Nano-capsules of jade ear mushroom polysaccharide-gelatin loaded with catechins and preparation method thereof

Abstract

The present invention discloses a nano-capsule of auricularia polytricha polysaccharide-gelatin loaded with catechin and a preparation method thereof. The preparation method includes: dissolving auricularia polytricha polysaccharide and gelatin in ultrapure water to obtain an auricularia polytricha polysaccharide solution and a gelatin solution; adjusting the pH; dissolving catechin in absolute ethanol to obtain a catechin solution; adding the catechin solution to the auricularia polytricha polysaccharide solution and stirring at room temperature to obtain an auricularia polytricha polysaccharide-catechin solution; dropping the auricularia polytricha polysaccharide-catechin solution into the gelatin solution and performing ultrasonic treatment in the dark and an ice-water bath to obtain a nano-capsule suspension of auricularia polytricha polysaccharide-gelatin loaded with catechin. The preparation method of the present invention is simple and convenient, the process is safe and controllable, and by using auricularia polytricha polysaccharide and gelatin as a composite wall material to synergistically exert physical and chemical properties, the efficient delivery and sustained release of catechin are realized, providing new ideas for the application of auricularia polytricha polysaccharide and improving the bioavailability of catechin.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide-based nanocapsule materials and sustained-release preparations of active substances, and particularly relates to a nano-capsule of Tremella aurantialba polysaccharide-gelatin loaded with catechins and a preparation method thereof. Background Art

[0002] Consumers' demand for nutritional products is increasing day by day, and bioactive compounds derived from natural foods have received extensive attention. Catechin is a phenolic active substance, mainly extracted from natural plants such as tea leaves, and has significant antioxidant, free radical scavenging and antibacterial activities. Many studies have reported that catechin can be used as a promising drug for anti-tumor, anti-platelet aggregation, anti-inflammation and anti-aging, and it is considered to have potential application prospects and commercial value in the fields of functional foods and medicines. However, the oral bioavailability of catechin is low, and its astringency, bitterness and extremely poor physicochemical stability severely limit its application in drugs and foods. Therefore, effective methods are urgently needed to solve these problems.

[0003] Drug delivery systems with nanostructures such as liposomes, emulsions and particles have received extensive attention in past studies due to their advantages such as small carrier volume, large encapsulation amount, and good physical and chemical stability; they have also been proven to improve the stability and bioavailability of catechins. Delivery systems based on nano-encapsulation systems are considered a viable option to protect catechins from harsh environments, thereby improving stability. Generally, drug-loaded nano-systems can protect the core material from adverse environmental conditions, delay its degradation in the body fluid environment, and easily break through various barriers to reach the target organ, thereby controlling the release of the drug and improving the bioavailability of the active substance. Tremella aurantialba is a common edible mushroom, which has received extensive attention in recent years due to its rich nutritional components and potential medicinal value. Tremella aurantialba polysaccharide is mainly composed of monosaccharides such as glucose, mannose and galactose, and is considered to have various biological activities such as immunomodulation, anti-tumor and antioxidant, and has broad application potential in the fields of food, health products and medicines. However, there are few studies on the application of Tremella aurantialba polysaccharide in the field of nano-scale carrier materials. Gelatin is a natural polymer material hydrolyzed from collagen, and is widely used in drug delivery due to its excellent biocompatibility, biodegradability and good processing performance.

[0004] Currently, gelatin is mainly prepared into microspheres or nanoparticles through methods such as emulsification and spray drying for encapsulating drugs, or hydrogels are prepared based on gelatin for controlling the drug release rate, which can adjust its swelling property according to environmental changes (such as pH, temperature, etc.), thereby achieving intelligent drug release. However, the development of gelatin-based nanocapsule materials has rarely been carried out. Due to the good biocompatibility, biodegradability, and biological activity of Auricularia polytricha polysaccharide and gelatin, realizing the sustainable controlled release of catechin active substances based on these two biological macromolecules is a feasible way to improve the bioavailability of the encapsulated nanosystem. Summary of the Invention

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

[0006] Another object of the present invention is to provide a preparation method of Auricularia polytricha polysaccharide-gelatin nanocapsules loaded with catechin, which is simple and convenient, the process is safe and controllable, and continuous and low-cost green production can be achieved.

[0007] Another object of the present invention is to provide Auricularia polytricha polysaccharide-gelatin nanocapsules loaded with catechin, which synergistically exert physical and chemical properties by using Auricularia polytricha polysaccharide and gelatin as composite wall materials to achieve efficient delivery and slow release of catechin, providing an effective idea for expanding the application of Auricularia polytricha polysaccharide and improving the bioavailability of catechin.

[0008] According to these objects and other advantages of the present invention, a preparation method of Auricularia polytricha polysaccharide-gelatin nanocapsules loaded with catechin is provided, which specifically includes the following steps:

[0009] 1) Dissolve Auricularia polytricha polysaccharide and gelatin separately in ultrapure water to prepare an Auricularia polytricha polysaccharide solution and a gelatin solution with a concentration of 0.2 - 0.5% w / v;

[0010] 2) Adjust the pH of the Auricularia polytricha polysaccharide solution and the gelatin solution to 2.5 - 4.0;

[0011] 3) Dissolve catechin in absolute ethanol to prepare a catechin solution with a concentration of 0.2 - 0.4% w / v;

[0012] 4) Add the catechin solution to the Auricularia polytricha polysaccharide solution and magnetically stir at a constant speed of 600 - 800 rpm at room temperature for 20 - 35 min to prepare an Auricularia polytricha polysaccharide-catechin solution; wherein, in the Auricularia polytricha polysaccharide-catechin solution, the concentration of catechin is 0.05 - 0.2% w / v;

[0013] 5) The tremella aurantialba polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1.0-4.0, and ultrasonic treatment was carried out in the dark and an ice-water bath to obtain a nano-capsule suspension of tremella aurantialba polysaccharide-gelatin loaded with catechin.

[0014] Preferably, between step 4) and step 5), a dialysis step is further included, including:

[0015] Select a dialysis bag with a molecular weight cut-off range of 1000-3000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80 °C for 30 min, and then rinse it thoroughly with ultrapure water;

[0016] Load the tremella aurantialba 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 tremella aurantialba polysaccharide-catechin solution in ultrapure water for dialysis, and control the volume ratio of ultrapure water to the tremella aurantialba polysaccharide-catechin solution at 100:1-200:1; use a magnetic stirrer to stir the outer dialysis solution, and the stirring speed is 200-300 rpm; place the dialysis device in an incubator and keep the temperature at 4-8 °C; replace the dialysis water every 4-6 h, and detect the conductivity of the outer dialysis solution with a conductivity meter before replacement. If the decrease in conductivity is less than 10%, increase the replacement frequency of the outer dialysis solution to once every 2 h; continue dialysis for 24-48 h.

[0017] Preferably, between step 3) and step 4), an antioxidant step is further included, including:

[0018] First, adjust the temperature of the prepared catechin solution to 20-25 °C, add ascorbic acid with a mass fraction of 0.025-0.05%, and stir at a speed of 200-300 rpm for 10-15 min; at the same time, introduce nitrogen into the solution, and the gas flow rate is 0.5-1 L / min;

[0019] Increase the stirring speed to 230-260 rpm, add ascorbic acid with a mass fraction of 0.025-0.05% again, and stir for 4-6 min; maintain the nitrogen introduction, and adjust the flow rate to 1-1.5 L / min to obtain the treated catechin solution.

[0020] Preferably, in step 1), the concentrations of the auricularia auricula polysaccharide solution and the gelatin solution are both 0.5% w / v; in step 2), the pH values of the jade auricularia auricula 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 jade auricularia auricula polysaccharide-catechin solution to the gelatin solution is 1:1.

[0021] Preferably, in step 5), the conditions of the ultrasonic treatment are: the frequency is 20 kHz, the power is 600 - 1000 W, the ultrasonic rotation speed is 600 - 800 rpm, and the ultrasonic time is 1.5 - 2.5 h.

[0022] Preferably, in step 5), the conditions of the ultrasonic treatment are: the frequency is 20 kHz, the power is 600 W, the ultrasonic rotation speed is 700 rpm, and the ultrasonic time is 2.0 h.

[0023] Preferably, in step 2), 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution are used to adjust the pH.

[0024] Preferably, the jade auricularia auricula polysaccharide is extracted from the jade auricularia auricula fruiting body.

[0025] Preferably, the extraction process specifically includes: crushing the dried jade auricularia auricula fruiting body, screening through a 160-mesh sieve to obtain powder, using choline chloride / urea-based natural deep eutectic solvent as the 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 the crude jade auricularia auricula polysaccharide, concentrating the supernatant to 1 / 5 - 1 / 4 of its volume using a rotary evaporator, then adding 4 times the volume of absolute ethanol and standing at 4°C for 12 h, centrifuging at 4000 r / min for 10 min, collecting the precipitate, removing proteins from the precipitate using the Sevag method to obtain a concentrate, and freeze-drying the concentrate at -80°C for 12 h to obtain the jade auricularia auricula polysaccharide.

[0026] The object of the present invention can also be further achieved by the nano-capsules of jade auricularia auricula polysaccharide-gelatin loaded with catechin prepared by the described preparation method.

[0027] The present invention has at least the following beneficial effects:

[0028] The present invention provides a method for preparing nano-capsules of auricularia polytricha polysaccharide-gelatin loaded with catechins, which is simple and convenient to operate, safe and controllable in the process, and can achieve continuous and low-cost green production. By using auricularia polytricha polysaccharide and gelatin as composite wall materials to synergistically exert their physical and chemical properties, efficient encapsulation, delivery and slow release of catechins are realized, providing an effective idea for expanding the application of auricularia polytricha polysaccharide and improving the bioavailability of catechins.

[0029] Other advantages, objectives and features of the present invention will be partially reflected by the following description, and partially will be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a comparison chart of the catechin encapsulation efficiency in the final substances prepared in Examples 1-5 and Comparative Examples 2-15 of the present invention;

[0031] Figure 2 It is a comparison chart of the bioavailability of the final substances prepared in Examples 1-5 and Comparative Examples 1-15 of the present invention in simulated in vitro digestion;

[0032] Figure 3 It is a comparison chart of the retention rates of the final substances prepared in Examples 1-5 and Comparative Examples 1-15 of the present invention after light treatment;

[0033] Figure 4 It is a comparison chart of the retention rates of the final substances prepared in Examples 1-5 and Comparative Examples 1-15 of the present invention after heat treatment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The following further describes the present invention in detail with reference to the drawings, so that those skilled in the art can implement it according to the description in the specification.

[0035] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0036] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0037] It should be noted that, for the convenience of comparison, in the following examples, a high-intensity ultrasonic processor (SHL-2A, Kunshan Ultrasonic Instruments Co., Ltd., Jiangsu), a spectrophotometer (UV-2600, Shimadzu Corporation), a magnetic stirrer (Model 79-1 magnetic stirrer, Shanghai Sile Instruments), an ultraviolet lamp (10 cm, Thermo Fisher Scientific), an LGJ-10 vacuum freeze dryer (Beijing Songyuan Huaxing Technology Development Co., Ltd.), tremella polysaccharide (extracted from tremella fruiting bodies), gelatin (Shanghai Aladdin Biochemical Technology Co., Ltd.), catechin (Shanghai Aladdin Biochemical Technology Co., Ltd.), β-cyclodextrin (Shanghai Aladdin Biochemical Technology Co., Ltd.), arabic gum (Shanghai Aladdin Biochemical Technology Co., Ltd.) are used, but are not limited thereto.

[0038] Example 1

[0039] A nano-capsule of tremella polysaccharide-gelatin loaded with catechin, and its preparation method comprises the following steps:

[0040] 1) Dissolve tremella polysaccharide and gelatin separately in ultrapure water to prepare a 0.5% w / v tremella polysaccharide solution and a gelatin solution;

[0041] 2) Adjust the pH of the tremella polysaccharide solution and the gelatin solution to 4.0 with 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide solution;

[0042] 3) Dissolve catechin in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0043] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25 °C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; meanwhile, introduce nitrogen gas into the solution at a gas flow rate of 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen gas introduction, and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0044] 5) Add the treated catechin solution to the tremella polysaccharide solution so that the catechin concentration is 0.1% w / v, and magnetically stir at a constant speed of 700 rpm with a high-intensity ultrasonic device at room temperature for 30 min to prepare a tremella polysaccharide-catechin solution;

[0045] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly with ultrapure water; fill the tremella aurantialba polysaccharide-catechin solution into the pretreated dialysis bag, ensuring that the volume of the solution in the dialysis bag does not exceed 80% of its maximum capacity; place the dialysis bag containing the tremella aurantialba polysaccharide-catechin solution in ultrapure water for dialysis, and control the volume ratio of ultrapure water to the tremella aurantialba polysaccharide-catechin solution at 150:1; use a magnetic stirrer to stir the external dialysis solution at a stirring speed of 250 rpm; place the dialysis device in an incubator and maintain the temperature at 6 °C; change the dialysis water every 5 h. Before changing, use a conductivity meter to detect the conductivity of the external dialysis solution. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution, and increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0046] 7) The tremella aurantialba polysaccharide-catechin solution is added dropwise to the gelatin solution at a volume ratio of 1:1. In the dark and in an ice-water bath, use a high-intensity ultrasonic device to perform ultrasonic treatment at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of tremella aurantialba polysaccharide-gelatin loaded with catechin. Among them, the working conditions of the high-intensity ultrasonic device are: the probe diameter is 10 mm, the frequency is 20 kHz, the power is 600 W (the maximum power is 1000 W), and the on-off time is 5 s.

[0047] Among them, the extraction process of tremella aurantialba polysaccharide: crush the dried tremella aurantialba fruiting bodies and screen them through a 160-mesh sieve to obtain powder. Use choline chloride / urea-based natural deep eutectic solvent (water content 30%, 1:2 molar ratio) as the extraction solvent, with a material ratio of 1:53 g / mL, and extract at 65 °C for 90 min. Centrifuge the extract at 4000 r / min for 10 min, collect the supernatant containing crude tremella aurantialba polysaccharide, use a rotary evaporator to concentrate the crude polysaccharide solution to 1 / 5 - 1 / 4 of its original volume, then add 4 times the volume of absolute ethanol and let it stand at 4 °C for 12 h, and then centrifuge at 4000 r / min for 10 min to collect the precipitate. Use the Sevag method to remove proteins from the precipitate. The concentrate is frozen at -80 °C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare tremella aurantialba polysaccharide (APP).

[0048] Example 2

[0049] A nano-capsule of tremella aurantialba polysaccharide-gelatin loaded with catechin, and its preparation method includes the following steps:

[0050] 1) Dissolve tremella aurantialba polysaccharide and gelatin in ultrapure water respectively to prepare 0.3% w / v tremella aurantialba polysaccharide solution and gelatin solution;

[0051] 2) Adjust the pH of the tremella aurantialba polysaccharide solution and the gelatin solution to 4.0 with 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide solution;

[0052] 3) Dissolve catechin in absolute ethanol to prepare 0.2% w / v catechin solution;

[0053] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25 °C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; meanwhile, introduce nitrogen into the solution with a gas flow rate of 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen introduction and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0054] 5) Add the catechin solution to the tremella aurantialba polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stir at a constant speed of 700 rpm for 30 min at room temperature with a high-intensity ultrasonic device to prepare tremella aurantialba polysaccharide-catechin solution;

[0055] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly with ultrapure water; put the tremella aurantialba 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 tremella aurantialba polysaccharide-catechin solution in ultrapure water for dialysis, and control the volume ratio of ultrapure water to the tremella aurantialba polysaccharide-catechin solution at 150:1; use a magnetic stirrer to stir the external dialysis solution at a stirring speed of 250 rpm; place the dialysis device in an incubator and keep the temperature at 6 °C; change the dialysis water every 5 h, and detect the conductivity of the external dialysis solution with a conductivity meter before replacement. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0056] 7) The polysaccharide-catechin solution of Auricularia auricula-judae was added dropwise to the gelatin solution at a volume ratio of 1:1. In the dark and ice-water bath, it was ultrasonically treated with a high-intensity ultrasonic device at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of polysaccharide-catechin-loaded Auricularia auricula-judae-gelatin. Among them, the working conditions of the high-intensity ultrasonic device were: the probe diameter was 10 mm, the frequency was 20 kHz, the power was 600 W (the maximum power was 1000 W), and the on-off time was 5 s.

[0057] Among them, the extraction process of polysaccharide from Auricularia auricula-judae: The dried fruiting bodies of Auricularia auricula-judae were crushed and screened through a 160-mesh sieve to obtain powder. Using choline chloride / urea-based natural deep eutectic solvent (water content 30%, molar ratio 1:2) as the extraction solvent, at a material ratio of 1:53 g / mL, it was extracted at 65 °C for 90 min. The extract was centrifuged at 4000 r / min for 10 min, and the supernatant containing crude polysaccharide from Auricularia auricula-judae was collected. The crude polysaccharide solution was concentrated to 1 / 5 - 1 / 4 of its original volume using a rotary evaporator, then 4 times the volume of absolute ethanol was added and left to stand at 4 °C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. The Sevag method was used to remove proteins from the precipitate. The concentrate was frozen at -80 °C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare polysaccharide from Auricularia auricula-judae (APP).

[0058] Example 3

[0059] A nano-capsule of polysaccharide-catechin-loaded Auricularia auricula-judae-gelatin, the preparation method thereof comprises the following steps:

[0060] 1) Dissolve polysaccharide from Auricularia auricula-judae and gelatin separately in ultrapure water to prepare a 0.5% w / v polysaccharide solution of Auricularia auricula-judae and a gelatin solution;

[0061] 2) Adjust the pH of the polysaccharide solution of Auricularia auricula-judae and the gelatin solution to 4.0 using 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide solution;

[0062] 3) Dissolve catechin in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0063] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25 °C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; meanwhile, introduce nitrogen gas into the solution at a gas flow rate of 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen gas introduction, and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0064] 5) Add the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.1% w / v. At room temperature, use a high-intensity ultrasonic device to magnetically stir at a constant speed of 700 rpm for 30 min to prepare the Auricularia auricula polysaccharide-catechin solution;

[0065] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly 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 control the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution at 150:1; use a magnetic stirrer to stir the external dialysis solution, and the stirring speed is 250 rpm; place the dialysis device in an incubator and keep the temperature at 6 °C; change the dialysis water every 5 h. Before changing, use a conductivity meter to detect the conductivity of the external dialysis solution. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0066] 7) Add the Auricularia auricula polysaccharide-catechin solution dropwise to the gelatin solution at a volume ratio of 4:1. In the dark and ice-water bath, use a high-intensity ultrasonic device to perform ultrasonic treatment at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of Auricularia auricula polysaccharide-gelatin loaded with catechin. Among them, the working conditions of the high-intensity ultrasonic device are: the probe diameter is 10 mm, the frequency is 20 kHz, the power is 600 W (the maximum power is 1000 W), and the on and off times are 5 s.

[0067] Among them, the extraction process of Auricularia auricula polysaccharide: Crush the dried Auricularia auricula fruiting body and screen it through a 160-mesh sieve to obtain powder. Use choline chloride / urea-based natural deep eutectic solvent (water content 30%, 1:2 molar ratio) as the extraction solvent, with a material ratio of 1:53 g / mL, and extract at 65 °C for 90 min. Centrifuge the extract at 4000 r / min for 10 min, collect the supernatant containing crude Auricularia auricula polysaccharide, use a rotary evaporator to concentrate the crude polysaccharide solution to 1 / 5 - 1 / 4 of its original volume, then add 4 times the volume of absolute ethanol and let it stand at 4 °C for 12 h, and then centrifuge at 4000 r / min for 10 min to collect the precipitate. Use the Sevag method to remove proteins from the precipitate. The concentrate is frozen at -80 °C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare Auricularia auricula polysaccharide (APP).

[0068] Example 4

[0069] A nano-capsule of jade ear mushroom polysaccharide-gelatin loaded with catechins, and its preparation method comprises the following steps:

[0070] 1) Dissolve jade ear mushroom polysaccharide and gelatin separately in ultrapure water to prepare a 0.5% w / v jade ear mushroom polysaccharide solution and a gelatin solution;

[0071] 2) Adjust the pH of the jade ear mushroom polysaccharide solution and the gelatin solution to 2.5 with 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution;

[0072] 3) Dissolve catechins in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0073] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25 °C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; at the same time, introduce nitrogen into the solution with a gas flow rate of 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen introduction and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0074] 5) Add the catechin solution to the jade ear mushroom polysaccharide solution to make the catechin concentration 0.1% w / v, and magnetically stir at a constant speed of 700 rpm with a high-intensity ultrasonic device at room temperature for 30 min to prepare a jade ear mushroom polysaccharide-catechin solution;

[0075] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly with ultrapure water; put the jade ear mushroom 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 jade ear mushroom polysaccharide-catechin solution in ultrapure water for dialysis, and control the volume ratio of ultrapure water to the jade ear mushroom polysaccharide-catechin solution at 150:1; use a magnetic stirrer to stir the external dialysis solution at a stirring speed of 250 rpm; place the dialysis device in an incubator and keep the temperature at 6 °C; change the dialysis water every 5 h, and detect the conductivity of the external dialysis solution with a conductivity meter before replacement. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution, and increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0076] 7) The Tremella aurantialba polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:1. In the dark and in an ice-water bath, it was ultrasonically treated with a high-intensity ultrasonic device at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of Tremella aurantialba polysaccharide-gelatin loaded with catechin. Among them, the working conditions of the high-intensity ultrasonic device were: the probe diameter was 10 mm, the frequency was 20 kHz, the power was 600 W (the maximum power was 1000 W), and the on and off times were 5 s.

[0077] Among them, the extraction process of Tremella aurantialba polysaccharide: The dried fruiting bodies of Tremella aurantialba were crushed and screened through a 160-mesh sieve to obtain powder. Using choline chloride / urea-based natural deep eutectic solvent (water content 30%, molar ratio 1:2) as the extraction solvent, at a material ratio of 1:53 g / mL, it was extracted at 65 °C for 90 min. The extract was centrifuged at 4000 r / min for 10 min, and the supernatant containing crude Tremella aurantialba polysaccharide was collected. The crude polysaccharide solution was concentrated to 1 / 5 - 1 / 4 of its original volume using a rotary evaporator, then 4 times the volume of absolute ethanol was added and it was left to stand at 4 °C for 12 h, and then centrifuged at 4000 r / min for 10 min to collect the precipitate. The Sevag method was used to remove proteins from the precipitate. The concentrate was frozen at -80 °C for 12 h in an LGJ-10 vacuum freeze dryer and then freeze-dried to prepare Tremella aurantialba polysaccharide (APP).

[0078] Example 5

[0079] A nano-capsule of Tremella aurantialba polysaccharide-gelatin loaded with catechin, and its preparation method includes the following steps:

[0080] 1) Dissolve Tremella aurantialba polysaccharide and gelatin separately in ultrapure water to prepare a 0.5% w / v Tremella aurantialba polysaccharide solution and a gelatin solution;

[0081] 2) Adjust the pH of the Tremella aurantialba polysaccharide solution and the gelatin solution to 4.0 using 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide solution;

[0082] 3) Dissolve catechin in absolute ethanol to prepare a 0.4% w / v catechin solution;

[0083] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25 °C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; meanwhile, pass nitrogen into the solution, and the gas flow rate is 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen input, and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0084] 5) Add the catechin solution to the Auricularia auricula polysaccharide solution to make the catechin concentration 0.2% w / v. At room temperature, use a high-intensity ultrasonic device to magnetically stir at a constant speed of 700 rpm for 30 min to prepare the Auricularia auricula polysaccharide-catechin solution;

[0085] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly 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 control the volume ratio of ultrapure water to the Auricularia auricula polysaccharide-catechin solution at 150:1; use a magnetic stirrer to stir the external dialysis solution, and the stirring speed is 250 rpm; place the dialysis device in an incubator and keep the temperature at 6 °C; change the dialysis water every 5 h, and detect the conductivity of the external dialysis solution with a conductivity meter before changing. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution, and increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0086] 7) Dropwise add the Auricularia auricula polysaccharide-catechin solution to the gelatin solution at a volume ratio of 1:1. In the dark and ice-water bath, use a high-intensity ultrasonic device to perform ultrasonic treatment at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of Auricularia auricula polysaccharide-gelatin loaded with catechin. Among them, the working conditions of the high-intensity ultrasonic device are: the probe diameter is 10 mm, the frequency is 20 kHz, the power is 600 W (the maximum power is 1000 W), and the on and off times are 5 s.

[0087] Among them, the extraction process of Auricularia auricula polysaccharide: Crush the dried Auricularia auricula fruiting body and screen it through a 160-mesh sieve to obtain powder. Use choline chloride / urea-based natural deep eutectic solvent (water content 30%, 1:2 molar ratio) as the extraction solvent, with a material ratio of 1:53 g / mL, and extract at 65 °C for 90 min. Centrifuge the extract at 4000 r / min for 10 min, collect the supernatant containing crude Auricularia auricula polysaccharide, use a rotary evaporator to concentrate the crude polysaccharide solution to 1 / 5 - 1 / 4 of its original volume, then add 4 times the volume of absolute ethanol and let it stand at 4 °C for 12 h, and then centrifuge at 4000 r / min for 10 min to collect the precipitate. Use the Sevag method to remove proteins from the precipitate. The concentrate is freeze-dried in an LGJ-10 vacuum freeze dryer after freezing at -80 °C for 12 h to prepare Auricularia auricula polysaccharide (APP).

[0088] Comparative Example 1

[0089] Single catechin without any nano - capsule embedding treatment.

[0090] Comparative Example 2

[0091] A polysaccharide - catechin solution of Tremella aurantialba, and its preparation method includes the following steps:

[0092] 1) Dissolve polysaccharide of Tremella aurantialba fully in ultrapure water to prepare a 0.5% w / v polysaccharide solution of Tremella aurantialba;

[0093] 2) Adjust the pH of the polysaccharide solution of Tremella aurantialba to 4.0 with 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution;

[0094] 3) Dissolve catechin in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0095] 4) Add the catechin solution to the polysaccharide solution of Tremella aurantialba so that the catechin concentration is 0.2% w / v, and stir magnetically at a constant speed of 700 rpm for 30 min at room temperature to prepare the polysaccharide - catechin solution of Tremella aurantialba.

[0096] Comparative Example 3

[0097] A gelatin - catechin solution, and its preparation method includes the following steps:

[0098] 1) Dissolve gelatin fully in ultrapure water to prepare a 0.5% w / v gelatin solution;

[0099] 2) Adjust the pH of the gelatin solution to 4.0 with 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution;

[0100] 3) Dissolve catechin in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0101] 4) Add the catechin solution to the gelatin solution so that the catechin concentration is 0.2% w / v, and stir magnetically at a constant speed of 700 rpm for 30 min at room temperature to prepare the gelatin - catechin solution.

[0102] Comparative Example 4

[0103] The difference from Example 1 is:

[0104] In step 1), dissolve polysaccharide of Tremella aurantialba and gelatin separately in ultrapure water to prepare a 0.05% w / v polysaccharide solution of Tremella aurantialba and a 0.05% w / v gelatin solution.

[0105] Comparative Example 5

[0106] The difference from Example 1 is as follows:

[0107] In step 5), the Auricularia cornea polysaccharide-catechin solution was added dropwise to the gelatin solution at a volume ratio of 1:3. At room temperature, using a high-intensity ultrasonic device, with an ultrasonic rotation speed of 700 rpm, ultrasonic treatment was carried out for 2.0 h to obtain a suspension of Auricularia cornea polysaccharide-gelatin nanocapsules loaded with catechin.

[0108] Comparative Example 6

[0109] The difference from Example 1 is as follows:

[0110] In step 3), catechin was dissolved in absolute ethanol to prepare a catechin solution with a concentration of 0.2% w / v.

[0111] In step 4), the catechin solution was added to the Auricularia cornea polysaccharide solution to make the catechin concentration 0.04% w / v, and magnetic stirring was carried out at a constant speed of 700 rpm for 30 min at room temperature to prepare an Auricularia cornea polysaccharide-catechin solution.

[0112] Comparative Example 7

[0113] The difference from Example 1 is as follows:

[0114] Steps 4) and 6) are not included.

[0115] Comparative Example 8

[0116] The difference from Example 1 is as follows:

[0117] Step 4) is not included.

[0118] Comparative Example 9

[0119] The difference from Example 1 is as follows:

[0120] Step 6) is not included.

[0121] Comparative Example 10

[0122] The difference from Example 1 is as follows:

[0123] The Auricularia cornea polysaccharide was replaced with β-cyclodextrin.

[0124] Comparative Example 11

[0125] The difference from Example 1 is as follows:

[0126] The gelatin was replaced with arabic gum.

[0127] Comparative Example 12

[0128] A catechin-loaded Auricularia auricula polysaccharide-gelatin nanocapsule, which is different from Example 1 in that its preparation method comprises the following steps:

[0129] 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;

[0130] 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;

[0131] 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;

[0132] 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;

[0133] 5) Dissolving catechins in anhydrous ethanol to prepare a 0.2% w / v catechin solution;

[0134] 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;

[0135] 7) Add the catechin solution to the Auricularia auricula polysaccharide-gelatin solution to make the catechin concentration 0.1% w / v. In the dark and an ice-water bath, use a high-intensity ultrasonic device to perform ultrasonic treatment at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of Auricularia auricula polysaccharide-gelatin loaded with catechin;

[0136] Among them, the extraction process of Auricularia auricula polysaccharide is the same as that in Example 1.

[0137] Comparative Example 13

[0138] A nano-capsule of Auricularia auricula polysaccharide-gelatin loaded with catechin, which is different from that obtained in Example 1, and its preparation method includes the following steps:

[0139] 1) Dissolve Auricularia auricula polysaccharide and gelatin separately in ultrapure water to prepare a 0.5% w / v Auricularia auricula polysaccharide solution and a 0.5% w / v gelatin solution;

[0140] 2) Use 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution to adjust the pH of the Auricularia auricula polysaccharide solution and gelatin solution to 4.0;

[0141] 3) Dissolve catechin in absolute ethanol to prepare a 0.2% w / v catechin solution;

[0142] 4) Antioxidation: Adjust the temperature of the catechin solution prepared in step 3) to 25°C, add ascorbic acid with a mass fraction of 0.05%, and stir at a speed of 230 rpm for 13 min; at the same time, introduce nitrogen into the solution, and the gas flow rate is 1 L / min; increase the stirring speed to 260 rpm, add ascorbic acid with a mass fraction of 0.05% again, and stir for 4 min; maintain the nitrogen introduction, and adjust the flow rate to 1.5 L / min to obtain the treated catechin solution;

[0143] 5) Add the catechin solution to the gelatin solution to make the catechin concentration 0.1% w / v, and use a high-intensity ultrasonic device to magnetically stir at a constant speed of 700 rpm for 30 min to obtain a gelatin-catechin solution;

[0144] 6) Select a dialysis bag with a molecular weight cut-off 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 °C for 30 min, and then rinse it thoroughly with ultrapure water; fill the gelatin-catechin solution into the pre-treated dialysis bag, ensuring that the volume of the solution in the dialysis bag does not exceed 80% of its maximum capacity; place the dialysis bag containing the gelatin-catechin solution in ultrapure water for dialysis, with the volume ratio of ultrapure water to the gelatin-catechin solution controlled at 150:1; use a magnetic stirrer to stir the external dialysis solution at a stirring speed of 250 rpm; place the dialysis device in an incubator and maintain the temperature at 6 °C; change the dialysis water every 5 h. Before replacement, use a conductivity meter to detect the conductivity of the external dialysis solution. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 36 h;

[0145] 7) Slowly add the gelatin-catechin solution drop by drop to the Auricularia cornea polysaccharide solution at a volume ratio of 1:1. In the dark and in an ice-water bath, use a high-intensity ultrasonic device to perform ultrasonic treatment at a rotation speed of 700 rpm for 2.0 h to obtain a nano-capsule suspension of Auricularia cornea polysaccharide-gelatin loaded with catechin;

[0146] Among them, the extraction process of Auricularia cornea polysaccharide is the same as that in Example 1.

[0147] Comparative Example 14

[0148] The difference from Example 1 is that:

[0149] In step 2), 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution were used to adjust the pH of the Auricularia cornea polysaccharide solution and the gelatin solution to 9.0.

[0150] Comparative Example 15

[0151] The difference from Example 1 is that:

[0152] In step 2), 0.1 mol / L hydrochloric acid and 0.1 mol / L NaOH solution were used to adjust the pH of the Auricularia cornea polysaccharide solution and the gelatin solution to 7.0.

[0153] Performance Experiment

[0154] I. Encapsulation efficiency of catechin:

[0155] Using the nano-capsule suspension of epicatechin-loaded Auricularia polytricha polysaccharide-gelatin prepared in Examples 1 to 5 of the present invention, 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, ultrasonicated for 20 min, and then centrifuged at 2500 rpm for 10 min. The content of epicatechin in the supernatant was analyzed using a UV-2600 spectrophotometer. 1 mL of epicatechin standard solutions with different concentrations were prepared, successively mixed with 1% w / v vanillin methanol solution and 25% v / v H2SO4 solution, reacted for 15 min in a dark environment at 30 °C, and the absorbance value was measured at 500 nm. The content of epicatechin in the test samples was determined according to the same steps as above, and the content of epicatechin in the test samples was determined based on the calibration curve of the epicatechin standard. The calculation formulas for the encapsulation efficiency and delivery rate of epicatechin in the test samples are as follows:

[0156] Encapsulation efficiency (%) = A1 / A2 × 100

[0157] where A1 is the content of encapsulated epicatechin and A2 is the total content of epicatechin.

[0158] The test results are as Figure 1 shown, from Figure 1It can be seen that, compared with single Auricularia auricula-judae polysaccharide or gelatin-embedded delivery of catechin (Comparative Example 2, Comparative Example 3), the nano-capsules prepared from Auricularia auricula-judae polysaccharide and gelatin have a higher encapsulation efficiency for catechin. It should be noted that the concentrations of Auricularia auricula-judae polysaccharide and gelatin working solutions, the ratio of the content of Auricularia auricula-judae polysaccharide to gelatin, the environmental pH value, and the concentration of catechin are all important influencing factors affecting the encapsulation efficiency of catechin. The increase in the contents of Auricularia auricula-judae polysaccharide, gelatin, and catechin is positively correlated with the improvement of the catechin encapsulation rate. A higher proportion of Auricularia auricula-judae polysaccharide in the two is helpful to increase the encapsulation rate of catechin, which may be closely related to the good viscoelasticity and adsorption of Auricularia auricula-judae polysaccharide. In addition, comparative studies have found that the nano-capsules formed by Auricularia auricula-judae polysaccharide and gelatin are more conducive to the encapsulation of catechin in an acidic environment, which may be caused by the easy decomposition of catechin in an alkaline environment. When β-cyclodextrin is used to replace Auricularia auricula-judae polysaccharide and arabic gum is used to replace gelatin as wall materials respectively (Comparative Example 10, Comparative Example 11), the prepared β-cyclodextrin-gelatin nano-capsules loaded with catechin and Auricularia auricula-judae polysaccharide-arabic gum nano-capsules loaded with catechin are found to have a lower encapsulation rate than the Auricularia auricula-judae polysaccharide-gelatin nano-capsules loaded with catechin prepared with Auricularia auricula-judae polysaccharide and gelatin as wall materials. It should be noted that as two different embedding materials, the addition order of gelatin and Auricularia auricula-judae polysaccharide will affect the encapsulation efficiency of catechin (Comparative Example 12, Comparative Example 13). For example, in Comparative Example 13, gelatin binds to catechin first, and then the addition of Auricularia auricula-judae polysaccharide may further affect the structure or stability of gelatin, thus reducing the encapsulation efficiency. Through the comparison of Comparative Examples 7, 8, 9 and Example 1, it can be concluded that without antioxidant treatment, catechin may be partially oxidized during the preparation process. The binding ability of oxidized catechin to Auricularia auricula-judae polysaccharide becomes weaker, resulting in a poorer encapsulation effect of the nano-capsules on catechin. Without dialysis treatment, there may be small molecule impurities and unbound catechin in the nano-capsules, which will affect the structural stability and encapsulation efficiency of the nano-capsules. However, catechin itself has undergone antioxidant treatment and has a relatively stable structure. Under the dual influence of no antioxidant treatment, which makes catechin prone to oxidation, and no dialysis treatment to remove impurities and unbound catechin, the formation and encapsulation effect of the nano-capsules are severely damaged.

[0159] II. Bioavailability of Catechin:

[0160] Using the nano-capsule suspension of Auricularia auricula polysaccharide - gelatin loaded with catechins prepared in Examples 1 - 5 of the present invention and the final substances prepared in Comparative Examples 1 - 15 as test samples, a three-stage simulated gastrointestinal tract (GIT) model consisting of the oral cavity, stomach, and small intestine 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. Using 40 mg of free catechins (CA) as a control, the nano-capsule suspension of Auricularia auricula polysaccharide - gelatin loaded with 40 mg of catechins was mixed with simulated saliva (7.5 mL), and then the mixture was adjusted to pH = 6.8 and shaken at 90 rpm for 10 min. Simulated gastric juice 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 stage (15 mL) was mixed with 15 mL of simulated gastric juice. Then the pH of the mixture was 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. The simulated small intestine fluid containing bile extract solution (50 mg / mL), pancreatin (24 mg / mL), and physiological saline (7.5 M NaCl and 0.5 M CaCl2) was mixed with the digested sample. The pH value of the mixture was adjusted to 7.0, and intestinal digestion was carried out for 120 min. After the samples were processed by the GIT model, the bioavailability of catechins was determined. The raw chyme was collected and centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was collected because these micelle components contained catechins, which were easily absorbed by intestinal epithelial cells and thus represented the "bioavailable" components after the digestion process. The concentration of catechins in the supernatant was measured at 230 nm using an ultraviolet-visible spectrophotometer and calculated through the calibration curve of the catechin standard. The formula for calculating the bioavailability of catechins is as follows

[0161] Bioavailability (%) = C M / C I × 100

[0162] where C M and C I respectively represent the concentrations of catechins in the mixed micelle components and the initial samples at the end of the simulated GIT model.

[0163] The test results are as Figure 2 shown, and are from Figure 2It can be seen that the bioavailability of single catechin in the small intestine is the lowest, only reaching 18.39%. The nano-capsules prepared with single Tremella fuciformis polysaccharide, or single gelatin, or the nano-capsules prepared with Tremella fuciformis polysaccharide and gelatin can effectively promote the sustained release of catechin during digestion and absorption and improve its bioavailability. Increasing the concentration of Tremella fuciformis polysaccharide and gelatin from 0.3% to 0.5%, and increasing the content of catechin from 0.1% to 0.2% can effectively improve the bioaccessibility of catechin (Example 1, Example 2). In addition, increasing the proportion of Tremella fuciformis polysaccharide in the Tremella fuciformis polysaccharide-gelatin nano-capsules helps to improve the delivery and bioavailability of catechin. In addition, the environmental pH value is an important factor affecting the bioavailability of catechin. It is found by comparison that encapsulating catechin in an acidic environment is beneficial to improving its encapsulation rate and bioavailability during in vitro digestion and absorption. In addition, by using β-cyclodextrin to replace Tremella fuciformis polysaccharide and arabic gum to replace gelatin as wall materials respectively (Comparative Example 10, Comparative Example 11), the prepared β-cyclodextrin-gelatin nano-capsules loaded with catechin and the Tremella fuciformis polysaccharide-arabic gum nano-capsules loaded with catechin are found to have lower bioavailability of catechin than the Tremella fuciformis polysaccharide-gelatin nano-capsules loaded with catechin prepared with Tremella fuciformis polysaccharide-gelatin as the wall material. It should be noted that improper addition order of gelatin and Tremella fuciformis polysaccharide may change the release rate or distribution characteristics of catechin, thus affecting its bioavailability in vivo (Comparative Example 12, 13). For example, in Comparative Example 13, gelatin binds to catechin first, and then the addition of Tremella fuciformis polysaccharide may further affect the structure or stability of gelatin, thus reducing the bioavailability of catechin. Through the comparison of Comparative Examples 7, 8, 9 and Example 1, it can be concluded that catechin molecules contain multiple phenolic hydroxyl groups and are unstable, and are easily oxidized in the preparation, storage and in vivo environment. Step 4) created an antioxidant environment by adding ascorbic acid and introducing nitrogen, effectively reducing the oxidation of catechin. It is crucial to maintain the integrity of the catechin structure, because only the catechin with a complete structure can specifically bind to the corresponding targets in the body (such as specific receptors, enzymes, etc.), and then exert its biological activity. If catechin is oxidized and its chemical structure changes, it may not be normally absorbed and utilized by the human body, resulting in a decrease in its bioavailability. The antioxidant treatment makes catechin more stable during the preparation process of the nano-capsules and subsequent storage and transportation. The stable catechin can better maintain its loading state in the nano-capsules and reduce the loss before reaching the action site. When the nano-capsules enter the human body, the stable catechin is more likely to be released from the nano-capsules and effectively taken up by the absorption sites such as the gastrointestinal tract, thus increasing the absorption amount of catechin in the body and ultimately enhancing the bioavailability. The stable catechin is more evenly distributed in the nano-capsules and can be released from the nano-capsules more regularly in the in vivo environment.This slow and continuous release mode helps catechins maintain a certain concentration in the gastrointestinal tract, increasing the contact time and area with the absorption site, thus promoting the absorption of catechins. For example, on the surface of intestinal epithelial cells, stable catechins can better enter cells through passive diffusion, carrier-mediated transport, etc., and then enter the blood circulation, improving the bioavailability. Dialysis treatment can remove small molecule impurities and unbound catechins in the Auricularia polytricha polysaccharide-catechin solution, making the structure of the nanocapsules more regular and stable. A suitable nanocapsule structure is crucial for the encapsulation and release of catechins. A regular structure can more effectively encapsulate catechins and prevent their premature leakage before reaching the action site; at the same time, in the in vivo environment, it can release catechins in a timely manner according to physiological needs. For example, physical properties such as the particle size and surface charge of the nanocapsules are optimized during dialysis, which is more conducive to their interaction with biological membranes, promoting the absorption of catechins and thus improving their bioavailability. The small molecule 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 enhancing the bioavailability.

[0164] III. Light and heat stability tests:

[0165] Taking the nanocapsule suspensions of Auricularia polytricha polysaccharide-gelatin loaded with catechins prepared in Examples 1-5 of the present invention, the final substances prepared in Comparative Examples 1-15 as test samples, and an equal amount of catechin solution (4 mL) were respectively added into test tubes. Then, they were irradiated in front of a 10 cm ultraviolet lamp for 6 h, and the retention rate (RR) of catechins was calculated by the pH difference method. Catechins (4 mL) and each test sample were added into test tubes, and then the samples were heated in a water bath at 90°C. Then, they were quickly cooled to room temperature in ice, and the retention rate (RR) of catechins was calculated by the pH difference method. As described above, the calculation formula for the retention rate of catechins is as follows:

[0166] Catechin retention rate (%) = Catechin content (after light or heat treatment) / Total amount of catechins × 100

[0167] After light treatment, the determination results of the catechin retention rate are as Figure 3 shown. After heat treatment, the determination results of the catechin retention rate are as Figure 4 shown. From Figure 3 and Figure 4It is known that due to the instability of catechins, catechins are particularly sensitive to ultraviolet (UV) light during storage, especially UVB radiation, which can cause photoisomerization and photodegradation reactions. These reactions 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 form dimers or polymers with higher molecular weights. These oligomerization reactions are the main reasons for the instability of catechins at high temperatures. High temperature also promotes the oxidation reaction of catechins, leading to changes in their color and activity. For example, when the temperature is above 80 °C, the oxidation rate of catechins significantly increases. 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), encapsulating catechins can effectively enhance their stability under light and heat treatment. It is worth noting that increasing the content of Tremella aurantialba polysaccharide and gelatin does not significantly improve the stability of catechins, while neutral and alkaline pH environments enhance the instability of catechins. By using β-cyclodextrin to replace Tremella aurantialba polysaccharide and arabic gum to replace gelatin as wall materials respectively (Comparative Example 10, Comparative Example 11), the prepared β-cyclodextrin-gelatin nanocapsules loaded with catechins and Tremella aurantialba polysaccharide-arabic gum nanocapsules loaded with catechins were found to have lower light and heat stability than the Tremella aurantialba polysaccharide-gelatin nanocapsules loaded with catechins. It is worth noting that as two different encapsulating materials, the addition sequence of gelatin and Tremella aurantialba polysaccharide will affect the encapsulation efficiency of catechins (Comparative Examples 12, 13). For example, in Comparative Example 13, gelatin binds to catechins first, and then the addition of Tremella aurantialba polysaccharide may further affect the structure or stability of gelatin, thereby reducing the stability. Through the comparison of Comparative Examples 7, 8, 9 and Example 1, it can be concluded that in step 4), antioxidant treatment by adding ascorbic acid and introducing nitrogen can effectively reduce the degree of oxidation of catechins during the preparation process. During light treatment, catechins are prone to absorb light energy and react with oxygen to be oxidized. The chemically more stable catechins after antioxidant treatment can better maintain their integrity when forming a complex with Tremella aurantialba polysaccharide and finally being encapsulated into nanocapsules. In this way, after light treatment, the degree of oxidation and damage of catechins is reduced, thereby increasing the encapsulation rate after light treatment. Heat treatment accelerates the oxidation reaction rate of catechins. The antioxidant treatment in step 4) provides a certain protective barrier for catechins, making it more difficult for catechins to be oxidized under high-temperature conditions. During the formation of nanocapsules and subsequent heat treatment, stable catechins can be better encapsulated by Tremella aurantialba polysaccharide to form a stable encapsulation structure. Therefore, the encapsulation rate of catechins after antioxidant treatment is relatively high after heat treatment. The dialysis treatment in step 6) can remove small-molecule impurities and unbound catechins in the Tremella aurantialba polysaccharide-catechin solution, making the formation environment of nanocapsules purer.During light treatment, the pure nanocapsule structure 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, after dialysis treatment, the encapsulation rate of catechins in the nanocapsules will increase 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, enabling them to better withstand the temperature changes and thermal stress brought about by heat treatment. During heat treatment, the stable nanocapsule structure can effectively prevent the leakage of catechins, thus increasing the encapsulation rate after heat treatment.

[0168] IV. Antioxidant performance test:

[0169] Using the nanocapsule suspension of Auricularia polytricha polysaccharide - gelatin loaded with catechins prepared in Example 1 of the present invention and the final substances prepared in Comparative Examples 7, 8, and 9 as test samples, the antioxidant capacity of the Auricularia polytricha polysaccharide - gelatin nanocapsules loaded with catechins was detected by the DPPH free radical scavenging rate method, and the DPPH free radical scavenging rate (%) was calculated.

[0170] Analysis of test results: The DPPH free radical scavenging rate (%) of the nanocapsule suspension of Auricularia polytricha polysaccharide - gelatin loaded with catechins prepared in Example 1 was 85%, the DPPH free radical scavenging rate (%) of Comparative Example 7 was 60%, the DPPH free radical scavenging rate (%) of Comparative Example 8 was 70%, and the DPPH free radical scavenging rate (%) of Comparative Example 9 was 78%.

[0171] In step 4), adding ascorbic acid (which is 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 Auricularia polytricha polysaccharide - gelatin nanocapsules loaded with catechins, better protecting catechins from oxidation and maintaining their biological activity. Introducing nitrogen can remove oxygen in the solution, reducing the oxidation of catechins by oxygen. 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, enabling the nanocapsules to exert their antioxidant effect more persistently during application. An appropriate stirring speed helps catechins to mix fully with ascorbic acid, etc., making catechins disperse more evenly in the solution, which will affect the dispersion of catechins in the Auricularia polytricha polysaccharide - gelatin system during the subsequent formation of nanocapsules, facilitating the formation of well - dispersed nanocapsules, avoiding the aggregation of catechins, and thus improving the stability and performance consistency of the nanocapsules.

[0172] V. Particle size and distribution:

[0173] Using the nano-capsule suspension of Auricularia auricula polysaccharide-gelatin loaded with catechins prepared in Example 1 of the present invention and the final substances prepared in Comparative Examples 7, 8, and 9 as test samples, the average particle size and polydispersity index (PDI) of the nano-capsules were measured using a dynamic light scattering instrument (DLS).

[0174] Experimental results: The particle size in the nano-capsule suspension of Auricularia auricula polysaccharide-gelatin loaded with catechins 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 nano-capsules prepared in Comparative Example 7 are greatly affected, the average particle size increases significantly, the average particle size is 300 nm, and the PDI is 0.25; the average particle size of the nano-capsules 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 aggregation caused by the lack of antioxidant treatment, which in turn affects the formation of nano-capsules. The average particle size of the nano-capsules prepared in Comparative Example 9 is 220 nm and the PDI is 0.18; the increase in the average particle size may be due to the possible presence of some unreacted small molecules or impurities in the nano-capsules without dialysis, which affects the regularity of the nano-capsules.

[0175] In step 6) of Embodiments 1 to 5 of the present invention, during the dialysis process, factors such as the volume ratio of ultrapure water to Auricularia auricula polysaccharide-catechin solution, stirring speed, temperature, and dialysis time will affect the diffusion and exchange rates of small molecules. Appropriate conditions help control the degree of interaction between Auricularia auricula polysaccharide and catechin, preventing excessive molecular aggregation or growth, so that the finally formed nanocapsules have a moderate particle size and a uniform distribution. By detecting the conductivity to control the replacement frequency of the external dialysis solution, the ionic concentration and composition in the solution can be adjusted more precisely, further optimizing the formation environment of the nanocapsules, making the particle size distribution narrower, and improving the uniformity of the nanocapsules. This is very important for the application of nanocapsules in fields such as drug delivery and food preservation, and can ensure the stability and consistency of their performance. In addition, appropriate dialysis conditions can promote better encapsulation of catechin by Auricularia auricula polysaccharide, forming a stable Auricularia auricula polysaccharide-catechin complex, thereby increasing the encapsulation rate of catechin in the nanocapsules, reducing the loss of catechin during the preparation process, improving the loading efficiency of the nanocapsules for catechin, and enabling the nanocapsules to carry and protect catechin more effectively. If the dialysis process is properly controlled, the structure of the Auricularia auricula polysaccharide-catechin complex can be made more stable. After subsequent combination with gelatin to form nanocapsules, it can better prevent the leakage of catechin from the nanocapsules, improve the encapsulation stability of the nanocapsules, extend the release time of catechin, and enhance the effect of the nanocapsules in practical applications. Conditions such as dialysis temperature and stirring speed 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, which helps improve the physical stability of the nanocapsules after mixing with gelatin to form nanocapsules, making them less likely to aggregate, precipitate, etc. during storage and use. The precise control of the solution composition and environment during the dialysis process can reduce the possible impurities or unstable factors in the solution, reduce the possibility of adverse reactions between catechin and other substances, thereby improving the chemical stability of the nanocapsules and ensuring their good performance in different environments.

[0176] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. A preparation method of nano-capsules of jade ear mushroom polysaccharide - gelatin loaded with catechins, characterized in that, Specifically, it includes the following steps: 1) Dissolve tremella aurantialba polysaccharide and gelatin in ultrapure water respectively to prepare a tremella aurantialba polysaccharide solution and a gelatin solution with a concentration of 0.2 - 0.5% w / v; 2) Adjust the pH of the tremella aurantialba polysaccharide solution and the gelatin solution to 2.5 - 4.0; 3) Dissolve catechin in absolute ethanol to prepare a catechin solution with a concentration of 0.2 - 0.4% w / v; 4) Add the catechin solution to the tremella aurantialba polysaccharide solution and magnetically stir at a constant speed of 600 - 800 rpm for 20 - 35 min at room temperature to prepare a tremella aurantialba polysaccharide-catechin solution; among them, in the tremella aurantialba polysaccharide-catechin solution, the concentration of catechin is 0.05 - 0.2% w / v; 5) Gradually add the tremella aurantialba polysaccharide-catechin solution to the gelatin solution drop by drop according to a volume ratio of 1.0 - 4.0, and perform ultrasonic treatment in the dark and an ice-water bath to obtain a nanocapsule suspension of tremella aurantialba polysaccharide-gelatin loaded with catechin; Among them, between step 4) and step 5), there is also a dialysis step, including: Select a dialysis bag with a molecular weight cut-off range of 1000 - 3000 Da; soak the dialysis bag in a 10 mmol / L sodium bicarbonate solution containing 1 mmol / L ethylenediaminetetraacetic acid, treat it at 80°C for 30 min, and then rinse it thoroughly with ultrapure water; Put the tremella aurantialba 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 tremella aurantialba polysaccharide-catechin solution in ultrapure water for dialysis, and control the volume ratio of ultrapure water to the tremella aurantialba polysaccharide-catechin solution at 100:1 - 200:1; use a magnetic stirrer to stir the external dialysis solution, and the stirring speed is 200 - 300 rpm; place the dialysis device in an incubator and keep the temperature at 4 - 8°C; change the dialysis water every 4 - 6 h, and detect the conductivity of the external dialysis solution with a conductivity meter before replacement. If the decrease in conductivity is less than 10%, increase the replacement frequency of the external dialysis solution, and increase the replacement frequency of the external dialysis solution to once every 2 h; continue dialysis for 24 - 48 h.

2. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechins as described in claim 1, characterized in that, Between step 3) and step 4), there is also an antioxidant step, including: First, adjust the temperature of the prepared catechin solution to 20 - 25°C, add ascorbic acid with a mass fraction of 0.025 - 0.05%, and stir at a speed of 200 - 300 rpm for 10 - 15 min; at the same time, introduce nitrogen gas into the solution, and the gas flow rate is 0.5 - 1 L / min; Increase the stirring speed to 230 - 260 rpm, add ascorbic acid with a mass fraction of 0.025 - 0.05% again, and stir for 4 - 6 min; maintain the nitrogen gas introduction, and adjust the flow rate to 1 - 1.5 L / min to obtain the treated catechin solution.

3. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechins as described in claim 1, characterized in that, In step 1), the concentrations of the auricularia auricula polysaccharide solution and the gelatin solution are both 0.5% w / v; in step 2), the pH values of the jade auricularia auricula 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 jade auricularia auricula polysaccharide-catechin solution to the gelatin solution is 1:

1.

4. The method for preparing the nano-capsules of jade auricularia auricula polysaccharide-gelatin loaded with catechin as claimed in claim 1, in step 5), the conditions of ultrasonic treatment are: the frequency is 20 kHz, the power is 600 - 1000 W, the ultrasonic rotation speed is 600 - 800 rpm, and the ultrasonic time is 1.5 - 2.5 h.

5. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechins according to claim 4, characterized in that, In step 5), the conditions of ultrasonic treatment are: the frequency is 20 kHz, the power is 600 W, the ultrasonic rotation speed is 700 rpm, and the ultrasonic time is 2.0 h.

6. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechins as claimed in 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.

7. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechin according to claim 1, characterized in that, The jade auricularia auricula polysaccharide is extracted from the jade auricularia auricula fruit body.

8. The preparation method of the nano-capsule of jade ear polysaccharide-gelatin loaded with catechins according to claim 7, characterized in that, The extraction process specifically includes: crushing the dried jade auricularia auricula fruit body, screening through a 160-mesh sieve to obtain powder, using choline chloride / urea-based natural deep eutectic solvent as the 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 the crude jade auricularia auricula polysaccharide, concentrating the supernatant to 1 / 5 - 1 / 4 of its volume using a rotary evaporator, then adding 4 times the volume of absolute ethanol and standing at 4°C for 12 h, centrifuging at 4000 r / min for 10 min, collecting the precipitate, removing proteins from the precipitate using the Sevag method to obtain a concentrate, and freeze-drying the concentrate at -80°C for 12 h to obtain the jade auricularia auricula polysaccharide.

9. The nano-capsules of jade auricularia auricula polysaccharide-gelatin loaded with catechin prepared by the method for preparing the nano-capsules of jade auricularia auricula polysaccharide-gelatin loaded with catechin as claimed in any one of claims 1 - 8.

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