Catechin-loaded auricularia fuscosuccinea polysaccharide-gelatin nanocapsule and preparation method thereof

Abstract

The invention discloses a catechin-loaded auricularia fuscosuccinea polysaccharide-gelatin nanocapsule and a preparation method thereof. The preparation method comprises the following steps: dissolving auricularia fuscosuccinea polysaccharide and gelatin in ultrapure water to obtain an auricularia fuscosuccinea polysaccharide solution and a gelatin solution; adjusting the pH value; catechin is dissolved in absolute ethyl alcohol, and a catechin solution is obtained; adding the catechin solution into the auricularia fuscosuccinea polysaccharide solution, and stirring at room temperature to obtain an auricularia fuscosuccinea polysaccharide-catechin solution Dropwise adding the auricularia fuscosuccinea polysaccharide-catechin solution into a gelatin solution, and performing ultrasonic treatment in dark and ice-water bath to obtain catechin-loaded auricularia fuscosuccinea polysaccharide-gelatin nanocapsule suspension. The preparation method disclosed by the invention is simple and convenient, the process is safe and controllable, the physicochemical properties are synergistically exerted by taking the auricularia fuscosuccinea polysaccharide and the gelatin as the composite wall material, the efficient loading and slow release of the catechin are realized, and a new thought is provided for the application of the auricularia fuscosuccinea polysaccharide and the improvement of the bioavailability of the catechin.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide-based nanocapsule materials and active substance sustained-release preparations, and in particular to catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules and a preparation method thereof. Background Art

[0002] Consumers are increasingly demanding nutritional supplements, and bioactive compounds derived from natural foods have received widespread attention. Catechins are a type of phenolic active substance, mainly extracted from natural plants such as tea leaves, and have significant antioxidant, free radical scavenging and antibacterial activities. Many studies have reported that catechins can be used as a promising anti-tumor, platelet aggregation, inflammation and aging drug, and are considered to have potential application prospects and commercial value in the field of functional foods and medicine. However, the low oral bioavailability, astringency and bitterness, and extremely poor physicochemical stability of catechins severely limit their application in medicines and foods. Therefore, effective methods are urgently needed to address these problems.

[0003] Nanostructured drug delivery systems such as liposomes, emulsions, and particles have received extensive attention in past studies due to their advantages such as small carrier volume, large encapsulation volume, and good physical and chemical stability; they have also been shown to improve the stability and bioavailability of catechins. Delivery systems based on nanoencapsulation systems are considered to be a viable option that can protect catechins from harsh environments and thus improve stability. In general, drug-loaded nanosystems can protect core materials from adverse environmental conditions, delay their degradation in body fluid environments, and easily break through various barriers to reach target organs, thereby controlling the release of drugs and improving the bioavailability of active substances. Auricularia auricula is a common edible fungus that has received widespread attention in recent years for its rich nutritional components and potential medicinal value. Auricularia auricula polysaccharides are mainly composed of monosaccharides such as glucose, mannose, and galactose. They are believed to have multiple biological activities such as immunomodulation, anti-tumor, and antioxidant, and have broad application potential in the fields of food, health products, and medicines. However, there are few studies on the application of Auricularia auricula polysaccharides in the field of nanoscale carrier materials. Gelatin is a natural polymer material produced by the hydrolysis of collagen. It is widely used in drug delivery due to its excellent biocompatibility, biodegradability and good processing properties.

[0004] At present, gelatin is mainly prepared into microspheres or nanoparticles through emulsification, spray drying and other methods for drug encapsulation, or hydrogels based on gelatin are prepared to control drug release rate, which can adjust its swelling according to environmental changes (such as pH, temperature, etc.), thereby achieving smart drug release, but the development of gelatin-based nanocapsule materials is still rare. Since Auricularia auricula polysaccharides and gelatin have good biocompatibility, biodegradability and biological activity, the sustainable controlled release of catechin active substances based on these two biomacromolecules is a feasible way to improve the bioavailability of encapsulated nanosystems. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for preparing catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules, which is simple and convenient, has a safe and controllable process, and can achieve continuous and low-cost green production.

[0007] Still another object of the present invention is to provide a catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsule, which achieves efficient delivery and sustained release of catechins by using Auricularia auricularia polysaccharide and gelatin as composite wall materials to synergistically exert their physical and chemical properties, thereby providing an effective idea for expanding the application of Auricularia auricularia polysaccharide and improving the bioavailability of catechins.

[0008] According to these purposes and other advantages of the present invention, a method for preparing catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules is provided, which specifically comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare Auricularia auriculariae polysaccharide solution and gelatin solution with a concentration of 0.2-0.5% w / v; 2) Adjust the pH of the Auricularia auricula polysaccharide solution and gelatin solution to 2.5-4.0; 3) dissolving catechins in anhydrous ethanol to prepare a catechin solution with a concentration of 0.2-0.4% w / v; 4) adding the catechin solution to the Auricularia auriculariae polysaccharide solution, and magnetically stirring at a constant speed of 600 to 800 rpm for 20 to 35 min at room temperature to prepare an Auricularia auriculariae polysaccharide-catechin solution; wherein the concentration of catechin in the Auricularia auriculariae polysaccharide-catechin solution is 0.05 to 0.2% w / v; 5) The Auricularia auriculariae polysaccharide-catechin solution was added dropwise into the gelatin solution at a volume ratio of 1.0 to 4.0, and ultrasonic treatment was performed in the dark and in an ice water bath to obtain a suspension of Auricularia auriculariae polysaccharide-gelatin nanocapsules loaded with catechins.

[0009] Preferably, between step 4) and step 5), a dialysis step is further included, comprising: Select a dialysis bag with a molecular weight cutoff range of 1000-3000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L EDTA at 80°C for 30 min, and then rinse it with ultrapure water; The Auricularia auricularia polysaccharide-catechin solution was loaded into a pretreated dialysis bag so that the volume of the solution in the dialysis bag did not exceed 80% of its maximum carrying capacity; the dialysis bag containing the Auricularia auricularia polysaccharide-catechin solution was placed in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricularia polysaccharide-catechin solution was controlled at 100:1-200:1; a magnetic stirrer was used to stir the dialysis external fluid at a stirring speed of 200-300 rpm; the dialysis device was placed in a constant temperature box and the temperature was maintained at 4-8°C; the dialysis water was replaced every 4-6 hours, and the conductivity of the dialysis external fluid was tested with a conductivity meter before replacement. If the conductivity decreased by less than 10%, the replacement frequency of the dialysis external fluid was increased to once every 2 hours; the dialysis was continued for 24-48 hours.

[0010] Preferably, between step 3) and step 4), an anti-oxidation step is further included, comprising: First, the temperature of the prepared catechin solution is adjusted to 20-25°C, 0.025-0.05% of ascorbic acid is added, and the mixture is stirred at a speed of 200-300 rpm for 10-15 min; at the same time, nitrogen gas is introduced into the solution at a gas flow rate of 0.5-1 L / min; The stirring speed was increased to 230-260 rpm, and ascorbic acid with a mass fraction of 0.025-0.05% was added again, and the mixture was stirred for 4-6 min; nitrogen was kept flowing, and the flow rate was adjusted to 1-1.5 L / min to obtain a treated catechin solution.

[0011] Preferably, in step 1), the concentrations of the wood ear polysaccharide solution and the gelatin solution are both 0.5% w / v; in step 2), the pH of the wood ear polysaccharide solution and the gelatin solution are both 4.0; in step 3), the concentration of the catechin solution is 0.4% w / v; in step 4), the magnetic stirring speed is 700 rpm and the stirring time is 30 min; in step 5), the mixing ratio of the wood ear polysaccharide-catechin solution and the gelatin solution is 1:1.

[0012] Preferably, in step 5), the conditions for ultrasonic treatment are: frequency of 20 kHz, power of 600-1000 W, ultrasonic speed of 600-800 rpm, and ultrasonic time of 1.5-2.5 h.

[0013] Preferably, in step 5), the conditions for ultrasonic treatment are: frequency of 20 kHz, power of 600 W, ultrasonic speed of 700 rpm, and ultrasonic time of 2.0 h.

[0014] Preferably, in step 2), 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution are used to adjust the pH.

[0015] Preferably, the Auricularia auricularia polysaccharide is extracted from Auricularia auricularia fruiting bodies.

[0016] Preferably, the extraction process specifically comprises: crushing the dried Auricularia auricularia fruiting body, sieving through a 160-mesh sieve to obtain a powder, using a choline chloride / urea type natural deep eutectic solvent as an extraction solvent, with a material ratio of 1:53 g / mL, extracting the powder at 65°C for 90 min to obtain an extract, centrifuging the extract at 4000 r / min for 10 min, collecting the supernatant containing Auricularia auricularia crude polysaccharide, using a rotary evaporator to concentrate the supernatant to 1 / 5 to 1 / 4 of its volume, then adding 4 times the volume of anhydrous ethanol and standing at 4°C for 12 h, centrifuging at 4000 r / min for 10 min, collecting the precipitate, removing protein from the precipitate using the Sevag method to obtain a concentrate, and freeze-drying the concentrate at -80°C for 12 h to obtain Auricularia auricularia polysaccharide.

[0017] The purpose of the present invention can be further achieved by the catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules prepared by the preparation method.

[0018] The present invention has at least the following beneficial effects: The present invention provides a method for preparing catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules which is simple and convenient to operate, has a safe and controllable process, and can realize continuous and low-cost green production. The method realizes efficient encapsulation, delivery and sustained release of catechins by synergistically exerting the physical and chemical properties of Auricularia auricularia polysaccharide and gelatin as composite wall materials, thereby providing an effective idea for expanding the application of Auricularia auricularia polysaccharide and improving the bioavailability of catechins.

[0019] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of catechin encapsulation efficiency in the final materials prepared in Examples 1 to 5 of the present invention and Comparative Examples 2 to 15; Figure 2This is a comparison chart of the bioavailability of the final substances prepared in Examples 1 to 5 of the present invention and Comparative Examples 1 to 15 in simulated in vitro digestion; Figure 3 This is a comparison chart of the retention rates of the final substances prepared in Examples 1 to 5 and Comparative Examples 1 to 15 of the present invention after being treated with light; Figure 4 This is a comparison chart of the retention rates of the final substances prepared in Examples 1 to 5 of the present invention and Comparative Examples 1 to 15 after heat treatment. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0024] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0025] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0026] It is worth noting that, for the sake of comparison, in the following embodiments, high-intensity ultrasonic processor (SHL-2A, Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.), spectrophotometer (UV-2600, Shimadzu Corporation), magnetic stirrer (79-1 magnetic stirrer, Shanghai Silo Instrument), ultraviolet lamp (10 cm, Thermo Fisher Scientific), LGJ-10 vacuum freeze dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd.), Auricularia auricula polysaccharide (extracted from Auricularia auricula fruiting body), gelatin (Shanghai Aladdin Biological Reagent Co., Ltd.), catechin (Shanghai Aladdin Biological Reagent Co., Ltd.), β-cyclodextrin (Shanghai Aladdin Biological Reagent Co., Ltd.), gum arabic (Shanghai Aladdin Biological Reagent Co., Ltd.), but not limited to these.

[0027] Example 1 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the treated catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stirring at a constant speed of 700 rpm for 30 min using a high-intensity ultrasonicator at room temperature to prepare an Auricularia auricula polysaccharide-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auricula polysaccharide-catechin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auricula polysaccharide-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the replacement frequency of the dialysis external fluid to increase the replacement frequency of the dialysis external fluid to every 2 hours. h change once; dialysis continued for 36 h; 7) The Auricularia auricularia polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:1, and ultrasonic treatment was performed at 700 rpm for 2.0 h in the dark and in an ice water bath using a high-intensity ultrasonicator to obtain a suspension of Auricularia auricularia polysaccharide-gelatin nanocapsules loaded with catechins. The working conditions of the high-intensity ultrasonicator were as follows: probe diameter of 10 mm, frequency of 20 kHz, power of 600 W (maximum power of 1000 W), and on / off time of 5 s.

[0028] Among them, the extraction process of Auricularia auricula polysaccharide: the dried Auricularia auricula fruiting bodies were crushed and sieved through a 160-mesh sieve to obtain powder. Choline chloride / urea type natural deep eutectic solvent (water content 30%, 1:2 molar ratio) was used as the extraction solvent, and the material ratio was 1:53 g / mL, and the extraction was carried out at 65°C for 90 minutes. The extract was centrifuged at 4000 r / min for 10 min, and the supernatant containing Auricularia auricula crude polysaccharide was collected. The crude polysaccharide solution was concentrated to 1 / 5~1 / 4 of its original volume using a rotary evaporator, and then 4 times the volume of anhydrous ethanol was added and allowed to stand at 4°C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. Protein was removed from the precipitate using the Sevag method. The concentrate was frozen at -80°C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0029] Example 2 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.3% w / v Auricularia auriculariae polysaccharide solution and gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stirring the mixture at a constant speed of 700 rpm for 30 min using a high-intensity ultrasonicator at room temperature to prepare an Auricularia auricula polysaccharide-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auricula polysaccharide-catechin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auricula polysaccharide-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250 rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the frequency of replacement of the dialysis external fluid to increase the frequency of replacement of the dialysis external fluid to every 2 hours. h change once; dialysis continued for 36 h; 7) The Auricularia auricularia polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:1, and ultrasonic treatment was performed at 700 rpm for 2.0 h in the dark and in an ice water bath using a high-intensity ultrasonicator to obtain a suspension of Auricularia auricularia polysaccharide-gelatin nanocapsules loaded with catechins. The working conditions of the high-intensity ultrasonicator were as follows: probe diameter of 10 mm, frequency of 20 kHz, power of 600 W (maximum power of 1000 W), and on / off time of 5 s.

[0030] Among them, the extraction process of Auricularia auricula polysaccharide is as follows: the dried Auricularia auricula fruiting bodies are crushed and sieved through a 160-mesh sieve to obtain powder. Choline chloride / urea type natural deep eutectic solvent (water content 30%, 1:2 molar ratio) is used as the extraction solvent, and the material ratio is 1:53 g / mL, and the extraction is carried out at 65°C for 90 min. The extract is centrifuged at 4000 r / min for 10 min, and the supernatant containing Auricularia auricula crude polysaccharide is collected. The crude polysaccharide solution is concentrated to 1 / 5~1 / 4 of its original volume using a rotary evaporator, and then 4 times the volume of anhydrous ethanol is added and allowed to stand at 4°C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. Protein is removed from the precipitate using the Sevag method. The concentrate is frozen at -80°C in an LGJ-10 vacuum freeze dryer for 12 h and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0031] Example 3 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stirring the mixture at a constant speed of 700 rpm for 30 min using a high-intensity ultrasonicator at room temperature to prepare an Auricularia auricula polysaccharide-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auricula polysaccharide-catechin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auricula polysaccharide-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the frequency of dialysis external fluid replacement to every 2 hours. h change once; dialysis continued for 36 h; 7) The Auricularia auricularia polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 4:1, and ultrasonic treatment was performed at 700 rpm for 2.0 h in the dark and in an ice water bath using a high-intensity ultrasonicator to obtain a suspension of Auricularia auricularia polysaccharide-gelatin nanocapsules loaded with catechins. The working conditions of the high-intensity ultrasonicator were as follows: probe diameter 10 mm, frequency 20 kHz, power 600 W (maximum power 1000 W), and on / off time 5 s.

[0032] Among them, the extraction process of Auricularia auricula polysaccharide: the dried Auricularia auricula fruiting bodies were crushed and sieved through a 160-mesh sieve to obtain powder. Choline chloride / urea type natural deep eutectic solvent (water content 30%, 1:2 molar ratio) was used as the extraction solvent, and the material ratio was 1:53 g / mL, and the extraction was carried out at 65°C for 90 minutes. The extract was centrifuged at 4000 r / min for 10 min, and the supernatant containing Auricularia auricula crude polysaccharide was collected. The crude polysaccharide solution was concentrated to 1 / 5~1 / 4 of its original volume using a rotary evaporator, and then 4 times the volume of anhydrous ethanol was added and allowed to stand at 4°C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. Protein was removed from the precipitate using the Sevag method. The concentrate was frozen at -80°C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0033] Example 4 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 2.5; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stirring the mixture at a constant speed of 700 rpm for 30 min using a high-intensity ultrasonicator at room temperature to prepare an Auricularia auricula polysaccharide-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auricula polysaccharide-catechin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auricula polysaccharide-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the replacement frequency of the dialysis external fluid to increase the replacement frequency of the dialysis external fluid to every 2 hours. h change once; dialysis continued for 36 h; 7) The Auricularia auricularia polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:1, and ultrasonic treatment was performed at 700 rpm for 2.0 h in the dark and in an ice water bath using a high-intensity ultrasonicator to obtain a suspension of Auricularia auricularia polysaccharide-gelatin nanocapsules loaded with catechins. The working conditions of the high-intensity ultrasonicator were as follows: probe diameter of 10 mm, frequency of 20 kHz, power of 600 W (maximum power of 1000 W), and on / off time of 5 s.

[0034] Among them, the extraction process of Auricularia auricula polysaccharide is as follows: the dried Auricularia auricula fruiting bodies are crushed and sieved through a 160-mesh sieve to obtain powder. Choline chloride / urea type natural deep eutectic solvent (water content 30%, 1:2 molar ratio) is used as the extraction solvent, and the material ratio is 1:53 g / mL, and the extraction is carried out at 65°C for 90 min. The extract is centrifuged at 4000 r / min for 10 min, and the supernatant containing Auricularia auricula crude polysaccharide is collected. The crude polysaccharide solution is concentrated to 1 / 5~1 / 4 of its original volume using a rotary evaporator, and then 4 times the volume of anhydrous ethanol is added and allowed to stand at 4°C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. Protein is removed from the precipitate using the Sevag method. The concentrate is frozen at -80°C in an LGJ-10 vacuum freeze dryer for 12 h and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0035] Example 5 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.4% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.2% w / v, and magnetically stirring the mixture at a constant speed of 700 rpm using a high-intensity ultrasonicator for 30 min at room temperature to prepare an Auricularia auricula polysaccharide-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auricula polysaccharide-catechin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auricula polysaccharide-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the replacement frequency of the dialysis external fluid to increase the replacement frequency of the dialysis external fluid to every 2 hours. h change once; dialysis continued for 36 h; 7) The Auricularia auricularia polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:1, and ultrasonic treatment was performed at 700 rpm for 2.0 h in the dark and in an ice water bath using a high-intensity ultrasonicator to obtain a suspension of Auricularia auricularia polysaccharide-gelatin nanocapsules loaded with catechins. The working conditions of the high-intensity ultrasonicator were as follows: probe diameter of 10 mm, frequency of 20 kHz, power of 600 W (maximum power of 1000 W), and on / off time of 5 s.

[0036] Among them, the extraction process of Auricularia auricula polysaccharide: the dried Auricularia auricula fruiting bodies were crushed and sieved through a 160-mesh sieve to obtain powder. Choline chloride / urea type natural deep eutectic solvent (water content 30%, 1:2 molar ratio) was used as the extraction solvent, and the material ratio was 1:53 g / mL, and the extraction was carried out at 65°C for 90 minutes. The extract was centrifuged at 4000 r / min for 10 min, and the supernatant containing Auricularia auricula crude polysaccharide was collected. The crude polysaccharide solution was concentrated to 1 / 5~1 / 4 of its original volume using a rotary evaporator, and then 4 times the volume of anhydrous ethanol was added and allowed to stand at 4°C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. Protein was removed from the precipitate using the Sevag method. The concentrate was frozen at -80°C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0037] Comparative Example 1 Single catechin without any nanocapsule encapsulation treatment.

[0038] Comparative Example 2 A kind of Auricularia auricula polysaccharide-catechin solution, the preparation method of which comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide in ultrapure water to prepare a 0.5% w / v Auricularia auriculariae polysaccharide solution; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) The catechin solution was added to the Auricularia auriculariae polysaccharide solution to make the catechin concentration 0.2% w / v, and magnetic stirring was performed at a constant speed of 700 rpm at room temperature for 30 min to prepare an Auricularia auriculariae polysaccharide-catechin solution.

[0039] Comparative Example 3 A gelatin-catechin solution, the preparation method of which comprises the following steps: 1) Prepare 0.5% w / v gelatin solution by fully dissolving gelatin in ultrapure water; 2) Use 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution to adjust the pH of the gelatin solution to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) The catechin solution was added to the gelatin solution to make the catechin concentration 0.2% w / v, and the mixture was magnetically stirred at a constant speed of 700 rpm at room temperature for 30 min to prepare a gelatin-catechin solution.

[0040] Comparative Example 4 The difference between it and Example 1 is that: In step 1), Auricularia auriculariae polysaccharide and gelatin are fully dissolved in ultrapure water to prepare 0.05% w / v Auricularia auriculariae polysaccharide solution and 0.05% w / v gelatin solution, respectively.

[0041] Comparative Example 5 The difference between it and Example 1 is that: In step 5), the Auricularia auricula polysaccharide-catechin solution is added dropwise to the gelatin solution at a volume ratio of 1:3, and ultrasonic treatment is performed for 2.0 h at room temperature using a high-intensity ultrasonicator at a speed of 700 rpm to obtain a suspension of Auricularia auricula polysaccharide-gelatin nanocapsules loaded with catechins.

[0042] Comparative Example 6 The difference between it and Example 1 is that: In step 3), catechins are dissolved in anhydrous ethanol to prepare a 0.2% w / v catechin solution; In step 4), the catechin solution was added to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.04% w / v, and magnetic stirring was performed at a constant speed of 700 rpm at room temperature for 30 minutes to prepare the Auricularia auricula polysaccharide-catechin solution.

[0043] Comparative Example 7 The difference between it and Example 1 is that: Steps 4) and 6) are not included.

[0044] Comparative Example 8 The difference between it and Example 1 is that: Step 4 is not included).

[0045] Comparative Example 9 The difference between it and Example 1 is that: Step 6 is not included).

[0046] Comparative Example 10 The difference between it and Example 1 is that: The Auricularia auricula polysaccharide was replaced with β-cyclodextrin.

[0047] Comparative Example 11 The difference between it and Example 1 is that: Replace gelatin with gum arabic.

[0048] Comparative Example 12 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, which is different from Example 1 in that its preparation method comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and 0.5% w / v gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) The Auricularia auricula polysaccharide solution and the gelatin solution were fully mixed at a volume ratio of 1:1, and magnetically stirred at a constant speed of 700 rpm for 30 min using a high-intensity ultrasonicator at room temperature to obtain an Auricularia auricula polysaccharide-gelatin solution; 4) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the Auricularia auriculariae polysaccharide-gelatin solution into the pretreated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the Auricularia auriculariae polysaccharide-gelatin solution in ultrapure water for dialyzation, and the volume ratio of ultrapure water to the Auricularia auriculariae polysaccharide-gelatin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the replacement frequency of the dialysis external fluid to increase the replacement frequency of the dialysis external fluid to every 2 hours. h change once; dialysis continued for 36 h; 5) Dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 6) Antioxidation: The temperature of the catechin solution prepared in step 5) was adjusted to 25°C, 0.05% ascorbic acid by mass was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid by mass was added again, and the mixture was stirred for 4 min; the nitrogen was introduced at a flow rate of 1.5 L / min to obtain a treated catechin solution; 7) adding the catechin solution to the Auricularia auriculariae polysaccharide-gelatin solution to make the catechin concentration be 0.1% w / v, and ultrasonically treating the solution at 700 rpm for 2.0 h using a high-intensity ultrasonicator in the dark and in an ice water bath to obtain a suspension of Auricularia auriculariae polysaccharide-gelatin nanocapsules loaded with catechins; Among them, the extraction process of Auricularia auriculariae polysaccharide is the same as that in Example 1.

[0049] Comparative Example 13 A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, which is different from Example 1 in that its preparation method comprises the following steps: 1) Fully dissolving Auricularia auriculariae polysaccharide and gelatin in ultrapure water to prepare 0.5% w / v Auricularia auriculariae polysaccharide solution and 0.5% w / v gelatin solution respectively; 2) Using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution, the pH of the Auricularia auricula polysaccharide solution and the gelatin solution was adjusted to 4.0; 3) dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution; 4) Antioxidation: The temperature of the catechin solution prepared in step 3) was adjusted to 25°C, 0.05% ascorbic acid was added, and the mixture was stirred at 230 rpm for 13 min; at the same time, nitrogen was introduced into the solution at a gas flow rate of 1 L / min; the stirring speed was increased to 260 rpm, 0.05% ascorbic acid was added again, and the mixture was stirred for 4 min; the nitrogen was maintained at a flow rate of 1.5 L / min to obtain a treated catechin solution; 5) adding the catechin solution to the gelatin solution to make the catechin concentration 0.1% w / v, and magnetically stirring the solution at a constant speed of 700 rpm using a high-intensity ultrasonicator for 30 min to obtain a gelatin-catechin solution; 6) Select a dialysis bag with a molecular weight cutoff range of 2000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80℃ for 30 min, and then rinse it with ultrapure water; put the gelatin-catechin solution into the pre-treated dialysis bag so that the volume of the solution in the dialysis bag does not exceed 80% of its maximum carrying capacity; place the dialysis bag containing the gelatin-catechin solution in ultrapure water for dialysis, and the volume ratio of ultrapure water to gelatin-catechin solution is controlled at 150:1; use a magnetic stirrer to stir the dialysis external fluid at a stirring speed of 250rpm; place the dialysis device in a constant temperature box and maintain the temperature at 6℃; replace the dialysis water every 5 hours, and use a conductivity meter to detect the conductivity of the dialysis external fluid before replacement. If the conductivity decreases by less than 10%, increase the frequency of dialysis external fluid replacement to every 2 hours; continue dialysis for 36 hours; 7) The gelatin-catechin solution was added dropwise to the Auricularia auriculariae polysaccharide solution at a volume ratio of 1:1, and the solution was sonicated at 700 rpm for 2.0 h using a high-intensity ultrasonicator in the dark and in an ice water bath to obtain a suspension of Auricularia auriculariae polysaccharide-gelatin nanocapsules loaded with catechins; Among them, the extraction process of Auricularia auriculariae polysaccharide is the same as that in Example 1.

[0050] Comparative Example 14 The difference between it and Example 1 is that: In step 2), the pH of the Auricularia auricula polysaccharide solution and the gelatin solution is adjusted to 9.0 using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution.

[0051] Comparative Example 15 The difference between it and Example 1 is that: In step 2), the pH of the Auricularia auricula polysaccharide solution and the gelatin solution is adjusted to 7.0 using 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution.

[0052] Performance Experiment 1. Encapsulation efficiency of catechins: The catechin-loaded Auricularia polysaccharide-gelatin nanocapsule suspension prepared in Examples 1 to 5 of the present invention and the final substances prepared in Comparative Examples 2 to 15 were mixed with anhydrous methanol in a flask at a volume ratio of 1:9 as test samples, and after ultrasonic treatment for 20 min, centrifuged at 2500 rpm for 10 min. The supernatant was analyzed for catechin content using a UV-2600 spectrophotometer. 1 mL of catechin standards of different concentrations were prepared, mixed with 1% w / v vanillin methanol solution and 25% v / v H2SO4 solution successively, reacted for 15 min in a dark environment at 30°C, and the absorbance value was measured at 500 nm. The test sample was subjected to catechin content determination according to the same steps as above, and the catechin content in the test sample was obtained according to the calibration curve of the catechin standard. The encapsulation efficiency and delivery rate of catechins in the test sample are calculated as follows: Encapsulation efficiency (%) = A1 / A2×100 Wherein, A1 is the content of encapsulated catechins, and A2 is the total content of catechins.

[0053] The test results are as follows Figure 1 As shown by Figure 1It can be seen that compared with the single Auricularia auriculariae polysaccharide or gelatin encapsulation and delivery of catechins (Comparative Example 2, Comparative Example 3), the nanocapsules prepared by Auricularia auriculariae polysaccharide and gelatin have a higher encapsulation efficiency for catechins. It is worth noting that the concentration of Auricularia auriculariae polysaccharide and gelatin working solution, the ratio of Auricularia auriculariae polysaccharide to gelatin content, environmental pH value and catechin concentration are all important factors affecting the encapsulation efficiency of catechins. The increase in the content of Auricularia auriculariae polysaccharide, gelatin and catechins is positively correlated with the increase in the encapsulation rate of catechins. The higher proportion of Auricularia auriculariae polysaccharide in the two helps to increase the encapsulation rate of catechins, which may be closely related to the good viscoelasticity and adsorption of Auricularia auriculariae polysaccharides. In addition, comparative studies have found that the nanocapsules formed by Auricularia auriculariae polysaccharides and gelatin are more conducive to the encapsulation of catechins in an acidic environment, which may be caused by the easy decomposition of catechins in an alkaline environment. β-cyclodextrin-gelatin nanocapsules loaded with catechins and β-cyclodextrin-gelatin nanocapsules loaded with catechins were prepared by replacing Auricularia auricularia polysaccharide and gum arabic as wall materials (Comparative Example 10, Comparative Example 11), and it was found that their encapsulation efficiency was not as good as that of the catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules prepared with Auricularia auricularia polysaccharide-gelatin as wall material. It is worth noting that as two different encapsulation materials, gelatin and Auricularia auricularia polysaccharide, their addition order will affect the encapsulation efficiency of catechins (Comparative Example 12, Comparative Example 13). For example, in Comparative Example 13, gelatin preferentially combines with catechins, and then the addition of Auricularia auricularia polysaccharide may further affect the structure or stability of gelatin, thereby reducing the encapsulation efficiency. By comparing Comparative Examples 7, 8, and 9 with Example 1, it can be concluded that without antioxidant treatment, catechins may be partially oxidized during the preparation process, and the binding ability of oxidized catechins with Auricularia auricula polysaccharides becomes weak, making the encapsulation effect of nanocapsules on catechins worse. Without dialysis treatment, small molecular impurities and unbound catechins may exist in the nanocapsules, which will affect the structural stability and encapsulation efficiency of the nanocapsules. However, catechins themselves have undergone antioxidant treatment and are relatively stable in structure. Without antioxidant treatment, catechins are easily oxidized, and without dialysis treatment to remove impurities and unbound catechins, the formation and encapsulation effect of nanocapsules are seriously damaged under the influence of the dual factors.

[0054] 2. Bioavailability of Catechins: The catechin-loaded Auricularia polysaccharide-gelatin nanocapsule suspension prepared in Examples 1 to 5 of the present invention and the final substances prepared in Comparative Examples 1 to 15 were used as test samples, and a three-stage simulated gastrointestinal tract (GIT) model consisting of oral, stomach and small intestine phases was used to evaluate the bioavailability of catechins. All test samples were preheated at 37°C before mixing and maintained at 37°C throughout the digestion process. Preparation of simulated oral saliva, 1.594 mg / mL NaCl, 0.328 mg / mL NH4NO3, 0.636 mg / mL KH2PO4, 0.202 mg / mL KCl, 0.308 mg / mL K3C6H5O7·H2O, 0.021 mg / mL C5H3N4O3·Na, 0.198 mg / mL H2NCONH2, 0.146 mg / mL C3H5O3Na and 30 mg / mL mucin. The suspension of catechin-loaded Auricularia polysaccharide-gelatin nanocapsules with 40 mg of free catechin (CA) was mixed with simulated saliva (7.5 mL) and the mixture was then adjusted to pH = 6.8 and shaken at 90 rpm for 10 min. Simulated gastric fluid was prepared by adding NaCl (2 g / L), pepsin (3.2 g / L) and HCl (7 g / L) to distilled water. The sample obtained in the oral phase (15 mL) was mixed with 15 mL of simulated gastric fluid. The pH of the mixture was then adjusted to 1.5 and shaken at 90 rpm for 120 min to simulate gastric digestion. The pH of the simulated gastric phase digest was adjusted to 7.0. Simulated small intestinal fluid containing bile extract solution (50 mg / mL), pancreatin (24 mg / mL) and saline (7.5 M NaCl and 0.5 M CaCl2) was mixed with the digested sample. The pH of the mixture was adjusted to 7.0 and intestinal digestion was performed for 120 min. The bioavailability of catechins was determined after the samples were processed through the GIT model. The raw chyme was collected and centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was collected because these micellar fractions contain catechins, which are easily absorbed by intestinal epithelial cells and therefore represent the "bioavailable" fraction after the digestive process. The concentration of catechins in the supernatant was measured at 230 nm using a UV-Vis spectrophotometer and calculated using a catechin standard calibration curve. The bioavailability of catechins is calculated using the following formula: Bioavailability (%) = C M / C I × 100 Among them, C M and C I They represent the catechin concentrations in the mixed micelle components and the initial sample at the end of the simulated GIT model, respectively.

[0055] The test results are as follows Figure 2 As shown by Figure 2It can be seen that the bioavailability of a single catechin in the small intestine is the lowest, reaching only 18.39%. Nanocapsules prepared by single Auricularia auricula polysaccharide, single gelatin, or Auricularia auricula polysaccharide and gelatin can effectively promote the sustained release of catechins in digestion and absorption and improve their bioavailability. Increasing the concentration of Auricularia auricula polysaccharide and gelatin from 0.3% to 0.5%, and increasing the content of catechins from 0.1% to 0.2%, can effectively improve the bioaccessibility of catechins (Example 1, Example 2). In addition, increasing the proportion of Auricularia auricula polysaccharide in Auricularia auricula polysaccharide-gelatin nanocapsules helps to improve the delivery and bioavailability of catechins. In addition, the environmental pH value is an important factor affecting the bioavailability of catechins. By comparison, it was found that encapsulating catechins in an acidic environment is conducive to improving its encapsulation rate and bioavailability in in vitro digestion and absorption. In addition, β-cyclodextrin was used to replace the eurycoma polysaccharide and gum arabic was used to replace gelatin as the wall material (Comparative Example 10, Comparative Example 11), and the prepared β-cyclodextrin-gelatin nanocapsules loaded with catechins and the eurycoma polysaccharide-gum arabic nanocapsules loaded with catechins were found to have a lower bioavailability of catechins than the eurycoma polysaccharide-gelatin nanocapsules loaded with catechins prepared with eurycoma polysaccharide-gelatin as the wall material. It is worth noting that the improper order of adding gelatin and eurycoma polysaccharide may change the release rate or distribution characteristics of catechins, thereby affecting its bioavailability in vivo (Comparative Examples 12, 13). For example, in Comparative Example 13, gelatin preferentially binds to catechins, and then the addition of eurycoma polysaccharides may further affect the structure or stability of gelatin, thereby reducing the bioavailability of catechins. By comparing Comparative Examples 7, 8, 9 with Example 1, it can be concluded that the catechin molecule contains multiple phenolic hydroxyl groups, is unstable in nature, and is easily oxidized during preparation, storage and in vivo environments. Step 4) By adding ascorbic acid and passing nitrogen to create an antioxidant environment, the oxidation of catechins is effectively reduced. It is crucial to maintain the integrity of the catechin structure, because only catechins with a complete structure can specifically bind to the corresponding targets in the body (such as specific receptors, enzymes, etc.) and exert their biological activity. If catechins are oxidized, their chemical structure changes and they may not be normally absorbed and utilized by the human body, resulting in reduced bioavailability. Antioxidant treatment makes catechins more stable during the preparation process of nanocapsules and subsequent storage and transportation. Stable catechins can better maintain their loading state in nanocapsules and reduce losses before reaching the site of action. When the nanocapsules enter the human body, the stable catechins are more easily released from the nanocapsules and effectively taken up by absorption sites such as the gastrointestinal tract, thereby increasing the absorption of catechins in the body and ultimately improving bioavailability. Stable catechins are more evenly distributed in the nanocapsules and can be released more regularly from the nanocapsules in the in vivo environment.This slow and sustained release method helps catechins maintain a certain concentration in the gastrointestinal tract, increases their contact time and area with the absorption site, and thus promotes the absorption of catechins. For example, on the surface of intestinal epithelial cells, stable catechins can better enter the cells through passive diffusion, carrier-mediated transport, and then enter the blood circulation, thereby improving bioavailability. Dialysis treatment can remove small molecular impurities and unbound catechins in the Auricularia auricula polysaccharide-catechin solution, making the structure of the nanocapsule more regular and stable. Suitable nanocapsule structure is crucial for the encapsulation and release of catechins. The regular structure can more effectively encapsulate catechins and prevent them from leaking prematurely before reaching the site of action; at the same time, in the in vivo environment, catechins can be released in a timely manner according to physiological needs. For example, the physical properties of nanocapsules such as particle size and surface charge are optimized during the dialysis process, which is more conducive to their interaction with the biomembrane, promoting the absorption of catechins, and thus improving their bioavailability. The small molecular impurities removed by dialysis may interfere with the absorption and metabolism of catechins. These impurities may compete with catechins for absorption sites or affect the physiological environment of the gastrointestinal tract, thereby reducing the absorption efficiency of catechins. Through dialysis treatment, the presence of impurities is reduced, creating a more favorable absorption environment for catechins, increasing their absorption in the gastrointestinal tract, and thus improving bioavailability.

[0056] 3. Light and thermal stability test: The catechin-loaded Auricularia polysaccharide-gelatin nanocapsule suspension prepared in Examples 1 to 5 of the present invention and the final substance prepared in Comparative Examples 1 to 15 were used as test samples and an equal amount of catechin solution (4 mL) were added to test tubes. Then irradiate in front of a 10 cm ultraviolet lamp for 6 h, and calculate the retention rate (RR) of catechins by the pH difference method. Catechins (4 mL) and each test sample were added to a test tube, and then the sample was heated in a water bath at 90°C. Then it was quickly cooled to room temperature in ice, and the retention rate (RR) of catechins was calculated by the pH difference method. As mentioned above, the retention rate of catechins is calculated by the following formula: Catechin retention rate (%) = catechin content (after light or heating) / total catechin amount × 100 After light treatment, the catechin retention rate was determined as follows Figure 3 As shown in Figure 2, after heating treatment, the catechin retention rate was determined as follows: Figure 4 As shown by Figure 3 and Figure 4It can be seen that due to the instability of catechins, catechins are particularly sensitive to ultraviolet rays (UV) during storage, especially UVB radiation, which can cause photoisomerization and photolysis reactions. These reactions will produce yellow photodegradation products, such as dimeric catechins and superoxide anion radicals. Under high temperature conditions, catechins are prone to non-enzymatic oligomerization reactions to produce dimers or higher molecular weight polymers. These oligomerization reactions are the main reason for the instability of catechins at high temperatures. High temperatures also promote the oxidation reaction of catechins, causing changes in their color and activity. For example, above 80°C, the oxidation rate of catechins is significantly accelerated. During the heat treatment process, catechins may undergo epimerization, hydrolysis, and oxidation / condensation reactions, which further reduce their stability. Therefore, the stability of catechins is greatly affected by light and temperature. Compared with single catechins (Comparative Example 1), encapsulation of catechins can effectively enhance their stability under light and heat treatment. It is worth noting that increasing the content of Auricularia auriculariae polysaccharide and gelatin does not significantly improve the stability of catechins, while the neutral and alkaline pH environments enhance the instability of catechins. β-cyclodextrin was used to replace Auricularia auriculariae polysaccharide, and gum arabic was used to replace gelatin as wall materials (Comparative Example 10, Comparative Example 11), and the prepared β-cyclodextrin-gelatin nanocapsules loaded with catechins and Auricularia auriculariae polysaccharide-gum arabic nanocapsules loaded with catechins were found to have poor light and heat stability compared to the Auricularia auriculariae polysaccharide-gelatin nanocapsules loaded with catechins. It is worth noting that the order of addition of gelatin and Auricularia auriculariae polysaccharide as two different embedding materials will affect the embedding efficiency of catechins (Comparative Examples 12, 13). For example, in Comparative Example 13, gelatin first combines with catechins, and then the addition of Auricularia auriculariae polysaccharide may further affect the structure or stability of gelatin, thereby reducing stability. By comparing Comparative Examples 7, 8, 9 with Example 1, it can be concluded that step 4) can effectively reduce the degree of oxidation of catechins during the preparation process by adding ascorbic acid and passing nitrogen for antioxidant treatment. During light treatment, catechins easily absorb light energy and react with oxygen to be oxidized. The chemical structure of catechins treated with antioxidants is more stable, and when they are subsequently combined with Auricularia auriculariae polysaccharides to form a complex and finally encapsulated in nanocapsules, they can better maintain their integrity. In this way, after light treatment, the degree of oxidative damage of catechins is reduced, thereby improving the encapsulation rate after light treatment. Heat treatment will accelerate the oxidation reaction rate of catechins. The antioxidant treatment in step 4) provides a certain protective barrier for catechins, making catechins more difficult to be oxidized under high temperature conditions. During the formation of nanocapsules and the subsequent heat treatment process, stable catechins can be better encapsulated by Auricularia auriculariae polysaccharides to form a stable encapsulation structure. Therefore, the encapsulation rate of catechins treated with antioxidants will be relatively high after heat treatment. The dialysis treatment in step 6) can remove small molecular impurities and unbound catechins in the Auricularia auriculariae polysaccharide-catechin solution, making the formation environment of the nanocapsules purer.During light treatment, the structure of the pure nanocapsules is more stable and can better resist the influence of light. At the same time, the dialysis process can also adjust the internal structure and surface properties of the nanocapsules, enhancing their encapsulation ability for catechins. Therefore, the encapsulation rate of catechins in the dialysis-treated nanocapsules will be improved after light treatment. Dialysis treatment helps to optimize the structure of the nanocapsules, making them more stable during heat treatment. By removing impurities and adjusting the ion concentration, the thermal stability of the nanocapsules is enhanced, and they can better withstand the temperature changes and thermal stress caused by heat treatment. During the heat treatment process, the stable nanocapsule structure can effectively prevent the leakage of catechins, thereby improving the encapsulation rate after heat treatment.

[0057] 4. Antioxidant performance test: The catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsule suspension prepared in Example 1 of the present invention and the final substances prepared in Comparative Examples 7, 8 and 9 were used as test samples. The DPPH radical scavenging rate method was used to detect the antioxidant capacity of the catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules, and the DPPH radical scavenging rate (%) was calculated.

[0058] Analysis of test results: The DPPH radical scavenging rate (%) of the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule suspension prepared in Example 1 is 85%, the DPPH radical scavenging rate (%) of Comparative Example 7 is 60%, the DPPH radical scavenging rate (%) of Comparative Example 8 is 70%, and the DPPH radical scavenging rate (%) of Comparative Example 9 is 78%.

[0059] In step 4), adding ascorbic acid (itself an antioxidant) can enhance the antioxidant capacity of the catechin solution. After the subsequent formation of nanocapsules, it helps to improve the overall antioxidant performance of the catechin-loaded auricularia polysaccharide-gelatin nanocapsules, better protect the catechins from oxidation, and maintain their biological activity. The introduction of nitrogen can remove oxygen from the solution and reduce the oxidative effect of oxygen on catechins. By adding ascorbic acid twice and coordinating different stirring speeds and nitrogen flow rates, catechins can maintain a more stable antioxidant state in the solution, so that the nanocapsules can exert their antioxidant effects more persistently during the application process. The appropriate stirring speed helps catechins and ascorbic acid to be fully mixed, so that catechins are more evenly dispersed in the solution, which will affect the dispersion of catechins in the auricularia polysaccharide-gelatin system when the nanocapsules are subsequently formed, which is conducive to the formation of nanocapsules with good dispersion, avoiding catechin agglomeration, thereby improving the stability of the nanocapsules and the consistency of performance.

[0060] 5. Particle size and distribution: The catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule suspension prepared in Example 1 of the present invention and the final substances prepared in Comparative Examples 7, 8 and 9 were used as test samples, and the average particle size and polydispersity index (PDI) of the nanocapsules were measured using a dynamic light scattering (DLS).

[0061] Experimental results: The particle size of the suspension of the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules prepared in Example 1 of the present invention is moderate and evenly distributed, with an average particle size of 200 nm and a PDI of 0.15; the particle size and distribution of the nanocapsules prepared in Comparative Example 7 are greatly affected, and the average particle size is significantly increased, with an average particle size of 300 nm and a PDI of 0.25; the average particle size of the nanocapsules prepared in Comparative Example 8 is 250 nm and the PDI is 0.2; the increase in the average particle size may be due to partial agglomeration caused by the lack of antioxidant treatment, thereby affecting the formation of the nanocapsules. The average particle size of the nanocapsules prepared in Comparative Example 8 is 220 nm and the PDI is 0.18; the increase in the average particle size may be due to the lack of dialysis, which may cause some incompletely reacted small molecules or impurities in the nanocapsules, affecting the regularity of the nanocapsules.

[0062] In step 6 of Examples 1 to 5 of the present invention, during the dialysis process, factors such as the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution, stirring speed, temperature, and dialysis time will affect the diffusion and exchange rate of small molecules. Appropriate conditions help control the degree of interaction between Auricularia auricula polysaccharide and catechins, prevent excessive molecular aggregation or growth, so that the final nanocapsule has a moderate particle size and uniform distribution. Controlling the replacement frequency of the dialysis external solution by conductivity detection can more accurately adjust the ion concentration and composition in the solution, further optimize the formation environment of the nanocapsules, make the particle size distribution narrower, and improve the uniformity of the nanocapsules, which is very important for the application of nanocapsules in the fields of drug delivery and food preservation, and can ensure the stability and consistency of their performance. In addition, suitable dialysis conditions can promote catechins to be better encapsulated by Auricularia auricula polysaccharides to form a stable Auricularia auricula polysaccharide-catechin complex, thereby improving the encapsulation rate of catechins in nanocapsules, reducing the loss of catechins during the preparation process, and improving the loading efficiency of nanocapsules for catechins, so that nanocapsules can carry and protect catechins more effectively. If the dialysis process is properly controlled, the structure of the Auricularia auricula polysaccharide-catechin complex can be made more stable. After it is subsequently combined with gelatin to form nanocapsules, it can better prevent catechins from leaking from the nanocapsules, improve the encapsulation stability of the nanocapsules, prolong the release time of catechins, and enhance the effect of nanocapsules in practical applications. Dialysis temperature, stirring speed and other conditions will affect the physical state and intermolecular forces of the Auricularia auricula polysaccharide-catechin solution. Appropriate conditions can enable the molecules in the solution to form a stable structure. After mixing with gelatin to form nanocapsules, it helps to improve the physical stability of the nanocapsules, making it less likely to aggregate and precipitate during storage and use. Precise control of the solution composition and environment during the dialysis process can reduce possible impurities or unstable factors in the solution, reduce the possibility of adverse reactions between catechins and other substances, thereby improving the chemical stability of the nanocapsules and ensuring that they can maintain good performance under different environments.

[0063] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A method for preparing catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules, characterized in that: The specific steps include: 1) Fully dissolving Auricularia auricula polysaccharide and gelatin in ultrapure water to prepare Auricularia auricula polysaccharide solution and gelatin solution with a concentration of 0.2-0.5% w / v; 2) Adjust the pH of the Auricularia auricula polysaccharide solution and gelatin solution to 2.5-4.0; 3) dissolving catechins in anhydrous ethanol to prepare a catechin solution with a concentration of 0.2-0.4% w / v; 4) adding the catechin solution to the Auricularia auriculariae polysaccharide solution, and magnetically stirring at a constant speed of 600 to 800 rpm for 20 to 35 min at room temperature to prepare an Auricularia auriculariae polysaccharide-catechin solution; wherein the concentration of catechin in the Auricularia auriculariae polysaccharide-catechin solution is 0.05 to 0.2% w / v; 5) The Auricularia auriculariae polysaccharide-catechin solution was added dropwise into the gelatin solution at a volume ratio of 1.0 to 4.0, and ultrasonic treatment was performed in the dark and in an ice water bath to obtain a suspension of Auricularia auriculariae polysaccharide-gelatin nanocapsules loaded with catechins.

2. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 1, characterized in that: Between step 4) and step 5), a dialysis step is also included, including: Select a dialysis bag with a molecular weight cutoff range of 1000-3000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L EDTA at 80°C for 30 min, and then rinse it with ultrapure water; The Auricularia auricularia polysaccharide-catechin solution was loaded into a pretreated dialysis bag so that the volume of the solution in the dialysis bag did not exceed 80% of its maximum carrying capacity; the dialysis bag containing the Auricularia auricularia polysaccharide-catechin solution was placed in ultrapure water for dialysis, and the volume ratio of ultrapure water to the Auricularia auricularia polysaccharide-catechin solution was controlled at 100:1 to 200:1; a magnetic stirrer was used to stir the dialysis external fluid at a stirring speed of 200 to 300 rpm; the dialysis device was placed in a constant temperature box and the temperature was maintained at 4 to 8°C; the dialysis water was replaced every 4 to 6 hours, and the conductivity of the dialysis external fluid was tested with a conductivity meter before replacement. If the conductivity decreased by less than 10%, the replacement frequency of the dialysis external fluid was increased to once every 2 hours; the dialysis was continued for 24 to 48 hours.

3. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 2, characterized in that: Between step 3) and step 4), an antioxidant step is also included, including: First, the temperature of the prepared catechin solution is adjusted to 20-25°C, 0.025-0.05% of ascorbic acid is added, and the mixture is stirred at a speed of 200-300 rpm for 10-15 minutes; at the same time, nitrogen gas is introduced into the solution at a gas flow rate of 0.5-1 L / min; The stirring speed was increased to 230-260 rpm, and ascorbic acid with a mass fraction of 0.025-0.05% was added again, and the mixture was stirred for 4-6 min; nitrogen was kept flowing, and the flow rate was adjusted to 1-1.5 L / min to obtain a treated catechin solution.

4. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 1, characterized in that: In step 1), the concentrations of the wood ear polysaccharide solution and the gelatin solution are both 0.5% w / v; in step 2), the pH values ​​of the wood ear polysaccharide solution and the gelatin solution are both 4.0; in step 3), the concentration of the catechin solution is 0.4% w / v; in step 4), the magnetic stirring speed is 700 rpm, and the stirring time is 30 min; in step 5), the mixing ratio of the wood ear polysaccharide-catechin solution to the gelatin solution is 1:

1.

5. The method for preparing catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules according to claim 1, wherein in step 5), the conditions for ultrasonic treatment are: frequency of 20 kHz, power of 600-1000 W, ultrasonic speed of 600-800 rpm, and ultrasonic time of 1.5-2.5 h.

6. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 5, characterized in that: In step 5), the conditions for ultrasonic treatment are: frequency of 20 kHz, power of 600 W, ultrasonic speed of 700 rpm, and ultrasonic time of 2.0 h.

7. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 1, characterized in that: In step 2), 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution are used to adjust the pH.

8. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 1, characterized in that: Auricularia auricula polysaccharide is extracted from Auricularia auricula fruiting body.

9. The method for preparing the catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsules according to claim 8, characterized in that: The extraction process specifically includes: crushing the dried Auricularia auricularia fruiting body, sieving through a 160-mesh sieve to obtain powder, using choline chloride / urea type natural deep eutectic solvent as an extraction solvent, with a material ratio of 1:53 g / mL, extracting the powder at 65°C for 90 min to obtain an extract, centrifuging the extract at 4000 r / min for 10 min, collecting the supernatant containing Auricularia auricularia crude polysaccharide, using a rotary evaporator to concentrate the supernatant to 1 / 5~1 / 4 of its volume, and then adding 4 times the volume of anhydrous ethanol and standing at 4°C for 12 h, centrifuging at 4000 r / min for 10 min, collecting the precipitate, removing protein from the precipitate using the Sevag method to obtain a concentrate, and freeze-drying the concentrate at -80°C for 12 h to obtain Auricularia auricularia polysaccharide.

10. The catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules prepared by the method for preparing catechin-loaded Auricularia auricularia polysaccharide-gelatin nanocapsules according to any one of claims 1 to 9.

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