Quaternized cellulose / pectin / theaflavin compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food processing and biological medicine, and particularly relates to a quaternized cellulose / pectin / theaflavin compound as well as a preparation method and application thereof. The quaternized cellulose / pectin / theaflavin compound is prepared by self-assembling quaternized cellulose and pectin serving as wall materials and theaflavin serving as a loaded medicine. Experimental results show that the obtained composite system has efficient encapsulation efficiency and loading capacity on theaflavin, the constructed quaternized cellulose / pectin / theaflavin compound has strong oxidation resistance and pH responsiveness, and the strong oxidation resistance effectively protects theaflavin from oxidative damage in the delivery process and maintains the biological activity of theaflavin; due to the pH responsiveness, the compound realizes more efficient release of theaflavin in acidic environments such as tumor tissues or inflammation parts, so that the treatment pertinence and the treatment effect are improved. Moreover, the compound is simple in preparation method and good in repeatability, and is expected to provide a new reference method for development of a drug delivery system.

Description

Technical Field

[0001] The present invention belongs to the field of food processing technology and biomedicine, and more specifically, relates to a quaternary ammonium cellulose / pectin / theaflavin complex and a preparation method and application thereof. Background Art

[0002] Theaflavins (TFs), as an important component of black tea, have attracted extensive attention in the fields of food, cosmetics, medicine and health products in recent years due to their unique biological activities and pharmacological functions. Theaflavins not only have significant antioxidant, anti-inflammatory, antibacterial and anticancer activities, but also show a variety of health benefits such as lowering blood lipids, lowering blood sugar, and protecting cardiovascular health. However, theaflavins also face problems such as poor chemical stability and low bioavailability in practical applications, which seriously limit the full play of their efficacy.

[0003] In order to solve these problems, researchers began to explore the delivery system of theaflavins, aiming to improve its stability and bioavailability through scientific methods, so as to better realize its biological activity and pharmacological functions. The design principles of the delivery system mainly include protecting theaflavins from damage by the external environment, controlling its release rate, and improving its absorption and distribution efficiency in the body.

[0004] In the study of theaflavin delivery systems, a variety of materials and technologies are widely used. For example, nanotechnology provides a new idea for the delivery of theaflavin. By preparing theaflavin nanoparticles or loading them in nanocarriers, the stability of theaflavin can be significantly improved, while improving its biomembrane permeability and cellular uptake efficiency. However, most of the existing theaflavin delivery systems focus on the construction of single or binary complexes. For example, Chinese patent application CN117137142A discloses a theaflavin complex, which is prepared by compounding theaflavin on β-lactoglobulin through hydrogen bonds and hydrophobic interactions. The theaflavin complex can protect theaflavin from gastric acid and effectively ensure the stability of theaflavin in the human gastrointestinal tract. In addition, it can also improve the biological activity of theaflavin and increase its bioavailability in the human body. However, most traditional binary complexes (such as the theaflavin complex of the above patent) still have obvious deficiencies in stability, encapsulation efficiency and loading capacity, which not only affects the physical stability of the complex, but also limits its application potential in the fields of anti-oxidation, drug delivery and functional foods.

[0005] Therefore, there is an urgent need to construct a theaflavin delivery system with high stability, high encapsulation efficiency and high bioaccessibility. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the obvious deficiencies in stability, encapsulation efficiency and loading capacity of binary complexes used for theaflavin delivery, which not only affect the physical stability of the complex, but also limit its application potential in the fields of anti-oxidation, drug delivery and functional food, and provide a quaternized cellulose / pectin / theaflavin complex.

[0007] The object of the present invention is to provide a method for preparing the quaternary ammonium cellulose / pectin / theaflavin complex.

[0008] Another object of the present invention is to provide the application of the quaternary ammonium cellulose / pectin / theaflavin complex.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention protects a quaternary ammonium cellulose / pectin / theaflavin complex, comprising quaternary ammonium cellulose, pectin, and theaflavin. The quaternary ammonium cellulose / pectin / theaflavin complex uses quaternary ammonium cellulose and pectin as wall materials and theaflavin as a loaded drug, and the three are self-assembled;

[0011] The degree of esterification of the pectin is greater than 50%. Compared with low-ester pectin (degree of esterification less than 50%), high-ester pectin can also maintain good solubility in a low temperature environment. At the same time, under certain pH conditions, the complex prepared from high-ester pectin has a higher potential and can maintain its stability.

[0012] Quaternary ammonium cellulose is positively charged, while pectin and theaflavins are usually negatively charged. This charge difference enables them to be tightly bound through non-covalent bonds such as electrostatic interactions to form a stable quaternary ammonium cellulose / pectin / theaflavins complex. The network structure formed by quaternary ammonium cellulose and pectin can effectively encapsulate theaflavins and slow down the release of theaflavins through non-covalent bonds such as electrostatic interactions and physical barriers. This slow-release mechanism is of great significance for prolonging the action time of theaflavins and improving their effects in specific applications.

[0013] Furthermore, the self-assembly refers to the process in which basic structural units (such as molecules, nanomaterials, micrometer or larger scale substances) spontaneously assemble into polymers through the interaction of non-covalent bonds (such as electrostatic interaction forces, hydrogen bonds, van der Waals forces, hydrophobic effects, etc.).

[0014] Furthermore, the quaternary ammonium cellulose / pectin / theaflavin complex also includes a cross-linking agent. By adding a cross-linking agent, the stability of the complex can be further enhanced. The cross-linking agent can promote non-covalent bonding between the quaternary ammonium cellulose, pectin and theaflavin, thereby improving the structural strength of the resulting complex.

[0015] Preferably, the cross-linking agent comprises sucrose-hydrogen peroxide solution, sucrose-periodate solution or tannic acid.

[0016] Furthermore, the preparation method of the sucrose-hydrogen peroxide solution comprises the following steps: fully reacting sucrose with a hydrogen peroxide solution (the concentration of the hydrogen peroxide solution is preferably 0.3-1.5 vol%) at a solid-liquid ratio of 1: (2.5-10) g / mL to obtain the solution.

[0017] Preferably, the sufficient reaction time is 12 to 72 hours.

[0018] Specifically, the preparation method of the quaternized cellulose comprises the following steps: adding cellulose to a cellulose swelling promoter, filtering after sufficient infiltration, adding a quaternizing agent to the filtrate, and reacting sufficiently to obtain quaternized cellulose. The cellulose is chemically modified to introduce quaternary ammonium groups, thereby enhancing its positive charge. This step is crucial for the subsequent formation of a stable complex with negatively charged pectin and theaflavins.

[0019] Preferably, the cellulose swelling promoter includes one or more of sodium hydroxide, urea, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), tetraethylammonium hydroxide (TEAOH), ammonia water, and potassium hydroxide.

[0020] Preferably, the quaternizing agent includes one or more of 3-chloro-2-hydroxypropyl-trimethylammonium chloride (CHPTA), glycidyl trimethylammonium chloride (GTMAC), 2,3-epoxypropyl trimethylammonium chloride (GTAC), N,N,N',N'-tetramethyl-1,6-hexanediamine (TMHDA), triethylamine (TEA), and trimethylamine (TMA).

[0021] Preferably, the cellulose is selected from bacterial cellulose, plant cellulose or microcrystalline cellulose, more preferably bacterial cellulose. The quaternized cellulose obtained by quaternizing the above-mentioned types of cellulose can be used to prepare a relatively stable ternary complex with high-ester pectin.

[0022] Preferably, the mass ratio of the quaternized cellulose, pectin and theaflavins is 1:(10-250):(1-120).

[0023] More preferably, the mass ratio of the quaternized cellulose, pectin and theaflavins is 1:(25-100):(5-100).

[0024] More preferably, the mass ratio of the quaternized cellulose, pectin and theaflavins is 1:(25-100):(10-100).

[0025] Furthermore, the pectin is obtained by extracting a raw material rich in pectin using an acid extraction method.

[0026] Specifically, the preparation method of pectin comprises the following steps: mixing a raw material rich in pectin and an organic acid at a solid-liquid ratio of 1: (10-30) g / mL, fully extracting, filtering, mixing the filtrate with an ethanol solution, fully precipitating, filtering, and post-treating the precipitate.

[0027] Furthermore, the pectin-rich raw materials include one or more of pomelo peel sponge layer powder, potato peel, apple peel, and citrus peel. Pectins extracted from different sources are all high-ester pectins, and the prepared composites have substantially the same effect.

[0028] Furthermore, the pomelo peel sponge layer powder is obtained by crushing the white sponge layer (also known as the meridian or inner skin of pomelo) on the dried pomelo peel.

[0029] Furthermore, the organic acid includes one or more of citric acid, acetic acid, and tartaric acid.

[0030] Furthermore, the concentration of the ethanol solution is 30 to 99.5 vol%.

[0031] Furthermore, the mixing method is to use ultrasonic mixing.

[0032] The present invention also provides a method for preparing the quaternary ammonium cellulose / pectin / theaflavin complex, comprising the following steps:

[0033] S1. Theaflavin and quaternary ammonium cellulose are dispersed in water and mixed thoroughly to allow theaflavin to be fully adsorbed into the quaternary ammonium cellulose to obtain a theaflavin-quaternary ammonium cellulose complex;

[0034] S2. The theaflavins-quaternized cellulose complex obtained in step S1 is mixed with the pectin solution and allowed to react sufficiently to obtain a quaternized cellulose / pectin / theaflavins complex.

[0035] Furthermore, as a preferred method, the preparation method of the quaternary ammonium cellulose / pectin / theaflavin complex comprises the following steps:

[0036] S1. Theaflavin and quaternary ammonium cellulose are dispersed in water and mixed thoroughly to allow theaflavin to be fully adsorbed into the quaternary ammonium cellulose to obtain a theaflavin-quaternary ammonium cellulose complex;

[0037] S2. The theaflavins-quaternized cellulose complex obtained in step S1 is mixed with the pectin solution and the cross-linking agent and allowed to react sufficiently to obtain the quaternized cellulose / pectin / theaflavins complex.

[0038] Furthermore, in the above preparation method, an alkaline agent, an acidic agent or a buffer solution can be added to adjust the pH of the mixed system. The change in pH value will affect the charge state of quaternary ammonium cellulose, pectin and theaflavins, and further affect the interaction between them and the stability of the formed complex. Within a specific pH range, the charge state of these components is more conducive to the formation of a stable complex.

[0039] Furthermore, the pH range is 2-8, preferably the pH range is 2-6, and more preferably 2-4.

[0040] The above preparation can form a quaternary ammonium cellulose / pectin / theaflavin complex with high encapsulation rate and loading capacity under a wide range of pH conditions. This high encapsulation rate and loading capacity can ensure that more TFs are successfully encapsulated and delivered to the target site, thereby improving the therapeutic effect. Since theaflavin itself has better antioxidant capacity under acidic conditions, the antioxidant capacity of theaflavin solution alone decreases significantly with the increase in pH. After encapsulation, it still has a certain antioxidant capacity under alkaline conditions, and the decline in antioxidant capacity is significantly reduced.

[0041] Furthermore, the acidic reagent includes hydrochloric acid, sulfuric acid, acetic acid, and citric acid.

[0042] Furthermore, the alkaline reagent includes sodium hydroxide, potassium hydroxide, ammonium hydroxide, disodium hydrogen phosphate, and ammonia water.

[0043] Further, the buffer includes any one of acetate buffer (acetic acid-sodium acetate or acetic acid-ammonium acetate), citrate buffer (citric acid-sodium citrate), phosphate buffer (one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate), borate buffer (boric acid-potassium chloride, boric acid-calcium chloride, borax-calcium chloride or borax-sodium carbonate), disodium hydrogen phosphate-citric acid buffer, sodium carbonate-sodium bicarbonate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, and PBS buffer (sodium chloride-disodium hydrogen phosphate-potassium dihydrogen phosphate).

[0044] Furthermore, in the method for preparing the quaternary ammonium cellulose / pectin / theaflavin complex, the sufficient reaction time is 3 to 8 hours.

[0045] Preferably, the temperature of the sufficient reaction is 15° C. or below to avoid oxidation of theaflavins during the preparation process.

[0046] Furthermore, after the sufficient reaction, ultrasonic treatment can be performed to further disperse the obtained complex, so as to obtain more uniform and fine complex particles.

[0047] Preferably, the ultrasonic treatment time is 10 to 60 minutes.

[0048] Preferably, in step S1, in the theaflavin-quaternized cellulose complex, the concentration of theaflavin is 0.1-15 mg / mL (more preferably 0.5-10 mg / mL), and the concentration of the quaternized cellulose is 0.01-2 mg / mL (more preferably 0.05-1 mg / mL).

[0049] Preferably, in step S2, the concentration of the pectin solution is 5 to 15 mg / mL, more preferably 8 to 12 mg / mL).

[0050] Preferably, the amount of the cross-linking agent added is 10-20 vol%, more preferably 12-18 vol%, based on the volume percentage of the quaternary ammonium cellulose / pectin / theaflavin complex.

[0051] The present invention also protects the use of the quaternary ammonium cellulose / pectin / theaflavin complex in the preparation of medicines, health products or cosmetics.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The quaternary ammonium cellulose / pectin / theaflavin complex of the present invention uses quaternary ammonium cellulose and pectin as wall materials and theaflavin as loaded drugs, and the three are self-assembled. Experimental results show that the obtained composite system has a high encapsulation rate and loading capacity for theaflavin, and the constructed quaternary ammonium cellulose / pectin / theaflavin complex has strong antioxidant properties and pH responsiveness. The strong antioxidant capacity effectively protects theaflavin from oxidative damage during the delivery process and maintains its biological activity; pH ​​responsiveness enables the complex to achieve more efficient theaflavin release in acidic environments such as tumor tissues or inflammatory sites, thereby improving the targeted treatment and therapeutic effect. In addition, the preparation method of the complex is simple and reproducible, and is expected to provide a new reference method for the development of drug delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Appearance of CBC / TFs, PAPP / TFs, and CBC / PAPP / TFs when freshly prepared (left) and after 40 days (right).

[0055] Figure 2 The infrared spectra of homemade grapefruit peel sponge layer pectin (PAPP) and commercial pectin (PT).

[0056] Figure 3 The appearance of CBC / PAPP / TFs prepared at different pH values.

[0057] Figure 4The data of particle size (A), antioxidant capacity (B) and bioaccessibility (C) of CBC / PAPP / TFs prepared in Example 3 under the environment of simulated gastrointestinal digestion are statistically shown. (Note: Different lowercase letters in the figure indicate significant differences (p<0.05), and the same letters or no letters indicate no significant differences (p>0.05).) DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0059] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0060] Pomelo peel sponge layer powder: Take the white sponge layer on the commercially available dried pomelo peel (also known as the meridian or inner skin of the pomelo), and grind it into powder to obtain the pomelo peel sponge layer powder.

[0061] Commercial pectin (PT) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0062] Bacterial cellulose was purchased from Hainan Yide Food Co., Ltd.

[0063] Example 1 Preparation of quaternized bacterial cellulose

[0064] (1) Quaternization of bacterial cellulose: 0.06 g of bacterial cellulose was added to 300 mL of an aqueous solution containing 7 wt% NaOH and 12 wt% urea, homogenized at 10,000 rpm for 2 min, and then subjected to magnetic stirring for 1 h. The mixture was then frozen and stored. After being taken out, it was continued to be subjected to magnetic stirring for 1.5-5 h. The suspension was centrifuged at 8,000-10,000 r / min for 10-20 min to achieve solid-liquid separation, and the above steps were repeated for the solid part. CHPTAC (3-chloro-2-hydroxypropyltrimethylammonium chloride) was added to the clarified solution so that its concentration in the system was 1 wt%, and the mixture was continuously stirred at 30° C. for 24 h to complete the surface quaternization of the nanocellulose to obtain quaternized bacterial cellulose (CBC).

[0065] Example 2 Preparation of grapefruit peel derived pectin (esterification degree > 50%)

[0066] The pomelo peel sponge layer powder was mixed and diluted with a citric acid solution in a certain solid-liquid ratio (1 g: 30 mL), ultrasonicated for 10 min, heated in a water bath at a certain temperature and stirred for 12 min using a hot plate magnetic stirrer, and filtered for solid-liquid separation to obtain a filtrate; 95% ethanol and the obtained filtrate were mixed in a volume ratio of 1:1 to obtain a mixture, and the obtained mixture was allowed to stand at room temperature for 4 h before solid-liquid separation, the precipitate was taken and washed with 45% ethanol for 3 times, the ethanol was removed, and the mixture was dried using a vacuum freeze dryer to obtain pomelo peel-derived pectin (PAPP), whose esterification degree was greater than 50%, and it was high-ester pectin.

[0067] Example 3 Preparation of quaternized bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs

[0068] Preparation of PAPP solution: PAPP (prepared according to the method of Example 2) was dispersed in distilled water and stirred in a 30° C. water bath for 12 h to ensure its dissolution, to obtain a PAPP solution with a concentration of 1 wt % (ie, 10 mg / mL).

[0069] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0070] Preparation of quaternary bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs: Weigh CBC (prepared according to the method of Example 1) and TFs (theaflavin) and disperse them together in ultrapure water, and stir them in a water bath at 15°C for 2 hours to obtain a TFs-CBC dispersion (TFs concentration is 2 mg / mL, CBC concentration is 0.2 mg / mL), and then add the TFs-CBC dispersion to the above PAPP solution at a volume ratio of 1:1 under continuous stirring, add 15 vol% sucrose-hydrogen peroxide solution as a cross-linking agent, and perform ultrasonic treatment (add ice bags or ice to ensure that the water temperature is low, at 15°C or below) for 15 minutes, then the system is stirred for reaction in an ice water bath for 4 hours, and ultrasonic treatment is performed again for 30 minutes to obtain a quaternary bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs (the system pH is 3.58).

[0071] After the freshly prepared CBC / PAPP / TFs was placed under refrigeration conditions (4° C.) for 40 days (day, d), the obtained composite CBC / PAPP / TFs was named CBC / PAPP / TFs-40d.

[0072] Example 4 Preparation of quaternized bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs under different pH conditions

[0073] Preparation of PAPP solution: PAPP (prepared according to the method of Example 2) was dispersed in phosphate-citrate buffer at different pH values ​​(pH 2.2, 3.4, 4.0, 4.6, 5.2, 5.8, 7.0, 8.0), and stirred in a 30°C water bath for 12 h to ensure its dissolution, to obtain PAPP solutions with a concentration of 1 wt% (i.e., 10 mg / mL) at different pH values.

[0074] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0075] Preparation of quaternary ammonium bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs: CBC (prepared according to the method of Example 1) and TFs (theaflavin) were weighed and co-dispersed at different pH values ​​(pH The TFs-CBC dispersions with different pH values ​​were added into phosphate-citrate buffer (2.2, 3.4, 4.0, 4.6, 5.2, 5.8, 7.0, 8.0) and stirred in a water bath at 15°C for 2 h to obtain TFs-CBC dispersions with different pH values ​​(TFs concentration was 2 mg / mL and CBC concentration was 0.2 mg / mL). Then, under continuous stirring, the TFs-CBC dispersions with different pH values ​​were added into the PAPP solutions at the corresponding pH values ​​in a volume ratio of 1:1. A 15 wt % sucrose-hydrogen peroxide solution was added as a cross-linking agent and ultrasonic treatment was performed (adding ice bags or ice to ensure that the water temperature was low, at 15°C or below) for 15 min. The system was stirred for reaction in an ice-water bath for 4 h and ultrasonic treatment was performed again for 30 min to obtain quaternary ammonium bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs prepared under different pH values.

[0076] Example 5 Preparation of quaternized bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs loaded with different TFs contents

[0077] Preparation of PAPP solution: PAPP (prepared according to the method of Example 2) was dispersed in distilled water and stirred in a 30° C. water bath for 12 h to ensure its dissolution, to obtain a PAPP solution with a concentration of 1 wt % (ie, 10 mg / mL).

[0078] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0079] Preparation of quaternary ammonium bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs: Weigh CBC (prepared according to the method of Example 1) and different masses of TFs (theaflavin) were dispersed in ultrapure water and stirred in a water bath at 15°C for 2 h to obtain TFs-CBC dispersions with different TFs contents (TFs concentrations were 1, 2, 5, 10, and 20 mg / mL, respectively, and CBC concentration was 0.2 mg / mL). Then, under continuous stirring, TFs-CBC solutions with different TFs contents were added to the mixture at a volume ratio of 1:1. To the above PAPP solution (the final concentrations of TFs were 0.5, 1, 2.5, 5, and 10 mg / mL, respectively, and the final concentration here refers to the concentration of TFs in the system before the cross-linking agent was added), 15 vol% sucrose-hydrogen peroxide solution was added as a cross-linking agent, and ultrasonic treatment (adding ice bags or ice to ensure that the water temperature is low, at 15°C or below) was performed for 15 minutes. The system was stirred for reaction in an ice water bath for 4 hours, and ultrasonic treatment was performed again for 30 minutes to obtain quaternary ammonium bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs loaded with different TFs contents.

[0080] Example 6 Preparation of quaternized bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs with different wall material mass ratios

[0081] Preparation of PAPP solution: PAPP (prepared according to the method of Example 2) was weighed and dispersed in distilled water, and stirred in a 30° C. water bath for 12 h to ensure its dissolution, to obtain a PAPP solution with a concentration of 1 wt % (ie, 10 mg / mL).

[0082] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0083] Preparation of quaternary bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs: Different masses of CBC and TFs (theaflavin) were weighed and dispersed in ultrapure water, and stirred in a water bath at 15°C for 2 h to obtain TFs-CBC dispersions of different concentrations (TFs concentration was 2 mg / mL, and CBC concentrations were 0.05, 0.1, 0.2, 0.4, and 0.8 mg / mL, respectively). Then, under continuous stirring, the TFs-CBC solutions of different concentrations were added to the PAPP solution in a volume ratio of 1:1, and 15 vol% sucrose-hydrogen peroxide solution was added as a cross-linking agent. After ultrasonic treatment (adding ice bags or ice to ensure that the water temperature is low, at 15°C or below) for 15 min, the system was stirred for reaction in an ice-water bath for 4 h, and ultrasonic treatment was performed again for 30 min to obtain quaternary bacterial cellulose / pectin / theaflavin complex CBC / PAPP / TFs with different wall material mass ratios.

[0084] Comparative Example 1 Preparation of Pectin / Theaflavin Complex PAPP / TFs

[0085] Preparation of PAPP solution: PAPP (prepared according to the method of Example 2) was dispersed in distilled water and stirred in a 30° C. water bath for 12 h to ensure its dissolution, to obtain a PAPP solution with a concentration of 1 wt % (ie, 10 mg / mL).

[0086] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0087] Preparation of pectin / theaflavin complex PAPP / TFs: TFs was dispersed in PAPP solution and stirred in a 15°C water bath for 2 h to obtain a TFs-PAPP dispersion (TFs concentration was 1 mg / mL, PAPP concentration was 5 mg / mL), 15 vol% sucrose-hydrogen peroxide solution was added thereto as a cross-linking agent, and ultrasonic treatment was performed (adding ice bags or ice to ensure that the water temperature was low, at 15°C or below) for 15 min. The system was stirred in an ice water bath for 4 h, and ultrasonic treatment was performed again for 30 min to obtain a pectin / theaflavin complex PAPP / TFs.

[0088] After the freshly prepared PAPP / TFs were placed under refrigeration conditions (4° C.) for 40 days (day, d), the resulting composite PAPP / TFs was named PAPP / TFs-40d.

[0089] Comparative Example 2 Preparation of quaternized bacterial cellulose / theaflavin complex CBC / TFs

[0090] Preparation of sucrose-hydrogen peroxide solution: 20 g of sucrose was added to 100 mL of 0.6 vol% hydrogen peroxide solution for reaction.

[0091] Preparation of quaternary bacterial cellulose / theaflavin complex CBC / TFs: CBC and TFs were dispersed in ultrapure water and stirred in a 15°C water bath for 2 h to obtain a TFs-CBC dispersion (TFs concentration was 1 mg / mL, CBC concentration was 0.1 mg / mL). 15 vol% sucrose-hydrogen peroxide solution was added as a cross-linking agent and ultrasonically treated (adding ice bags or ice to ensure that the water temperature was low, at 15°C or below) for 15 min. The system was stirred in an ice water bath for 4 h and ultrasonically treated again for 30 min to obtain a quaternary bacterial cellulose / theaflavin complex CBC / TFs.

[0092] After the freshly prepared CBC / TFs were placed under refrigerated conditions (4° C.) for 40 days (day, d), the obtained composite CBC / TFs was named CBC / TFs-40d.

[0093] Comparative Example 3 Preparation of quaternized bacterial cellulose / chitosan / theaflavin complex CS / PAPP / TFs

[0094] The difference from Example 3 is that CBC is replaced by an equal amount of chitosan (CS) to obtain a quaternary ammonium bacterial cellulose / chitosan / theaflavin complex CS / PAPP / TFs.

[0095] Other conditions and parameters are consistent with those in Example 3.

[0096] Comparative Example 4 Preparation of bacterial cellulose / pectin / theaflavin complex BC / PAPP / TFs

[0097] The difference from Example 3 is that CBC is replaced by an equal amount of bacterial cellulose (BC) to obtain a bacterial cellulose / pectin / theaflavin complex BC / PAPP / TFs.

[0098] Other conditions and parameters are consistent with those in Example 3.

[0099] Comparative Example 5 Preparation of quaternized bacterial cellulose / carrageenan / theaflavin complex CBC / CG / TFs

[0100] The difference from Example 3 is that PAPP is replaced with an equal amount of carrageenan (CG) to obtain a quaternary ammonium bacterial cellulose / carrageenan / theaflavin complex CBC / CG / TFs.

[0101] Other conditions and parameters are consistent with those in Example 3.

[0102] Comparative Example 6 Preparation of quaternized bacterial cellulose / xanthan gum / theaflavin complex CBC / XG / TFs

[0103] The difference from Example 3 is that PAPP is replaced with an equal amount of xanthan gum (XG) to obtain a quaternary ammonium bacterial cellulose / xanthan gum / theaflavin complex CBC / XG / TFs.

[0104] Other conditions and parameters are consistent with those in Example 3.

[0105] Comparative Example 7 Preparation of quaternized bacterial cellulose / gellan gum / theaflavin complex CBC / GG / TFs

[0106] The difference from Example 3 is that PAPP is replaced with an equal amount of gellan gum (GG) to obtain a quaternary ammonium bacterial cellulose / gellan gum / theaflavin complex CBC / GG / TFs.

[0107] Other conditions and parameters are consistent with those in Example 3.

[0108] Comparative Example 8 Preparation of quaternized bacterial cellulose / pectin / theaflavin complex CBC / PT / TFs

[0109] The difference from Example 3 is that PAPP is replaced with an equal amount of commercial pectin (PT, with an esterification degree of <50%, which is low-ester pectin) to obtain a quaternary ammonium bacterial cellulose / pectin / theaflavin complex CBC / PT / TFs.

[0110] Other conditions and parameters are consistent with those in Example 3.

[0111] Comparative Example 9 Preparation of quaternized bacterial cellulose / pectin / tea polyphenols composite CBC / PAPP / TPs

[0112] The difference from Example 3 is that TFs is replaced by an equal amount of tea polyphenols (TPs) to obtain a quaternary ammonium bacterial cellulose / pectin / tea polyphenols complex CBC / PAPP / TPs.

[0113] Other conditions and parameters are consistent with those in Example 3.

[0114] Comparative Example 10 Preparation of quaternized bacterial cellulose / pectin / proanthocyanidin complex CBC / PAPP / Pas

[0115] The difference from Example 3 is that TFs is replaced by an equal amount of proanthocyanidins (Pas) to obtain a quaternary ammonium bacterial cellulose / pectin / proanthocyanidins complex CBC / PAPP / Pas.

[0116] Other conditions and parameters are consistent with those in Example 3.

[0117] Experimental Example 1 Determination of the physicochemical properties of the composite

[0118] 1. Experimental methods

[0119] (1) Determination of particle size and zeta potential

[0120] The particle size, Zeta potential and PDI value (polydispersity index) of the composite solution were measured using a nanoparticle size and Zeta potential instrument (Zetasizer nano ZS).

[0121] (2) Encapsulation efficiency and loading capacity of polyphenols

[0122] The polyphenols in the complex were quantified using the Folin phenol method. Taking theaflavin TFs as an example, 1 mL of the TFs-loaded complex dispersion was taken and centrifuged at 20,000 rpm for 20 min. 0.4 mL of the supernatant, 0.2 mL of Folin phenol reagent and 0.6 mL of sodium carbonate solution (20 wt%) were mixed and fixed to 10 mL. The mixture was placed in a 30 ° C water bath in the dark for 0.5 h and the absorbance at 765 nm was measured. The encapsulation efficiency and loading capacity of other polyphenols were basically the same as the above method, except that the polyphenol content was calculated using the corresponding standard curve for different polyphenols.

[0123] Determination of polyphenol content standard curve: first prepare 50mg / 10mL theaflavins (or tea polyphenols, anthocyanins) solution (then absorb 50, 40, 30, 20, 10, 0uL and finally add water to ensure that the system is 1000uL, and finally take 0.4mL in 1ml for testing), 0.2mL Folin phenol reagent and 0.6mL sodium carbonate solution (20wt%), mix, dilute to 10mL, 30℃ water bath away from light, stand for 0.5h, and measure the absorbance at 765nm. Substitute the absorbance value into the y value of the standard curve to calculate the x value, which is the theaflavins (or tea polyphenols, anthocyanins) content (mg / 10mL).

[0124] Tea polyphenols standard curve: y = 0.0909x-0.0019, R 2 =0.9955

[0125] Anthocyanin standard curve: y = 0.0599x-0.0005, R 2 =0.9998

[0126] Theaflavin standard curve: y = 0.1948x + 0.0046, R 2 =0.9987

[0127] Encapsulation efficiency (%) = (total amount of polyphenols added - free polyphenol content in the supernatant) / total amount of polyphenols added

[0128] ×100% Formula (1)

[0129] Loading capacity (mg / mL) = encapsulated polyphenol content (mg) / composite dispersion volume (mL) Formula (2)

[0130] The encapsulated polyphenol content = the total amount of polyphenol added - the free polyphenol content in the supernatant.

[0131] (3) Determination of antioxidant capacity (DPPH)

[0132] The composite dispersion liquid refers to samples such as juice and wine: the sample: nitrogen free radical extract liquid is shaken and mixed at a ratio of 1:9, centrifuged at 10000rpm for 10min, the supernatant is taken for standby use, and stored at 4℃ for future use. For specific test parameters, refer to Table 1.

[0133] Table 1 Test parameters

[0134] Nitrogen free radical extract DPPH Sample supernatant Anhydrous ethanol <![CDATA[Blank A 0 > 25uL 225uL / / <![CDATA[Sample A 样 > / 225uL 25uL - <![CDATA[Control A 对 > / / 25uL 225uL

[0135] DPPH free radical scavenging rate (%) = (A 0 -(A 样 -A 对 )) / A 0 ×100% Formula (3)

[0136] The particle size, potential, PDI, encapsulation efficiency, loading capacity and antioxidant capacity of the composite particles obtained in the present invention were measured according to the above method. The results are shown in Tables 2 to 9.

[0137] 2. Experimental results

[0138] (1) Comparison of the physicochemical properties of binary and ternary complexes

[0139] Table 2 Results of determination of physicochemical properties of binary and ternary complexes

[0140]

[0141] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0142] As shown in Table 2, the particle size (p<0.05), PDI and absolute value of potential (p<0.05) of the ternary composite prepared on the same day were better than those of the two binary composites.

[0143] For the binary complex CBC / TFs: The particle size of the CBC / TFs complex on the day of preparation was measured to be 1461.00±44.91nm, showing a relatively large particle size. However, after 40 days of storage, its particle size decreased significantly to 1197.00±170.89nm. This change is likely due to the significant depolymerization of the complex during storage. In addition, stratification of the dispersion was observed during long-term storage (see Figure 1 ), which is an intuitive evidence of the deagglomeration process. In addition, the PDI is still large, which reflects that during the deagglomeration process, the particle size distribution is wide, with a large number of small particles generated, and there may also be some incompletely deagglomerated large particles or newly formed aggregates.

[0144] For the binary complex PAPP / TFs: the particle size of the complex was 1105.40±133.21nm on the day of preparation, which decreased to 844.75±24.15nm after 40 days, and PDI increased from 0.500±0.030 to 0.580±0.050. This indicates that some of the large particles may have deagglomerated, but at the same time, there was particle aggregation, and the uneven distribution of particles increased, resulting in a decrease in particle size and an increase in PDI.

[0145] For the ternary complex CBC / PAPP / TFs: the complex may have slightly aggregated during storage, resulting in a slight increase in particle size, but the increase was not significant. The particle size was still at the nanometer level (<1000nm), and the particle distribution was still relatively uniform (PDI<0.5), maintaining a low level overall.

[0146] The higher the absolute value of the Zeta potential, the more stable the surface charge is, which helps prevent particle aggregation. In comparison, the absolute value of the Zeta potential of CBC / PAPP / TFs is the largest (34.65±2.24mV to 30.60±0.65mV), which has better stability and is not prone to aggregation (see Figure 1 ).

[0147] The encapsulation efficiency and loading capacity of all complexes decreased after storage, but the decrease in CBC / PAPP / TFs was the smallest and Figure 1 It can be seen that the composite system has a good embedding effect on theaflavins. After long-term storage, the composite system has neither stratification nor obvious discoloration, indicating that it has good long-term stability and can effectively protect theaflavins and prevent them from oxidative discoloration. Although the encapsulation rate and loading capacity of the binary complex CBC / TFs are not significantly different from those of the ternary complex CBC / PAPP / TFs before and after storage, Figure 1It can be seen that after a period of storage, obvious stratification occurs, indicating that the embedding system is unstable and cannot achieve stable encapsulation of theaflavins. In addition, after storage, the encapsulation rate and loading capacity of the binary complex PAPP / TFs decreased significantly, and the color of the system became significantly darker, indicating that more theaflavins were released from the embedding system, indicating that the long-term stability of the complex system is poor.

[0148] In summary, the CBC / PAPP / TFs complex showed better performance in terms of uniformity, stability, encapsulation efficiency and loading capacity, which was due to the fact that the composite structure composed of CBC, PAPP and TFs could better protect theaflavins from the influence of environmental factors.

[0149] (2) Comparison of the performance of ternary complexes prepared from pectins with different esterification degrees

[0150] The Fourier transform infrared spectrometer (FT-IR) was set in the wave number range of 4000 to 500 cm -1 The degree of esterification (DM) is calculated by integrating the area corresponding to the wavenumber range:

[0151] DM=A 1740 / (A 1630-1600 -A 1740 )*100%

[0152] Table 3 Determination results of CBC / PAPP / TFs and CBC / PT / TFs ternary complexes prepared from pectins with different esterification degrees

[0153]

[0154] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0155] The infrared spectra of homemade PAPP and commercial PT pectin were measured by FT-IR at 1740 cm -1 Nearby and 1630-1600cm -1 The spectral peaks near Figure 2) was integrated by area, and the esterification degree of PAPP was 54.55%, which was high-ester pectin (DM>50%); while the esterification degree of commercial PT was 15.36%, which was low-ester pectin (DM<50%). At the same time, it can be seen from Table 3 that by comparing the composites prepared by the two pectins, it can be seen that the CBC / PAPP / TFs prepared by high-ester pectin have a smaller particle size (672.00±68.38nm), PDI (0.404±0.012) and a larger absolute value of Zeta potential (-34.65±2.24mV), indicating that the CBC / PAPP / TFs prepared based on high-ester pectin have better uniformity and stability. The absolute value of the potential of CBC / PT / TFs prepared by low-ester pectin is lower than 15mV, the PDI value is also higher, the uniformity and stability are poor, and it is easy to stratify or flocculate, which is not conducive to long-term storage and subsequent further use.

[0156] (3) Comparison of the physicochemical properties of ternary complexes prepared using BC, CS, and CBC

[0157] Table 4 Results of determination of physicochemical properties of ternary complexes prepared using BC, CS and CBC

[0158] Group Compound Size(nm) PDI Zeta(mV) Encapsulation rate (%) Loading capacity (mg / mL) Example 3 CBC / PAPP / TFs <![CDATA[672.00±68.38 b ]]> <![CDATA[0.404±0.012 b ]]> <![CDATA[-34.65±2.24 a ]]> <![CDATA[83.41±2.85 a ]]> <![CDATA[0.7252±0.0248 a ]]> Comparative Example 3 CS / PAPP / TFs <![CDATA[776.00±3.07 a ]]> <![CDATA[0.527±0.017 a ]]> <![CDATA[-26.65±0.60 b ]]> <![CDATA[73.79±0.78 b ]]> <![CDATA[0.6417±0.0069 b ]]> Comparative Example 4 BC / PAPP / TFs <![CDATA[792.60±15.06 a ]]> <![CDATA[0.435±0.004 b ]]> <![CDATA[-24.10±0.77 b ]]> <![CDATA[74.03±0.03 b ]]> <![CDATA[0.6437±0.0003 b ]]>

[0159] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0160] The data of the three composites (CS / PAPP / TFs, BC / PAPP / TFs, and CBC / PAPP / TFs) can be analyzed from the aspects of particle size, polydispersity index (PDI), Zeta potential, encapsulation efficiency, and loading capacity. The results are shown in Table 4.

[0161] From the particle size and PDI data, CS / PAPP / TFs has the highest PDI value (0.527±0.017), indicating that its particle size distribution is relatively wide, and its stability and uniformity are poor. The PDI values ​​of BC / PAPP / TFs and CBC / PAPP / TFs are lower and similar (0.435±0.004 and 0.404±0.012, respectively), indicating that their particle size distribution is relatively more uniform, with better stability and consistency.

[0162] Judging from the potential data, the absolute value of Zeta potential of CBC / PAPP / TFs is the largest (34.65±2.24mV), which is significantly higher than the absolute values ​​of potential of BC / PAPP / TFs and CBC / PAPP / TFs, indicating that CBC / PAPP / TFs has better charge stability and more significant electrostatic interaction, which helps to prevent aggregation between particles and thus maintain the stable dispersion state of the complex.

[0163] Regarding the encapsulation efficiency and loading capacity, CBC / PAPP / TFs exhibited the highest encapsulation efficiency (83.41 ± 2.85%) and loading level, demonstrating its excellent ability to encapsulate target substances, while the loading capacities of CS / PAPP / TFs and BC / PAPP / TFs were relatively low.

[0164] In summary, TFs-CBC-PAPP showed better performance in terms of uniformity of particle size distribution, potential stability, encapsulation efficiency and loading capacity, and is a more advantageous complex for loading theaflavins.

[0165] (4) Comparison of the physicochemical properties of ternary complexes prepared under different anionic polysaccharides

[0166] Table 5 Results of the determination of the physicochemical properties of the ternary complexes prepared under different anionic polysaccharides

[0167]

[0168]

[0169] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0170] The physicochemical properties of the ternary complexes prepared under different anionic polysaccharides are shown in Table 5. The particle size of CBC / PAPP / TFs prepared by homemade pectin (PAPP) is the smallest (672.00±68.38nm), and the absolute value of potential is 34.65±2.24mV (absolute value of Zeta potential>25), indicating that it has good stability and uniformity. Although gellan gum (GG), carrageenan (CG) and xanthan gum (XG) have higher potential values, their particle sizes are larger and / or PDI values ​​are>0.5, the particle size is too large, and the uniformity is poor, which is not conducive to the digestion and absorption of the composite particles in the human gastrointestinal tract.

[0171] (5) Comparison of the physicochemical properties of ternary complexes loaded with different polyphenols

[0172] Table 6 Determination results of physicochemical properties of ternary complexes loaded with different polyphenols

[0173] Group Compound Size(nm) PDI Zeta(mV) Encapsulation rate (%) Loading capacity (mg / mL) Example 3 CBC / PAPP / TFs <![CDATA[672.00±68.38 b ]]> <![CDATA[0.404±0.012 b ]]> <![CDATA[-34.65±2.24 a ]]> <![CDATA[83.41±2.85 a ]]> <![CDATA[0.7252±0.0248 a ]]> Comparative Example 9 CBC / PAPP / TPs <![CDATA[772.70±17.31 c ]]> <![CDATA[0.424±0.002 a ]]> <![CDATA[-27.40±0.00 b ]]> <![CDATA[46.52±0.89 b ]]> <![CDATA[0.4652±0.0067 b ]]> Comparative Example 10 CBC / PAPP / Pas <![CDATA[864.50±24.76 a ]]> <![CDATA[0.435±0.014 a ]]> <![CDATA[-26.10±0.55 c ]]> <![CDATA[34.57±0.56 b ]]> <![CDATA[0.3748±0.0043 b ]]>

[0174] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0175] As shown in Table 6, the particle size of CBC / PAPP / TFs is 672.00±68.38nm, the Zeta potential is 34.65±2.24mv, the encapsulation rate reaches 83.41±2.85%, and the loading capacity reaches 0.7252±0.0248mg / mL. It has good potential stability, is not prone to coagulation, and has good encapsulation capacity for TFs. In the CBC / PAPP composite system, the encapsulation rate and loading capacity of CBC / PAPP / TFs are significantly better than those of the other two polyphenols TPs (tea polyphenols) or Pas (proanthocyanidins), indicating that the CBC / PAPP composite system is suitable for the encapsulation of theaflavins, and the encapsulation effect on other types of polyphenols is poor.

[0176] (6) Determination of TFs encapsulation efficiency and loading capacity of CBC / PAPP / TFs under different pH conditions

[0177] Table 7 Determination results of TFs encapsulation efficiency and loading capacity of CBC / PAPP / TFs under different pH conditions

[0178]

[0179]

[0180] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0181] As shown in Table 7, the ternary complex CBC / PAPP / TFs prepared at different pH values ​​(its appearance is shown in FIG. Figure 3 The encapsulation efficiency and loading capacity of the prepared solution (as shown) were above 76% and 0.66 mg / mL, respectively, and it had good encapsulation efficiency and loading level under a wide range of pH.

[0182] The test results of antioxidant properties showed that the ternary complex CBC / PAPP / TFs exhibited extremely strong antioxidant capacity in an acidic environment (especially at pH 2.2), and this strong antioxidant capacity can protect TFs from oxidative damage. Within 30 minutes, the DPPH clearance rate reached 86.46±1.20%, and increased to 93.16±1.50% after 12 hours. As the pH value increased, the antioxidant property gradually decreased. At pH 7.0 and 8.0, the antioxidant property was significantly reduced, and even negative clearance rates appeared (at pH 8.0, the DPPH clearance rate in 30 minutes was -11.57±0.21%). At the same time, compared with the antioxidant capacity of theaflavin aqueous solution, under the same pH conditions, the ternary complex CBC / PAPP / TFs has a long-lasting (12h) antioxidant capacity.

[0183] (7) Results of the determination of the physicochemical properties of the ternary complexes loaded with different amounts of TFs (Example 5)

[0184] Table 8 Determination results of physicochemical properties of ternary complexes loaded with different contents of TFs

[0185]

[0186]

[0187] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0188] As shown in Table 8, with the increase of TFs concentration, the particle size changes, but it is still a small particle size. When the theaflavins concentration is 1 mg / mL, the particle size is the smallest (672.00±68.38nm). The Zeta potential at all concentrations is negative, and the absolute value fluctuates between 30 and 35mV, indicating that the increase in concentration has no significant effect on the Zeta potential, indicating that CBC / PAPP / TFs with different TFs contents have good stability.

[0189] In addition, the encapsulation efficiency and loading capacity increased significantly with the increase of concentration, from 68.11±0.00% and 0.2973±0.0000mg / mL at 0.5mg / mL to 96.40±0.06% and 8.3825±0.0051mg / mL at 10mg / mL. This directly reflects that the CBC / PAPP composite system has a good encapsulation effect on TFs, and the encapsulation efficiency and loading capacity are improved with the increase of TFs concentration.

[0190] (8) Determination of the physical and chemical properties of ternary composites with different mass ratios of wall materials

[0191] Table 9 Determination results of physicochemical properties of ternary composites with different mass ratios of wall materials

[0192]

[0193] Note: Different lowercase letters in the same column indicate significant differences (p<0.05), while the same letters or no letters indicate insignificant differences (p>0.05).

[0194] As shown in Table 9, with the change of the wall material mass ratio, the particle size of the composite also changes, but the overall particle size is still at a small level. The absolute value of the Zeta potential at all concentrations is greater than 25mV (it is generally believed that the absolute value of the potential of the composite particle system>25mV has good charge stability), and all have good charge stability. Among them, when the mass ratio of CBC to PAPP is 1:25 and 1:50, the system is the most stable, and its absolute value of potential is 34.00±2.24mV and 34.08±1.27mV respectively.

[0195] When the mass ratio is 1:50, the complex has the maximum encapsulation efficiency (83.41±2.85%) and loading capacity (0.7252±0.0248mg / mL). As the mass ratio of CBC to PAPP decreases, the encapsulation efficiency and loading capacity of each complex increase first and then decrease, but the overall difference is not significant. This result shows that the change in the wall material mass ratio has limited effect on the encapsulation effect of TFs, that is, the ternary complexes prepared at different mass ratios have good encapsulation efficiency and loading capacity.

[0196] Experimental Example 2 Investigation of in vitro release and bioaccessibility of CBC / PAPP / TFs ternary complex

[0197] 1. Experimental methods

[0198] The CBC / PAPP / TFs ternary complex prepared in Example 3 was used as a representative test object to investigate its in vitro release and bioaccessibility.

[0199] (1) Preparation of TFs aqueous solution

[0200] An appropriate amount of TFs was dispersed in ultrapure water and stirred in a water bath at 15°C for 2 h. The obtained TFs aqueous solution was subjected to ultrasonic treatment (adding ice bags or ice to ensure that the water temperature is low, at 15°C or below) for 15 min. The system was stirred in an ice water bath for 4 h and ultrasonically treated again for 30 min to obtain a TFs aqueous solution with a concentration of 1 mg / mL.

[0201] (2) In vitro digestion experiment

[0202] a) Prepare 10 mL of 3.2 mg / mL pepsin solution (i.e. 32 mg pepsin / 10 mL artificial gastric juice, collectively known as SGF solution)

[0203] b) Prepare 10 mL of 0.8 mg / mL trypsin solution (i.e. 8 mg trypsin / 10 mL artificial intestinal fluid, collectively known as SIF solution)

[0204] c) Simulated gastric digestion: The ternary complex solution of Example 3 (or TFs aqueous solution) was mixed with the SGF solution at a volume ratio of 1:1, stirred in a 37°C water bath, and 2.5 mL was taken at 0, 30, 60, and 90 min, respectively, to measure the particle size and antioxidant capacity.

[0205] d) Simulated intestinal digestion: The mixed solution of the ternary complex CBC / PAPP / TFs (or TFs aqueous solution) of Example 3 and SGF solution was adjusted to pH 6.80±0.02, mixed with SIF solution at a volume ratio of 1:1, stirred in a 37°C water bath, and 2.5 mL was taken at 120, 150, 180 and 210 min, respectively, for the determination of particle size and antioxidant capacity.

[0206] e) After digestion is completed, the total theaflavins content in the simulated intestinal fluid is determined, and the bioaccessibility is calculated according to the following formula:

[0207] Bioaccessibility = m / m 0 ×100% Formula (4)

[0208] Where: m is the content of theaflavins in the intestinal fluid after simulated gastrointestinal digestion, mg; m 0 is the theaflavin content in the complex before digestion, mg.

[0209] 2. Experimental results

[0210] In order to explore the stability of nanoparticles in the gastrointestinal environment, the changes of CBC / PAPP / TFs ternary complex and TFs were compared in an environment simulating gastrointestinal fluid digestion. Figure 4 As shown. Figure 4As shown in Figure (A), after 90 minutes of digestion in simulated gastric juice, the average particle size of CBC / PAPP / TFs decreased from 672.00±68.38nm to 524.35±3.46nm, while the average particle size of TFs aqueous solution increased from 267.65±3.89nm to 1539.00±0.00nm, indicating that TFs have a certain degree of flocculation and aggregation in gastric juice, while CBC / PAPP / TFs has good stability, which shows that the composite CBC / PAPP / TFs can effectively resist harsh conditions such as gastric acid and protect TFs from being destroyed or aggregated. After continuing to digest in simulated intestinal juice for 120 minutes (i.e., 210 minutes of digestion in gastrointestinal juice), the average particle size of both decreased significantly, which is due to the dissociation of the complex caused by the action of digestive enzymes such as pancreatic enzymes, releasing TFs for intestinal absorption.

[0211] The bioavailability of bioactive compounds in the intestine is a key factor in determining whether they can be effectively utilized by the body. Figure 4 As shown in Figure (C), the bioaccessibility of the ternary complex CBC / PAPP / TFs (38.55%) increased by 18.52% compared to the bioaccessibility of unencapsulated TFs (20.03%). This is because the use of CBC and PAPP as carriers to encapsulate TFs not only improves the resistance of TFs to the gastrointestinal environment, but also promotes its release and absorption in the intestine, thereby improving the overall bioavailability. Higher bioaccessibility means that more active ingredients can be absorbed and utilized by the body, which is crucial to improving the effectiveness of drugs or nutritional supplements. This means that the complex CBC / PAPP / TFs can effectively carry and protect TFs, prevent it from being prematurely decomposed in the stomach, and allow it to be more absorbed in the intestine, thereby improving the stability and bioavailability of TFs.

[0212] Depend on Figure 4 As shown in Figure (B), the comparative experiment of the DPPH free radical scavenging ability of the ternary complex CBC / PAPP / TFs and TFs in the gastrointestinal environment shows that the complex CBC / PAPP / TFs can maintain a high antioxidant activity throughout the digestion process, while TFs significantly decreases after entering the intestinal environment. This further confirms the protective effect of the complex CBC / PAPP / TFs on TFs and its ability to exert biological activity at different digestion stages. This means that it can carry more TFs into the stomach. As the gastric juice gradually dissolves or degrades the complex, the encapsulated TFs can be slowly released for further absorption by the stomach and small intestine. Since the complex CBC / PAPP / TFs has good stability in an acidic environment, it can better maintain its structure in the gastric environment and gradually release TFs.

[0213] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A quaternary ammonium cellulose / pectin / theaflavin complex, It is characterized in that It includes quaternary ammonium cellulose, pectin and theaflavins. The quaternary ammonium cellulose / pectin / theaflavins complex uses quaternary ammonium cellulose and pectin as wall materials and theaflavins as loaded drugs, and the three are self-assembled; The esterification degree of the pectin is greater than 50%.

2. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 1, It is characterized in that The quaternized cellulose / pectin / theaflavin complex also includes a cross-linking agent.

3. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 1, It is characterized in that The preparation method of quaternized cellulose comprises the following steps: adding cellulose to a cellulose swelling promoter, filtering after fully soaking, adding a quaternizing agent to the filtrate, and reacting fully to obtain quaternized cellulose.

4. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 3, It is characterized in that The cellulose swelling promoter includes one or more of sodium hydroxide, urea, 1,8-diazabicyclo[5.4.0]undec-7-ene, tetraethylammonium hydroxide, ammonia water, and potassium hydroxide.

5. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 3, It is characterized in that The quaternizing agent includes one or more of 3-chloro-2-hydroxypropyl-trimethylammonium chloride, glycidyl trimethylammonium chloride, 2,3-epoxypropyl trimethylammonium chloride, N,N,N',N'-tetramethyl-1,6-hexanediamine, triethylamine, and trimethylamine.

6. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 3, It is characterized in that The cellulose is selected from bacterial cellulose, plant cellulose or microcrystalline cellulose.

7. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 1, It is characterized in that The mass ratio of the quaternary ammonium cellulose, pectin and theaflavins is 1:(10-250):(1-120).

8. The quaternary ammonium cellulose / pectin / theaflavin complex according to claim 1, It is characterized in that The pectin is obtained by extracting raw materials rich in pectin by using an acid extraction method.

9. The method for preparing the quaternary ammonium cellulose / pectin / theaflavin complex according to any one of claims 1 to 8, It is characterized in that The steps include: S1. Dispersing theaflavins and quaternary ammonium cellulose in water and mixing thoroughly to obtain theaflavins-quaternary ammonium cellulose complex; S2. The theaflavins-quaternized cellulose complex obtained in step S1 is mixed with the pectin solution and allowed to react sufficiently to obtain a quaternized cellulose / pectin / theaflavins complex.

10. Use of the quaternary ammonium cellulose / pectin / theaflavin complex according to any one of claims 1 to 8 in the preparation of medicines, health products or cosmetics.

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