -1 . After adding the mixed polyphenol MIX, these absorption peaks shift. The change in the position of the -OH peak in the sample indicates that there is a hydrogen bond interaction force between CS-TPS and MIX. CS is hydrophobic, and the mixed polyphenol MIX contains hydrophobic benzene rings. It is speculated that hydrophobic interaction may be involved in the formation of the nanocomposite. The characteristic absorption peaks in the range of 1000-1800 cm -1 observed in the spectrum of MIX disappear after encapsulation, and the infrared spectrum also shows that no new bonds are formed.

[0060] The above results indicate that the CS-TPS-MIX complex is formed through hydrogen bonds, electrostatic interactions, and hydrophobic interactions, and MIX has been successfully encapsulated in the CS-TPS copolymer.

[0061] 3. Differences in the encapsulation efficiency and loading rate of MIX

[0062] See Figure 5 , and the CS-TPS copolymer successfully encapsulates polyphenols with an encapsulation efficiency as high as 90%.

[0063] Further analysis showed that the encapsulation efficiency and loading efficiency of the four polyphenolic compounds in CS-TPS were different. The encapsulation efficiencies of GA, EGCG, and ECG in the mixture were 84.31%, 98.46%, and 95.59%, respectively, corresponding to loading capacities of 9.38%, 19.20%, and 6.69%, respectively. The encapsulation efficiency of GCG was the highest, approaching 100%, and the loading capacity was approximately 5.5%. The loading amounts of the four polyphenolic compounds were different. By analyzing the molecular structures, it was probably due to the different numbers of phenolic hydroxyl groups in the compounds. In fact, the more phenolic hydroxyl groups there are, the stronger the intermolecular hydrogen bond interactions. The particle size of the nano-complex formed by CS-TPS was about 100-200 nm, and there was a hydrophobic microenvironment inside. The planar rigid structures of EGCG and GCG were more likely to be embedded in the hydrophobic core of the copolymer, while GA molecules were smaller and had poor spatial adaptability, resulting in a decrease in encapsulation efficiency.

[0064] Multiple active ingredients in tea interact synergistically to provide various health benefits, such as antioxidant, anti-inflammatory, blood pressure lowering, and blood lipid lowering effects. A single active substance usually only has specific physiological activities. For example, tea polyphenols mainly exhibit antioxidant effects, and caffeine has the effect of refreshing the mind, etc. The CS-TPS-MIX nano-complex can mix multiple tea polyphenols together, enabling the complementation and enhancement of various effects and improving the overall health care effect of tea. Reasonably combining the active ingredients of tea and single active substances will bring consumers healthier and more delicious tea products.

[0065] The following is the functional analysis of the CS-TPS-MIX nano-complex through Comparative Example 1

[0066] Comparative Example 1:

[0067] The CS-MIX nano-complex was prepared by the self-assembly method.

[0068] Characterization:

[0069] (1) The CS-TPS-MIX ternary complex had a smaller and more uniform particle size

[0070] The particle sizes of the CS solution, the CS-MIX nano-complex in Comparative Example 1, and the CS-TPS-MIX ternary complex prepared in Example 1 were compared. The results are shown in Figure 6 , Figure 6 which was the statistical chart of the particle sizes and PDI of different nanoparticles. The particle sizes of CS, CS-MIX, and CS-TPS-MIX were mainly distributed between 100-300 nm. Compared with CS and CS-MIX nanoparticles, the particle size and PDI of the CS-TPS-MIX complex both decreased. Polyphenols usually contain multiple hydroxyl groups and can form hydrogen bonds or hydrophobic interactions with proteins and polysaccharides.

[0071] Polyphenols usually contain multiple hydroxyl groups, which can undergo complex interactions with proteins and polysaccharides, including forming hydrogen bonds or participating in hydrophobic interactions. When polyphenols are added to the system to form a ternary complex, polyphenols are very likely to act as cross-linking agents, further promoting the binding of proteins and polysaccharides, thus making the structure of the complex more compact. Specifically, the hydroxyl groups of polyphenols can form hydrogen bonds with the amino or carboxyl groups of proteins, or bind tightly to proteins through hydrophobic interactions. At the same time, the long-chain structure of polysaccharides often wraps proteins to form a core-shell structure, and the addition of polyphenols may fill the gaps between the core-shell structures, making the overall structure more dense. Tighter cross-linking may lead to a decrease in particle size, and the addition of polyphenols may introduce more interactions, reducing the aggregation between particles and forming more uniform nanoparticles, thereby reducing the PDI.

[0072] (2) The CS-TPS-MIX ternary complex has excellent thermal stability

[0073] As a thermal property technique, differential scanning calorimetry (DSC) is often used to explore the thermal behavior characteristics of the phase transition of biological materials. It can determine the temperature of sample pyrolysis and the exothermic or endothermic heat accompanying the pyrolysis reaction, and is an important indicator for evaluating the thermal decomposition of reaction products.

[0074] The DSC curves of different nanoparticles are as Figure 7 shown. It can be seen from the figure that:

[0075] ① After embedding the mixed polyphenol MIX, no endothermic peak of MIX appears in the CS-MIX and CS-TPS-MIX nanoparticles, indicating that MIX has changed from the crystalline state to the amorphous state, and the polyphenol has been successfully embedded;

[0076] ② In the temperature range of 50 - 220 °C, the DSC curve of CS has a relatively broad endothermic peak at about 193.71 °C, and that of the mixed polyphenol MIX has a relatively broad endothermic peak at about 97.66 °C, and its thermal stability is poor. After adding MIX to the CS and CS-TPS copolymers, the denaturation temperatures of CS-MIX and CS-TPS-MIX are increased to 195.43 °C and 201.92 °C respectively, both of which increase the peak temperature of MIX, thereby improving its thermal stability. It can be seen that the CS-TPS-MIX nanocomposite has better thermal stability.

[0077] (3) The CS-TPS-MIX ternary complex has a high free radical scavenging rate

[0078] The antioxidant activities of polyphenol MIX before and after encapsulation were evaluated by DPPH and ABTS free radical scavenging experiments. Specifically, the oxidation activities of the above CS-TPS-MIX nanocomposite powder were compared with those of CS, CS / TPS physical mixture solution, CS-TPS polymerization solution, and CS-MIX. The results are shown in Figure 8 and Figure 9 as follows.

[0079] Figure 8 and Figure 9 showed that the results of the scavenging of ABTS free radical activity by the above substances were similar to those of the scavenging of DPPH free radical activity. In the concentration gradient of 0.25 - 1.0 mg / mL, the DPPH and ABTS free radical scavenging abilities of CS, CS / TPS mixture, CS-TPS copolymer, CS-MIX, and CS-TPS-MIX all showed a dose-dependent phenomenon.

[0080] At about the concentration of 0.25 mg / mL, the free radical scavenging rate of the CS-TPS-MIX nanocomposite was much higher than that of CS-MIX, that is, the antioxidant activity of CS-TPS-MIX was still higher than that of CS-MIX.

[0081] The reasons for the high antioxidant ability of the CS-TPS-MIX nanocomposite are as follows:

[0082] ① The synergistic antioxidant effect of tea polysaccharide. Tea polysaccharide itself has certain antioxidant ability (by donating hydrogen or chelating metal ions). After forming a complex with casein, the overall antioxidant performance may be enhanced through the synergistic effect of multiple components;

[0083] ② The formation of reductones in the middle stage of the Maillard reaction. As an intermediate product of the Maillard reaction, reductones scavenge free radicals through hydrogen donors, electron transfer, and metal chelation mechanisms, inhibiting further oxidative damage;

[0084] ③ The melanoidins (casein - yellow big tea polysaccharide copolymer), the brown to black polymeric products formed in the final stage of the Maillard reaction. Melanoidins are composed of nitrogen-containing heterocyclic compounds, polycyclic aromatic hydrocarbons, and sugar - amino acid cross-linking products. The formation of new bonds enhances the thermal stability and antioxidant ability of the copolymer, compensating for the loss of polyphenol activity decline;

[0085] ④ The nanostructure of the CS-TPS-MIX nanocomposite is more stable, making the release of polyphenols more controllable, while exposing more active sites. The core-shell system formed by the CS-TPS copolymer provides better protection for the polyphenol substances inserted therein, reducing their degradation or inactivation during the measurement process.

[0086] (4) Sustained release of the CS-TPS-MIX ternary complex

[0087] The CS-TPS-MIX nanocomposite was digested in SGF for 120 min and then quickly transferred to SIF for continued digestion for 120 min. The sustained-release rate of EGCG was measured at different time points, and the results are as Figure 10 shown Figure 10 in the gastrointestinal tract simulated digestion statistical charts of MIX and CS-TPS-MIX nanocomposites.

[0088] As can be seen from the figure, after 2 h of enzymatic hydrolysis in SGF, the release rate of the mixed polyphenol MIX was 54.06%, while the release rate after encapsulation was 40.38%. After continuous digestion in SIF for 4 h, the release rate of CS-TPS-MIX reached 89.09%, and the release rate of the free polyphenol MIX was 98.33%.

[0089] It can be seen that the polyphenol release rate of the CS-TPS-MIX complex in the gastric stage was significantly lower than that of the free MIX. CS-TPS had a protective effect on the mixed polyphenol MIX in the stomach and promoted its release in the intestine. In SGF, the CS-TPS-MIX nanocomposite hindered the digestion of the mixed polyphenol MIX by pepsin, resulting in a smaller release of the mixed polyphenol MIX. When the CS-TPS-MIX nanocomposite was transferred to simulated intestinal fluid, the change in pH weakened the interaction between CS and TPS. Subsequently, trypsin destroyed the protein structure to promote the release of the mixed polyphenol MIX.

[0090] Compared with the free mixed polyphenol MIX, the CS-TPS-MIX system formed by encapsulation with CS-TPS showed unique release characteristics. The time required for complete release of CS-TPS-MIX was significantly prolonged, which fully demonstrated that CS-TPS effectively protected the mixed polyphenol MIX. And in the intestinal digestion stage, the polyphenol release rate in CS-TPS-MIX was significantly accelerated, and its bioaccessibility was significantly improved compared with the free MIX. This phenomenon strongly indicates that the CS-TPS-MIX complex has the ability to precisely regulate the release of polyphenols, can reasonably adjust the release rhythm of polyphenols according to the physiological environment of the specific intestinal stage, and thus greatly improves the bioavailability of polyphenols.

[0091] The casein delivery system constructed by modification through the Maillard reaction based on casein in the present invention can not only significantly enhance the stability of bioactive substances in the gastrointestinal tract and resist the degradation by gastric acid, digestive enzymes, etc., but also achieve the targeted release of bioactive substances at specific sites through precise design. This characteristic greatly improves the effectiveness and safety of bioactive substances, reduces the required dosage, and reduces potential side effects. At the same time, it provides important technical support for the optimization and innovation of bioactive substance delivery systems.

[0092] Through the casein-tea polysaccharide-polyphenol composite nanoparticles and the preparation method thereof in this embodiment, further improvement of the oxidation activity of the polyphenol delivery system can be achieved, and the structure of the system is stable, the release of polyphenols is more controllable, and at the same time, more active sites are exposed, which can better protect polyphenolic substances and ensure their biological activities.

[0093] The above embodiments are only exemplary embodiments of the present application and are not used to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions within the essence and protection scope of the present application, and such modifications or equivalent substitutions should also be regarded as falling within the protection scope of the present application.

Claims

1. A preparation method of casein-tea polysaccharide-polyphenol composite nanoparticles, characterized in that, It includes the following steps: Mix yellow big tea polysaccharide with casein solution to obtain CS / TPS mixture, and carry out glycosylation modification on casein in the CS / TPS mixture by Maillard reaction to prepare casein-polysaccharide copolymer solution; Mix several kinds of catechins and dissolve them in ultrapure water to obtain a mixture stock solution. Mix the mixture stock solution with the casein-polysaccharide copolymer solution at a volume ratio of 1:16, stir for 1 h for self-assembly, and place the stirred solution at -80 °C for pre-freezing and freeze-dry for 48 h to obtain CS-TPS-MIX nanocomposite powder.

2. The preparation method of a casein-tea polysaccharide-polyphenol composite nanoparticle according to claim 1, characterized in that, The Maillard reaction includes the following steps: Adjust the pH of the CS / TPS mixture to 7.4, heat and react at 80 °C for 1.5 h. After the reaction ends, quickly cool it in an ice-water bath and store it at 4 °C for standby.

3. The preparation method of a casein-tea polysaccharide-polyphenol composite nanoparticle according to claim 1, characterized in that, The preparation steps of the casein solution are as follows: Dissolve casein in ultrapure water, continuously stir until completely dissolved, adjust the pH to 7.4 to make the final concentration of casein 8 mg / mL to prepare the casein solution; Store the prepared casein solution at 4 °C until completely hydrated.

4. The preparation method of a casein-tea polysaccharide-polyphenol composite nanoparticle according to claim 1, characterized in that, The catechin is selected from any one of EGCG, ECG, GCG, and GA.

5. The preparation method of a casein-tea polysaccharide-polyphenol composite nanoparticle according to claim 1, characterized in that The preparation method of the yellow big tea polysaccharide is as follows: Using yellow big tea as the raw material, extract and purify the yellow big tea polysaccharide through the steps of water extraction, alcohol precipitation, protein removal, and pigment removal.

6. A casein-tea polysaccharide-polyphenol composite nanoparticle, which is prepared by the preparation method of the casein-tea polysaccharide-polyphenol composite nanoparticle according to any one of claims 1-5.

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