Preparation method and application of a kind of bird's nest peptide for mucosa and wound healing repair and immunomodulation

Abstract

The application discloses a preparation method and application of a bird's nest peptide for mucosa and wound healing repair and immune regulation, and belongs to the technical field of bioactive peptides. The application adopts a pure physical extraction process of ultramicro crushing, soaking and vibration loosening, high-temperature and high-pressure heating hydrolysis extraction, multi-layer precision filtration, ceramic ultrafiltration membrane purification, low-temperature vacuum concentration and spray powder drying, and does not add enzyme preparations and chemical reagents in the whole process, so that the bitter and astringent taste of traditional enzymolysis process is effectively avoided, and active ingredients in the bird's nest are reserved. Compared with the prior art, the obtained bird's nest peptide can repair damaged tissues by promoting cell mitosis and migration, promote mucosa surface fold protein formation to repair mucosa, and form a mucopolysaccharide protein immune protection layer on the mucosa surface, and has the triple effects of mucosa repair, wound healing and immune regulation. The product is high in safety and pure in flavor, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a method for preparing and applying bird's nest peptides for mucosal and wound healing and immune regulation. Background Technology

[0002] Bird's nest is the nest built by swiftlets of the Apodidae family and other related species using a mixture of saliva and down feathers. It is rich in protein, sialic acid, epidermal growth factor, and other active ingredients, possessing physiological functions such as anti-oxidation, anti-aging, and immune enhancement. In recent years, with the deepening research on bioactive peptides, bird's nest peptides, due to their small molecular weight, easy absorption, and high bioactivity, have become a research hotspot in the field of bird's nest processing.

[0003] Patent CN121944073A discloses an ion-modified bird's nest peptide complex that enhances bone density and promotes growth and development, along with its preparation method and applications. This complex improves the binding capacity of bird's nest peptides to metal ions through metal ion modification, giving it both antioxidant activity and the ability to promote osteoblast differentiation. However, this technical solution mainly focuses on the regulatory effect of metal ion modification on bone metabolism, without addressing the application of bird's nest peptides in mucosal repair and immune regulation. Furthermore, the introduction of metal ions increases the safety risks of the product. Patent CN121248719A discloses a preparation method for bird's nest peptides and their applications. The amino acid sequence of this bird's nest peptide is KPAPPKPEPK, exhibiting strong resistance to UV damage. However, this bird's nest peptide achieves its anti-UV effect through screening specific sequences, resulting in a single-directional effect, and its preparation method does not adequately address the taste issue of the bird's nest peptide product. Patent CN120665149A discloses a bird's nest peptide and a key preparation method for bird's nest peptides used in pregnant women. This bird's nest peptide can significantly increase the proliferation rate of nerve cells. However, this method requires simulating the in vitro digestion process and screening specific peptides, which is complex, time-consuming, and the product is a single peptide with a specific sequence, making it difficult to retain the synergistic effects of multiple active ingredients in bird's nest.

[0004] In summary, existing bird's nest peptide technologies have the following main shortcomings: First, most preparation methods employ enzymatic hydrolysis, introducing exogenous enzymes and chemical substances, resulting in products with a sour and bitter taste, and potentially damaging the bound active ingredients of sialic acid in the stable glycoprotein structure of bird's nest. Second, current technologies lack sufficient research on the systemic effects of bird's nest peptides in mucosal repair, wound healing, and immune regulation, and there is a lack of bird's nest peptide products that can simultaneously achieve these multiple effects. Third, existing bird's nest peptide separation and purification processes mostly rely on organic solvents or complex chromatographic techniques, which are costly and inefficient, making it difficult to meet the needs of large-scale production. Therefore, there is an urgent need to develop a bird's nest peptide preparation method that does not require the addition of enzymes and chemical reagents throughout the entire process, retains the natural active ingredients of bird's nest, and possesses multiple effects including mucosal repair, wound healing, and immune regulation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for preparing and applying bird's nest peptides for mucosal and wound healing and immune regulation.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a bird's nest peptide that promotes mucosal and wound healing and repair as well as immune regulation is as follows:

[0008] Step 1, Raw material ultrafine grinding: Select dried bird's nest as raw material, grind it to obtain bird's nest ultrafine powder;

[0009] Step 2, Soaking and Vibrating to Loosen: Mix the bird's nest ultrafine powder obtained in Step 1 with deionized water, soak and vibrate to loosen, and obtain bird's nest suspension;

[0010] Step 3, High-temperature and high-pressure hydrolysis extraction: The bird's nest suspension obtained in step 2 is subjected to high-temperature and high-pressure hydrolysis extraction to obtain bird's nest glycoprotein peptide hydrolysate.

[0011] Step 4, Multi-stage precision filtration: After the bird's nest glycoprotein peptide hydrolysate obtained in Step 3 is naturally cooled to room temperature, it is passed through three-stage precision filtration in sequence, and the clear coarse filtrate is collected.

[0012] Step 5, Ceramic ultrafiltration membrane purification: The clarified coarse filtrate obtained in step 4 is purified by cross-flow ultrafiltration through a ceramic ultrafiltration membrane, and the permeate is collected to obtain the purified bird's nest glycoprotein peptide solution.

[0013] Step 6, Low-temperature vacuum concentration and spray drying: The purified bird's nest glycoprotein peptide solution obtained in step 5 is concentrated under vacuum and then spray dried. The resulting dried powder is aseptically packaged to obtain the bird's nest peptide for mucosal and wound healing repair and immune regulation.

[0014] The pulverization process involves pulverizing under a negative pressure of -0.07 to -0.09 MPa and passing the powder through a 200-400 mesh sieve to obtain bird's nest ultrafine powder with a particle size D90 of less than 50 μm.

[0015] The soaking and vibration loosening process involves mixing the bird's nest ultrafine powder obtained in step 1 with deionized water at a mass ratio of 1:10-15, placing it in a constant temperature shaking water bath, and soaking it for 1-3 hours at 20-40℃ and a shaking frequency of 50-100rpm, so that the bird's nest ultrafine powder can fully absorb water and expand to 2-4 times its original volume.

[0016] The high-temperature and high-pressure heating hydrolysis extraction is carried out under the condition of stirring speed of 100-200 rpm, first heating to 50-70℃ and holding for 10-50 min to allow the bird's nest glycoprotein to fully swell, then heating to 110-130℃ at a rate of 1-3℃ / min and pressure inside the vessel of 0.1-0.15MPa, and holding for hydrolysis for 100-300 min. After the hydrolysate naturally cools down to below 80℃, the exhaust valve is slowly opened to release pressure to atmospheric pressure.

[0017] The three-stage precision filtration involves sequentially passing the material through a 50-200 mesh stainless steel screen, a 100-300 mesh silk cloth filter, and a 0.3-0.6 μm microfiltration membrane.

[0018] The cross-flow ultrafiltration purification is performed through a ceramic ultrafiltration membrane at 30-50℃, transmembrane pressure of 0.15-0.25MPa, and membrane surface flow rate of 1-3m / s.

[0019] The vacuum concentration process involves controlling the material temperature to ≤60℃ until the solid content is 15-30%.

[0020] The powder spraying dryer controls the inlet air temperature to be 150-180℃ and the outlet air temperature to be 60-90℃.

[0021] The ceramic ultrafiltration membrane is prepared as follows:

[0022] S1. Dissolve zirconium oxychloride and yttrium nitrate hexahydrate in deionized water at a zirconium-yttrium molar ratio of 90-95:5-10 to prepare a mixed solution with a total metal ion concentration of 0.3-0.8 mol / L. Add polyethylene glycol as a dispersant at 0.8-1.2% of the theoretically generated total mass of metal oxides. Under stirring at 70-90℃, add 0.5-2 mol / L sodium hydroxide solution dropwise to adjust the pH to 9-11 and react for 2-6 hours. After cooling, wash the resulting gel repeatedly with deionized water until the conductivity of the filtrate is ≤10 μS / cm. Then dry at 50-70℃, redisperse in deionized water, and adjust the pH to 3-5 with 0.5-2 mol / L dilute nitric acid to obtain a sol.

[0023] S2. First, the dopant is dissolved in deionized water to prepare an aqueous solution with a concentration of 0.3-0.8 mol / L. Under the conditions of room temperature and stirring speed of 200-400 rpm, the above-mentioned dopant aqueous solution is added dropwise to the sol obtained in S1 at a rate of 1-3 drops per second. After the addition is completed, stirring is continued for 1-3 hours, followed by ultrasonic dispersion for 20-40 minutes to obtain a composite sol. The amount of dopant added is 1-3% of the theoretical solid content of metal oxide in the sol. Subsequently, silica sol accounting for 3-6% of the theoretical solid content of metal oxide, polyvinyl alcohol 1-3% and hydroxypropyl methylcellulose 0.3-0.8% are added to the composite sol. After vacuum degassing, a coating solution is obtained. The porous alumina ceramic, which has been cleaned with deionized water and sterilized at high temperature, is then coated with the coating solution. After immersing the support in the coating solution for 10-50 seconds, it is vertically pulled up at a speed of 3-8 cm / min and dried at 50-90℃ for 1-3 hours. The above immersion, pulling up, and drying operations are repeated 1-3 times to control the membrane thickness at 5-8 μm. Then, the temperature is increased to 280-320℃ at a rate of 0.5-2℃ / min and held for 40-80 minutes. The temperature is then increased to 480-520℃ at a rate of 0.5-2℃ / min and held for 40-80 minutes. Finally, the temperature is increased to 800-900℃ at a rate of 0.5-2℃ / min and calcined for 1-5 hours. After naturally cooling to room temperature, the membrane module is boiled in deionized water for 1-3 hours and sterilized with high-temperature steam at 110-130℃ for 10-50 minutes to obtain the ceramic ultrafiltration membrane.

[0024] The dopant is at least one of aluminum nitrate nonahydrate, titanium sulfate tetrahydrate, magnesium nitrate hexahydrate, and cerium nitrate hexahydrate.

[0025] Preferably, the dopant is titanium oxysulfate tetrahydrate.

[0026] Preferably, the amount of the dopant added is 1.8-2.2% of the theoretical solid content of the metal oxide in the sol.

[0027] The application of the aforementioned bird's nest peptides with mucosal and wound healing repair and immune regulation in the preparation of food, health products, medicines or cosmetics with mucosal repair, wound healing and immune regulation effects.

[0028] During the research and development process, this invention discovered that the hydrolyzed extract of bird's nest glycoprotein peptides contains both target small molecule peptides and large molecule glycoprotein impurities. When purified using a ceramic ultrafiltration membrane, the membrane surface is easily contaminated by glycoproteins, leading to rapid flux decay. In existing technologies, the hydrophilic network on the surface of ceramic membranes modified with a single silica sol is incomplete, resulting in insufficient stability of the hydration layer and limited antifouling durability. To address these issues, this invention introduces both silica sol and titanium oxysulfate tetrahydrate as composite modifiers into the ceramic ultrafiltration membrane coating solution. After high-temperature calcination, titanium dioxide and silica cross-link through covalent bonds to form a three-dimensional inorganic hydrophilic network, significantly enhancing the stability of the membrane hydration layer and the charge repulsion effect. This effectively inhibits the adsorption and deposition of glycoproteins on the membrane surface, extending the continuous operation cycle of the membrane module.

[0029] During the research and development process, this invention further discovered that different metal oxide dopants exhibit significant differences in their ability to construct the hydrophilic network of ceramic ultrafiltration membranes. While oxides of aluminum, magnesium, and cerium can improve the hydrophilicity of the membrane surface to some extent, the covalent cross-linked network they form with silica has weak hydration capacity, or its surface charge characteristics in neutral feed solutions are unfavorable for antifouling. To address these issues, this invention, through comparative screening, determined titanium oxysulfate tetrahydrate as the preferred dopant. The hydrophilic network formed by the covalent cross-linking of titanium dioxide and silica generated by its calcination is the most dense, and the titanium dioxide nanoparticles can effectively refine the zirconium oxide grains and optimize the membrane pore structure, thereby improving both antifouling performance and membrane separation accuracy.

[0030] During the research and development process, it was also discovered that more dopant is not necessarily better. Insufficient dopant results in sparse distribution of titanium dioxide in the film, incomplete coverage of the covalently cross-linked hydrophilic network, and an inability to fully realize the synergistic strengthening effect. Excessive dopant leads to localized enrichment of titanium dioxide nanoparticles in the film, increasing surface roughness and causing uneven pore size distribution, which in turn weakens the density of the hydration layer and separation accuracy. To address these issues, this invention optimizes the dopant dosage to achieve a continuous and uniform distribution at the zirconia grain boundaries, achieving optimal synergy between the integrity of the hydrophilic network coverage and the uniformity of the film structure, thereby simultaneously achieving the best results in both anti-fouling performance and separation accuracy.

[0031] The bird's nest peptide obtained in this invention repairs damaged tissues by promoting cell mitosis and migration, repairs and smooths the mucosal barrier by promoting the formation of folded proteins on the mucosal surface, and forms a mucopolysaccharide protein immune protective layer on the mucosal surface to enhance local immune function, thereby achieving the triple effects of mucosal repair, wound healing and immune regulation.

[0032] Compared with the prior art, the present invention has the following beneficial technical effects:

[0033] 1) This invention adopts a pure physical extraction process, which includes ultra-fine pulverization, soaking and vibration loosening, high temperature and high pressure heating and hydrolysis extraction, multi-layer precision filtration, ceramic ultrafiltration membrane purification and spray drying. No enzyme preparations or chemical reagents are added throughout the process, which effectively avoids the sour and bitter taste produced by traditional enzymatic hydrolysis processes, and retains the active ingredients in bird's nest glycoproteins. The product has high safety and pure flavor.

[0034] 2) This invention introduces silica sol and tetrahydrate titanium oxysulfate for synergistic modification into yttrium-stabilized zirconia ceramic ultrafiltration membranes. After calcination, titanium dioxide and silica cross-link through covalent bonds to form a three-dimensional inorganic hydrophilic network, which significantly enhances the stability and antifouling ability of the membrane surface hydration layer, effectively inhibits the adsorption and deposition of bird's nest glycoprotein peptides on the membrane surface, and extends the continuous operation cycle of the membrane module.

[0035] 3) This invention optimizes the doping amount of tetrahydrate titanium oxysulfate to form a continuous and uniform distribution of titanium dioxide in the membrane layer, achieving the optimal synergy between the integrity of the hydrophilic network coverage and the uniformity of the membrane structure. At the same time, it uses a ceramic ultrafiltration membrane with a specific pore size to perform cross-flow purification of the bird's nest glycoprotein peptide hydrolysate, effectively retaining large molecular impurities and enriching small molecular active peptides. The resulting bird's nest peptide product has nutritional effects such as mucosal repair, wound healing and immune regulation. Detailed Implementation

[0036] Some material sources or parameters:

[0037] Dried bird's nest: Edible bird's nest is the nest built by swiftlets of the Apodidae family using a mixture of saliva and down feathers. It is crescent-shaped or boat-shaped in appearance, ivory white or slightly yellow in color, with a water content of no more than 15% and a protein content of no less than 50%. It is used as the raw material of this invention after being manually cleaned of feathers and impurities.

[0038] Zirconium oxychloride: Zirconium dichloride octahydrate, CAS No. 13520-92-8, molecular weight 322.25.

[0039] Yttrium nitrate hexahydrate: CAS No. 13494-98-9, molecular weight 383.01.

[0040] Polyethylene glycol: CAS No. 25322-68-3, average molecular weight approximately 4000.

[0041] Aluminum nitrate nonahydrate: CAS No. 7784-27-2, molecular weight 375.13.

[0042] Titanium oxysulfate tetrahydrate: CAS No. 13825-74-6, molecular weight is 231.97.

[0043] Magnesium nitrate hexahydrate: CAS No. 13446-18-9, molecular weight 256.41.

[0044] Cerium nitrate hexahydrate: CAS No. 10294-41-4, molecular weight 434.22.

[0045] Porous alumina ceramic support: Alpha-type alumina porous ceramic support with an average pore size of 0.1 to 1.0 micrometers, a porosity of 30%-50%, an outer diameter of 10-30 mm, a wall thickness of 2-4 mm, and a bending strength of not less than 30 MPa, serving as the mechanical carrier for ceramic ultrafiltration membranes.

[0046] Silica sol: The product has a solid content of 20%-30%, a silica particle size of 10-30nm, a pH of 8.0-10.0, and an appearance of colorless transparent to semi-transparent liquid.

[0047] Polyvinyl alcohol: CAS No. 9002-89-5, average degree of polymerization approximately 1700, degree of alcoholysis 87%-89%.

[0048] Hydroxypropyl methylcellulose: CAS No. 9004-65-3, methoxy content 19%-24%, hydroxypropyl content 4%-12%.

[0049] All raw materials used in the embodiments of this invention are commercially available products.

[0050] Example 1

[0051] A method for preparing a bird's nest peptide that promotes mucosal and wound healing and repair as well as immune regulation is as follows:

[0052] Step 1: Ultrafine grinding of raw materials: Select dried bird's nest as raw material, place it in an ultrafine airflow pulverizer, grind it under a negative pressure of -0.08MPa and pass it through a 300-mesh sieve to obtain bird's nest ultrafine powder with a particle size D90 of less than 50μm;

[0053] Step 2, Soaking and Vibrating to Loosen: Mix the bird's nest ultrafine powder obtained in Step 1 with deionized water at a mass ratio of 1:12, place it in a constant temperature shaking water bath, and soak it for 2 hours at 30℃ and shaking frequency of 80rpm to allow the bird's nest ultrafine powder to fully absorb water and expand to 3 times its original volume, thus obtaining a bird's nest suspension.

[0054] Step 3, High-temperature and high-pressure heating and hydrolysis extraction: The bird's nest suspension obtained in Step 2 is transferred into a sealed stainless steel reactor equipped with a mechanical stirrer. The staged heating and hydrolysis extraction is carried out under the condition of stirring speed of 150 rpm. First, the temperature is raised to 60℃ and kept at 30 min to allow the bird's nest glycoprotein to fully swell. Then, the temperature is raised to 121℃ at a rate of 2℃ / min and the pressure inside the reactor is 0.12 MPa. The hydrolysis is kept at this temperature for 180 min. After the hydrolysate naturally cools down to below 80℃, the exhaust valve is slowly opened to release the pressure to atmospheric pressure to obtain the bird's nest glycoprotein peptide hydrolysate extract.

[0055] Step 4, Multi-layer precision filtration: After the bird's nest glycoprotein peptide hydrolysate obtained in Step 3 is naturally cooled to room temperature, it is then subjected to three-stage precision filtration through a 100-mesh stainless steel sieve, a 200-mesh silk cloth filter, and a 0.45μm microfiltration membrane, and the clarified coarse filtrate is collected.

[0056] Step 5, Ceramic ultrafiltration membrane purification: The clarified coarse filtrate obtained in step 4 is purified by cross-flow ultrafiltration through a ceramic ultrafiltration membrane at 40℃, transmembrane pressure of 0.20MPa and membrane surface flow rate of 2m / s. The permeate is collected to obtain the purified bird's nest glycoprotein peptide solution.

[0057] Step 6, Low-temperature vacuum concentration and spray drying: The purified bird's nest glycoprotein peptide solution obtained in step 5 is concentrated under vacuum pressure, with the material temperature controlled at ≤60℃. After the solid content is 20%, it is spray dried by spray drying machine, with the inlet air temperature controlled at 170℃ and the outlet air temperature controlled at 75℃. The resulting dried powder is aseptically packaged to obtain the bird's nest peptide for mucosal and wound healing repair and immune regulation.

[0058] The ceramic ultrafiltration membrane is prepared as follows:

[0059] S1. Zirconium oxychloride and yttrium nitrate hexahydrate were dissolved in deionized water at a zirconium-yttrium molar ratio of 92:8 to prepare a mixed solution with a total metal ion concentration of 0.5 mol / L. Polyethylene glycol was added as a dispersant at 1.0% of the total mass of the theoretically generated metal oxides. The pH was adjusted to 10 by adding 1 mol / L sodium hydroxide solution dropwise under stirring at 80°C and reacted for 4 hours. After cooling, the resulting gel was repeatedly washed with deionized water until the conductivity of the filtrate was ≤10 μS / cm. It was then dried at 60°C, redispersed in deionized water, and the pH was adjusted to 4 by 1 mol / L dilute nitric acid to obtain a sol.

[0060] S2. First, the dopant is dissolved in deionized water to prepare an aqueous solution with a concentration of 0.5 mol / L. At room temperature and a stirring speed of 300 rpm, the dopant aqueous solution is added dropwise to the sol obtained in S1 at a rate of 2 drops per second. After the addition is complete, stirring continues for 2 hours, followed by ultrasonic dispersion for 30 minutes to obtain a composite sol. The amount of dopant added is 1% of the theoretical solid content of the metal oxide in the sol. Subsequently, silica sol (5% of the theoretical solid content of the metal oxide), polyvinyl alcohol (2% of the theoretical solid content), and hydroxypropyl methylcellulose (0.5% of the hydroxypropyl methylcellulose) are added to the composite sol. After vacuum degassing, a coating solution is obtained. The solution is then purified by deionized water... After cleaning and high-temperature sterilization, the porous alumina ceramic support is immersed in the coating solution for 30 seconds, then vertically pulled at a speed of 5 cm / min and dried at 80℃ for 2 hours. The above immersion, pulling, and drying operations are repeated 3 times to control the membrane thickness at 7 μm. Then, the temperature is increased to 300℃ at a rate of 1℃ / min and held for 60 minutes. The temperature is then increased to 500℃ at a rate of 1℃ / min and held for 60 minutes. Finally, the temperature is increased to 850℃ at a rate of 1℃ / min and calcined for 3 hours. After naturally cooling to room temperature, the membrane module is boiled in deionized water for 2 hours and sterilized by high-temperature steam at 121℃ for 30 minutes to obtain the ceramic ultrafiltration membrane.

[0061] The dopant is titanium oxysulfate tetrahydrate.

[0062] Example 2

[0063] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that the dopant in the preparation method of the ceramic ultrafiltration membrane is aluminum nitrate nonahydrate.

[0064] Example 3

[0065] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that the dopant in the preparation method of the ceramic ultrafiltration membrane is magnesium nitrate hexahydrate.

[0066] Example 4

[0067] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that the dopant in the preparation method of the ceramic ultrafiltration membrane is cerium nitrate hexahydrate.

[0068] Example 5

[0069] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that the amount of dopant added in the preparation method of the ceramic ultrafiltration membrane is 2% of the theoretical solid content of metal oxide in the sol.

[0070] Example 6

[0071] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that the amount of dopant added in the preparation method of the ceramic ultrafiltration membrane is 3% of the theoretical solid content of metal oxide in the sol.

[0072] Comparative Example 1

[0073] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that silica sol is not added in the preparation method of the ceramic ultrafiltration membrane.

[0074] Comparative Example 2

[0075] The preparation method of a bird's nest peptide for mucosal and wound healing repair and immune regulation is basically the same as that in Example 1, except that no dopant is added in the preparation method of the ceramic ultrafiltration membrane.

[0076] Test Example 1

[0077] Anti-fouling flux stability test:

[0078] Test method: The ceramic ultrafiltration membranes prepared in each embodiment and comparative example were cut into pieces with an effective filtration area of ​​50 cm². 2 The membrane was installed in a cross-flow filtration device; the clarified coarse filtrate of bird's nest glycoprotein peptides obtained in step 4 was used as the test solution, and the device was continuously cross-flowed for 120 minutes under constant conditions of solution temperature 40℃, transmembrane pressure 0.20MPa, and membrane surface flow velocity 2m / s; the flux attenuation rate was calculated according to the formula: Flux attenuation rate (%) = (J0 - J 120 ) / J0×100%, where J0 is the initial flux (L·m -2 ·h -1 ), J 120 The flux at the end of 120 minutes of operation (L·m) -2 ·h -1 The test results are shown in Table 1.

[0079] Table 1

[0080] Example 1 17.2 Example 2 20.8 Example 3 22.6 Example 4 21.5 Example 5 10.8 Example 6 13.5 Comparative Example 1 31.6 Comparative Example 2 25.3

[0081] Test Example 2

[0082] Recovery rate and membrane separation accuracy testing:

[0083] Test method: Molecular weight distribution was determined by high performance gel size exclusion chromatography (HPLC). The permeate from each example and comparative example was taken after 60 min of running in step 5, with the feed solution from step 5 used as a control. The chromatographic column used was a TSKgel G3000SWXL (7.8 mm × 300 mm, size exclusion limit 50000 Da), and the mobile phase was 0.1 mol / L phosphate buffer (pH 10.5). The standard parameters were: flow rate 0.5 mL / min, detection wavelength 280 nm, injection volume 20 μL, and column temperature 25 °C. Small molecule peptide recovery was calculated using 10000 Da as the cutoff: Small molecule peptide recovery (%) = Peak area of ​​molecules <10000 Da in the permeate / Peak area of ​​molecules <10000 Da in the feed solution × 100%. Large molecule rejection was calculated: Large molecule rejection (%) = (Peak area of ​​molecules >10000 Da in the feed solution - Peak area of ​​molecules >10000 Da in the permeate) / Peak area of ​​molecules >10000 Da in the feed solution × 100%. Relevant test data are summarized in Table 2.

[0084] Table 2

[0085] Example 1 81.5 96.2 Example 2 77.2 95.6 Example 3 74.8 95.1 Example 4 76.1 95.4 Example 5 87.6 96.8 Example 6 84.3 96.5 Comparative Example 1 67.5 93.2 Comparative Example 2 71.4 94.5

[0086] Test Example 3

[0087] Mucosal repair efficacy test:

[0088] Human gastric mucosal epithelial cells were seeded into 6-well plates and cultured until monolayer confluence. After vertical scratching with a sterile pipette tip, detached cells were washed away. Serum-free culture medium containing 400 μg / mL of the bird's nest peptide obtained in each example and comparative example was added, while the control group was added only to serum-free culture medium. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. Images were taken at the same location under an inverted microscope at 0 h and 24 h, and the scratch area was measured using image analysis software. The scratch healing rate was calculated using the formula: Scratch healing rate (%) = (A... 0h -A 24h ) / A 0h ×100%;

[0089] In the formula, A 0h A is the scratch area at 0h. 24h The area of ​​the scratch after 24 hours.

[0090] The test results are shown in Table 3.

[0091] Table 3

[0092] Example 1 78.6 Example 2 72.4 Example 3 69.5 Example 4 70.8 Example 5 85.3 Example 6 82.1 Comparative Example 1 58.7 Comparative Example 2 62.3

[0093] The results of Test Example 3 show that the bird's nest peptides obtained in each example and comparative example have a significant promoting effect on the healing of scratches on human gastric mucosal epithelial cells, and the scratch healing rate increases with the enrichment degree of small molecule bird's nest glycoprotein peptides. Among them, Example 5 has the highest scratch healing rate due to the best separation precision of the ceramic ultrafiltration membrane and the most sufficient enrichment of small molecule active peptides. Comparative Example 1 has the lowest scratch healing rate due to insufficient membrane separation precision and more residual large molecule impurities caused by the lack of silica sol synergistic modification. This confirms that the present invention can significantly enhance the mucosal repair efficacy of bird's nest peptides by optimizing the ceramic ultrafiltration membrane preparation process to improve the purity of small molecule bird's nest glycoprotein peptides.

[0094] Comparative Example 1, without the addition of silica sol, lacked a silica-oxygen hydrophilic network on the membrane surface, failing to form a stable hydration barrier. Glycoprotein peptides from bird's nest easily adsorbed and deposited on the hydrophobic membrane surface, gradually forming a fouling layer, leading to rapid decay of membrane flux. Comparative Example 2, without the addition of dopants, although the membrane contained silica components introduced by the silica sol, lacked heterogeneous metal oxides to synergize with it, failing to form a dense covalently cross-linked hydrophilic network. The stability and antifouling durability of the surface hydration layer were insufficient. Example 1 simultaneously introduced silica sol and titanium oxysulfate tetrahydrate. After calcination, titanium dioxide and silicon dioxide formed a three-dimensional inorganic hydrophilic network through covalent cross-linking, significantly enhancing the stability of the membrane hydration layer and the charge repulsion effect, thereby effectively inhibiting glycoprotein adsorption and deposition.

[0095] In Examples 1 to 4, Example 1 used titanium oxysulfate tetrahydrate as a dopant. The titanium dioxide generated by its calcination is covalently cross-linked with silicon dioxide, constructing the densest hydrophilic network. Furthermore, the titanium dioxide nanoparticles effectively refined the zirconia grains and optimized the pore tortuosity. Example 2 used aluminum nitrate nonahydrate. The covalently cross-linked network formed by aluminum oxide and silicon dioxide has weak hydration capacity, and aluminum oxide has a high isoelectric point, resulting in a positively charged surface in neutral solutions. This leads to electrostatic attraction with negatively charged glycoprotein peptides, which is detrimental to antifouling. Example 3 used magnesium nitrate hexahydrate. While magnesium oxide can improve hydrophilicity, it tends to produce trace amounts of inorganic deposition on the film surface. Example 4 used cerium nitrate hexahydrate. The synergistic hydrophilic effect of cerium oxide and silicon dioxide, as well as its effect on refining zirconia grains, are not as good as those of titanium dioxide. Therefore, Example 1 is more effective.

[0096] In Example 5, the dopant content was the optimal value. At this point, the ratio of titanium dioxide generated from the calcination of tetrahydrated titanium oxysulfate to silicon dioxide generated from silica sol was appropriate, resulting in the most complete covalently cross-linked hydrophilic network coverage. The refinement of zirconia grains and the uniformity of pore size distribution were optimal, and the synergistic effect of anti-fouling performance and separation accuracy was best achieved. In Example 1, the dopant content was too low, resulting in insufficient titanium dioxide and incomplete covalently cross-linked network coverage, thus failing to fully realize the synergistic strengthening effect. In Example 6, the dopant content was too high, leading to excessive accumulation of titanium dioxide nanoparticles in the film layer, increasing surface roughness and causing uneven pore size distribution, which in turn weakened the density of the hydrated layer and the separation accuracy.

Claims

1. A method for preparing bird's nest peptides for mucosal and wound healing repair and immune regulation, characterized in that, The method is as follows: Step 1, Raw material ultrafine grinding: Select dried bird's nest as raw material, grind it to obtain bird's nest ultrafine powder; Step 2, Soaking and Vibrating to Loosen: Mix the bird's nest ultrafine powder obtained in Step 1 with deionized water, soak and vibrate to loosen, and obtain bird's nest suspension; Step 3, High-temperature and high-pressure hydrolysis extraction: The bird's nest suspension obtained in step 2 is subjected to high-temperature and high-pressure hydrolysis extraction to obtain bird's nest glycoprotein peptide hydrolysate. Step 4, Multi-stage precision filtration: After the bird's nest glycoprotein peptide hydrolysate obtained in Step 3 is naturally cooled to room temperature, it is passed through three-stage precision filtration in sequence, and the clear coarse filtrate is collected. Step 5, Ceramic ultrafiltration membrane purification: The clarified coarse filtrate obtained in step 4 is purified by cross-flow ultrafiltration through a ceramic ultrafiltration membrane, and the permeate is collected to obtain the purified bird's nest glycoprotein peptide solution. Step 6, Low-temperature vacuum concentration and spray drying: The purified bird's nest glycoprotein peptide solution obtained in step 5 is concentrated under vacuum and then spray dried. The resulting dried powder is aseptically packaged to obtain the bird's nest peptide for mucosal and wound healing repair and immune regulation.

2. The preparation method according to claim 1, characterized in that, The pulverization process involves pulverizing under a negative pressure of -0.07 to -0.09 MPa and passing the powder through a 200-400 mesh sieve to obtain bird's nest ultrafine powder with a particle size D90 of less than 50 μm. The soaking and vibration loosening process involves mixing the bird's nest ultrafine powder obtained in step 1 with deionized water at a mass ratio of 1:10-15, placing it in a constant temperature shaking water bath, and soaking it for 1-3 hours at 20-40℃ and a shaking frequency of 50-100rpm, so that the bird's nest ultrafine powder can fully absorb water and expand to 2-4 times its original volume.

3. The preparation method according to claim 1, characterized in that, The high-temperature and high-pressure heating hydrolysis extraction is carried out under the condition of stirring speed of 100-200 rpm, first heating to 50-70℃ and holding for 10-50 min to allow the bird's nest glycoprotein to fully swell, then heating to 110-130℃ at a rate of 1-3℃ / min and pressure inside the vessel of 0.1-0.15MPa, and holding for hydrolysis for 100-300 min. After the hydrolysate naturally cools down to below 80℃, the exhaust valve is slowly opened to release pressure to atmospheric pressure.

4. The preparation method according to claim 1, characterized in that, The three-stage precision filtration involves sequentially passing the filter through a 50-200 mesh stainless steel screen, a 100-300 mesh silk cloth filter, and a 0.3-0.6 μm microfiltration membrane. The cross-flow ultrafiltration purification is performed through a ceramic ultrafiltration membrane at 30-50℃, transmembrane pressure of 0.15-0.25MPa, and membrane surface flow rate of 1-3m / s.

5. The preparation method according to claim 1, characterized in that, The vacuum concentration process involves controlling the material temperature to ≤60℃ until the solids content reaches 15-30%. The powder spraying dryer controls the inlet air temperature to be 150-180℃ and the outlet air temperature to be 60-90℃.

6. The preparation method according to claim 1, characterized in that, The ceramic ultrafiltration membrane is prepared as follows: S1. Dissolve zirconium oxychloride and yttrium nitrate hexahydrate in deionized water at a zirconium-yttrium molar ratio of 90-95:5-10 to prepare a mixed solution with a total metal ion concentration of 0.3-0.8 mol / L. Add polyethylene glycol as a dispersant at 0.8-1.2% of the theoretically generated total mass of metal oxides. Under stirring at 70-90℃, add 0.5-2 mol / L sodium hydroxide solution dropwise to adjust the pH to 9-11 and react for 2-6 hours. After cooling, wash the resulting gel repeatedly with deionized water until the conductivity of the filtrate is ≤10 μS / cm. Then dry at 50-70℃, redisperse in deionized water, and adjust the pH to 3-5 with 0.5-2 mol / L dilute nitric acid to obtain a sol. S2. First, the dopant is dissolved in deionized water to prepare an aqueous solution with a concentration of 0.3-0.8 mol / L. Under the conditions of room temperature and stirring speed of 200-400 rpm, the above-mentioned dopant aqueous solution is added dropwise to the sol obtained in S1 at a rate of 1-3 drops per second. After the addition is completed, stirring is continued for 1-3 hours, followed by ultrasonic dispersion for 20-40 minutes to obtain a composite sol. The amount of dopant added is 1-3% of the theoretical solid content of metal oxide in the sol. Subsequently, silica sol accounting for 3-6% of the theoretical solid content of metal oxide, polyvinyl alcohol 1-3% and hydroxypropyl methylcellulose 0.3-0.8% are added to the composite sol. After vacuum degassing, a coating solution is obtained. The porous alumina ceramic, which has been cleaned with deionized water and sterilized at high temperature, is then coated with the coating solution. After immersing the support in the coating solution for 10-50 seconds, it is vertically pulled at a speed of 3-8 cm / min and dried at 50-90℃ for 1-3 hours. The above immersion, pulling and drying operations are repeated 1-3 times to control the membrane thickness at 5-8 μm. Then, the temperature is increased to 280-320℃ at a rate of 0.5-2℃ / min and held for 40-80 minutes. The temperature is then increased to 480-520℃ at a rate of 0.5-2℃ / min and held for 40-80 minutes. Finally, the temperature is increased to 800-900℃ at a rate of 0.5-2℃ / min and calcined for 1-5 hours. After naturally cooling to room temperature, the membrane module is boiled in deionized water for 1-3 hours and sterilized by high-temperature steam at 110-130℃ for 10-50 minutes to obtain the ceramic ultrafiltration membrane. The dopant is at least one of aluminum nitrate nonahydrate, titanium sulfate tetrahydrate, magnesium nitrate hexahydrate, and cerium nitrate hexahydrate.

7. The preparation method according to claim 6, characterized in that, The dopant is titanium oxysulfate tetrahydrate.

8. The preparation method according to claim 7, characterized in that, The amount of dopant added is 1.8-2.2% of the theoretical solid content of the metal oxide in the sol.

9. A bird's nest peptide for mucosal and wound healing repair and immune regulation, characterized in that, It is prepared by the preparation method described in any one of claims 1-8.

10. The use of the bird's nest peptide as described in claim 9 in the preparation of food, health products, pharmaceuticals or cosmetics with mucosal repair, wound healing and immune regulation effects.