Cordyceps sinensis-derived small molecular glycopeptide composition, preparation method and application thereof

Abstract

This invention relates to the field of glycopeptide composition technology, specifically to a small molecule glycopeptide composition derived from bird's nest, its preparation method, and its applications. A method for preparing a small molecule glycopeptide composition derived from bird's nest includes: cleaning, soaking, and extracting the bird's nest raw material; enzymatic hydrolysis; enrichment and impurity removal; and ultrafiltration purification. In the purification stage, this invention uses a modified covalent organic framework as an adsorbent. Boric acid groups can target and capture glycopeptides, eliminating impurities without cis-diol structures, solving the problems of poor selectivity and high loss rate in traditional methods, and reducing the load on subsequent steps. Chitosan modification prevents framework aggregation, and its polar groups synergistically enhance adsorption capacity and specificity with the framework groups, improving product purity. Proline-modified chitosan can break intermolecular hydrogen bonds, ensuring uniform coating. Through multiple effects, it strengthens adsorption specificity and capacity, effectively enriching glycopeptides and reducing impurities, laying the foundation for high product yield, high purity, and effective biological function.

Description

Technical Field

[0001] This invention relates to the field of glycopeptide composition technology, specifically to a small molecule glycopeptide composition derived from bird's nest, its preparation method, and its application. Background Technology

[0002] Bird's nest, a traditional and precious tonic, primarily contains glycopeptides as its main active ingredients. These components, composed of amino acid residues forming a peptide backbone combined with a sugar chain structure, possess various biological functions such as antioxidation, immune regulation, and mucosal repair, showing broad application prospects in health products, cosmetics, and biomedicine. With the increasing market demand for deep-processed bird's nest products, preparing small-molecule glycopeptide compositions from bird's nest through enzymatic hydrolysis can significantly improve its bioavailability and meet the application needs of different scenarios. Therefore, the preparation technology of small-molecule glycopeptide compositions from bird's nest has become a research hotspot.

[0003] Currently, the preparation of small molecule glycopeptide compositions derived from bird's nest is mostly based on bird's nest raw materials, and is obtained through steps such as pretreatment, enzymatic hydrolysis, purification, and separation. Among them, the enzymatic hydrolysis step can degrade the large molecule proteins and glycoproteins in bird's nest into small molecule glycopeptides. However, the composition of the enzymatic hydrolysate is extremely complex. In addition to the target small molecule glycopeptides, it also contains a large number of free amino acids, sugar-free peptides, impurities, enzymatic hydrolysis residues, and trace impurities. The presence of these impurities not only interferes with subsequent separation and purification operations, but also reduces the purity and bioactivity of the final product, affecting the yield of glycopeptides. Therefore, the purification and enrichment step is the core link that determines the quality of the product.

[0004] Existing technologies for purifying and enriching glycopeptides in bird's nest enzymatic hydrolysates mainly include ultrafiltration, gel chromatography, ion exchange chromatography, and traditional adsorption methods. However, these methods have many technical drawbacks in practical applications, making it difficult to simultaneously achieve high enrichment efficiency, selectivity, and yield of glycopeptides. Ultrafiltration and gel chromatography rely on molecular weight differences for separation, exhibiting poor selective recognition of glycopeptides and impurities, easily leading to co-separation of glycopeptides and impurities, resulting in high glycopeptide loss rates. Furthermore, they have high load and long separation cycles when directly processing complex enzymatic hydrolysates, making it difficult to achieve efficient concentration of glycopeptides. Ion exchange chromatography is significantly affected by the pH value of the system and has limited specific adsorption capacity for glycopeptides, resulting in low product yields.

[0005] Therefore, this application develops a bird's nest-derived small molecule glycopeptide composition, its preparation method, and its application, which can solve the above-mentioned technical pain points, achieve efficient and highly selective enrichment of glycopeptides, and thus effectively improve product yield. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a small molecule glycopeptide composition derived from bird's nest, its preparation method, and its application.

[0007] This invention provides a method for preparing a small molecule glycopeptide composition derived from bird's nest, comprising: S1: The bird's nest raw material is pretreated by washing and soaking in water, then extracted and centrifuged to obtain a crude extract solution of bird's nest glycoprotein; S2: The crude extract of bird's nest glycoprotein was first hydrolyzed with pepsin at a pH of 2.0-3.0, an enzyme-to-protein ratio of 1:90-110, a hydrolysis temperature of 35-40℃, and a hydrolysis time of 1-3 hours. Then, it was hydrolyzed with trypsin at a pH of 7.0-8.0, an enzyme-to-protein ratio of 1:90-110, a hydrolysis temperature of 45-55℃, and a hydrolysis time of 3-6 hours. Finally, the enzyme was inactivated and centrifuged to obtain the hydrolysate. S3: The enzymatic hydrolysate is first enriched and impurities removed by a modified covalent organic framework to obtain a pretreated enzymatic hydrolysate; S4: The pretreated enzymatic hydrolysate is passed through an ultrafiltration membrane system with a molecular weight cutoff of 1000-3000 Da, and then purified by gel chromatography. The purified target component solution is concentrated and freeze-dried to obtain a small molecule glycopeptide composition from bird's nest. The preparation of the above-mentioned modified covalent organic framework includes: adding 20-30 parts by weight of 2,4,6-tricarboxypyrogallol, 60-70 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 10-15 parts by weight of 4-aminophenylboronic acid to anhydrous ethanol for reaction to obtain a covalent organic framework; then modifying the covalent organic framework with modified chitosan to obtain a modified covalent organic framework. The preparation of the modified chitosan includes: first, dissolving sodium hydroxide and proline in water and stirring, adding tert-butanol and stirring at high speed, and then adding di-tert-butyl dicarbonate dropwise under controlled temperature to obtain a mixed solution; after extraction, pH adjustment, washing and drying, pretreated amino acids are obtained; then, a swollen chitosan mixture and an amino acid mixture are prepared, and the two are mixed and reacted, followed by washing, system reaction, dialysis and freeze drying to obtain modified chitosan.

[0008] As one preferred aspect, the specific steps include: S1: After washing the bird's nest raw material with deionized water, soak it in 10-15 times its volume of deionized water for 2-4 hours. Then, stir and extract it at 50-60℃ for 1-2 hours. Centrifuge at 4000-8000rpm for 10-20 minutes, take the supernatant, and then concentrate it under reduced pressure to obtain a crude extract solution of bird's nest glycoprotein. S2: Adjust the pH of the crude extract of bird's nest glycoprotein to 2.0-3.0 with 1M HCl, then add pepsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90-110, and react at 35-40℃ for 1-3 hours. Then adjust the pH to 7.0-8.0, add trypsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90-110, and continue to react at 45-55℃ for 3-6 hours. After the enzymatic hydrolysis is completed, heat in a water bath at 85-100℃ for 5-15 minutes, then centrifuge to obtain a clear enzymatic hydrolysate. S3: Mix the enzymatic hydrolysate and the loading solution at a volume ratio of 1:9 to obtain a mixed solution. Then, add 50-60 mg / mL of modified covalent organic framework to the mixed solution and react in a constant temperature shaker at 37℃ and 300-400 rpm for 60-70 min. After the reaction is complete, centrifuge at 10000-12000 rpm for 5-10 min. Then, wash the precipitate with the elution buffer at 37℃ for 20-30 min and centrifuge at 10000-12000 rpm for 5-10 min to obtain the pretreated enzymatic hydrolysate. S4: The pretreated enzymatic hydrolysate is passed through an ultrafiltration membrane system with a molecular weight cutoff of 1000-3000 Da and ultrafiltration is performed under a nitrogen pressure of 0.3-0.5 MPa to obtain an ultrafiltration permeate. The ultrafiltration permeate is purified by gel chromatography, and the purified target component solution is concentrated and freeze-dried to obtain a bird's nest-derived small molecule glycopeptide composition.

[0009] As a preferred aspect, the preparation of the modified covalent organic framework in step S3 specifically includes the following steps: A1: Add 20-30 parts by weight of 2,4,6-tricarboxypyrogallol, 60-70 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 10-15 parts by weight of 4-aminophenylboronic acid to 200-300 parts by weight of anhydrous ethanol, and then react at 80-90℃ and 300-500 rpm for 4-5 h. After the reaction is completed, cool to room temperature, then centrifuge at 10000-12000 rpm for 10-20 min. Then wash the precipitate 3-5 times by centrifugation with N,N-dimethylformamide and anhydrous ethanol, and finally vacuum dry to obtain a covalent organic framework. A2: Add 5-8 parts by weight of modified chitosan to 30-40 parts by weight of deionized water, and add 1-2 parts by weight of glacial acetic acid. Stir and mix, then add 100-120 parts by weight of deionized water and mix evenly to obtain a modified chitosan solution. Disperse 3-5 parts by weight of covalent organic framework in 100 parts by weight of deionized water to obtain a covalent organic framework dispersion. A3: Mix the covalent organic framework dispersion and the modified chitosan solution at a volume ratio of 1:2-3, stir and react for 2-3 hours, filter after the reaction is complete, and freeze dry to obtain the modified covalent organic framework.

[0010] As a preferred aspect, the preparation of the above-mentioned modified chitosan specifically includes the following steps: a1: Add 2-3 parts by weight of sodium hydroxide to 40-50 parts by weight of deionized water, then add 8-10 parts by weight of proline, stir and mix at 200-300 rpm for 20-30 min, then add 40-50 parts by weight of tert-butanol, stir at 500-800 rpm for 15-20 min, then add 10-12 parts by weight of ditert-butyl dicarbonate dropwise at 25-28℃ for 20-30 min, then react for 50-60 min, then raise the temperature to 33-35℃ and react for 20-24 h to obtain the reaction mixture; a2: Extract the reaction mixture with petroleum ether 2-3 times, combine the aqueous phases, then adjust the pH to 1-1.2 with dilute sulfuric acid in an ice-water bath, then extract with diethyl ether 3-5 times, combine the diethyl ether layers, then wash the diethyl ether layer with deionized water and dry with anhydrous sodium sulfate for 10-12 hours, filter, concentrate, and obtain the pretreated amino acids. a3: Add 5-8 parts by weight of chitosan to 25-30 parts by weight of DMF, stir and mix for 2-3 hours to obtain a swollen chitosan mixture. Add 8-10 parts by weight of pretreated amino acids to 25-30 parts by weight of DMF, then add 0.01-0.03 parts by weight of 4-dimethylaminopyridine. Then, under ice bath conditions, add 10-12 parts by weight of N,N-dicyclohexylcarbodiimide, stir and mix to obtain a mixed solution. a4: Mix the mixed solution and the swollen chitosan mixture, then react in an ice bath for 2-3 hours, and then react at 25-28℃ for 20-24 hours. After filtration, wash with anhydrous ethanol 3-5 times. Place the washed product in a 25wt% trifluoroacetic acid DCM system and react for 2-3 hours. After the reaction is complete, wash with ethanol, then dialyze with deionized water, and freeze-dry to obtain modified chitosan.

[0011] As a preferred aspect, the loading solution in step S3 consists of an acetonitrile:deionized water:trifluoroacetic acid volume ratio of 85:14.9:0.1.

[0012] As a preferred aspect, the eluent in step S3 consists of an acetonitrile:deionized water:trifluoroacetic acid volume ratio of 35:64.5:0.5.

[0013] As a preferred aspect, in step S4, an ultrafiltration membrane system with a molecular weight cutoff of 2000 Da is preferred.

[0014] As a preferred aspect, the gel chromatography in step S4 uses a Sephadex G-15 column.

[0015] The present invention also provides a bird's nest-derived small molecule glycopeptide composition, which is prepared by any of the methods described in the present invention for preparing a bird's nest-derived small molecule glycopeptide composition.

[0016] The above-described bird's nest-derived small molecule glycopeptide composition is used in the preparation of drugs, health foods, and food additives for promoting nerve cell development, improving memory, treating insomnia, and delaying neurodegenerative diseases.

[0017] The present invention has the following advantages: 1. This invention introduces a modified covalent organic framework as an adsorbent during the purification stage. The covalent organic framework is prepared through the polycondensation reaction of 2,4,6-tricarboxypyrogallol, 2,5-diamino-1,4-benzenedisulfonic acid, and 4-aminophenylboronic acid, introducing borate functional groups. The borate groups can specifically react with the cis-diol groups on the glycopeptide sugar chains to form reversible five- or six-membered cyclic borate ester bonds, achieving targeted capture of glycopeptides. Impurities without cis-diol structures, such as free amino acids and peptides without sugar chains, are excluded. This specific recognition mechanism solves the pain points of traditional purification methods, namely "poor selectivity and high glycopeptide loss rate," effectively improving the enrichment efficiency and selectivity of glycopeptides. This greatly reduces the load on subsequent ultrafiltration and gel chromatography steps, making the separation target more specific. The widely dispersed glycopeptide components are pre-concentrated on the covalent organic framework material, and after elution, a "pretreated enzymatic hydrolysate" is obtained, in which the relative concentration of the target glycopeptide is significantly increased, laying the foundation for obtaining high-yield products downstream.

[0018] 2. The covalent organic framework in this invention is modified with chitosan. Chitosan's molecular chains can coat the surface of the covalent organic framework through hydrogen bonds and van der Waals forces, creating steric hindrance between the covalent organic framework particles. This effectively prevents the covalent organic framework from agglomerating, ensuring its uniform dispersion in the reaction system. This modification ensures the effective utilization of the functional groups of the covalent organic framework, guaranteeing efficient adsorption of glycopeptides in the enzymatic hydrolysate, thus guaranteeing a high yield of the final composition. Furthermore, the chitosan molecular chains contain polar groups such as amino groups, which, after coating the surface of the covalent organic framework, can form multiple interactions with glycopeptides, including hydrogen bonds and ionic bonds. These interactions, along with the sulfonic acid and boric acid groups of the covalent organic framework itself, further enhance the adsorption capacity and specificity for glycopeptides. The modified covalent organic framework exhibits significantly improved adsorption selectivity for glycopeptides, further removing trace amounts of impurities in the enzymatic hydrolysate, improving the purity of the pretreated enzymatic hydrolysate, and ultimately obtaining a higher purity small molecule glycopeptide composition.

[0019] 3. In this invention, proline-modified chitosan is used. Proline, as a natural amino acid, can be covalently grafted onto the chitosan molecular chain via pyrrolidine groups and carboxyl groups, disrupting the hydrogen bonds between chitosan molecules and significantly improving the dispersibility of chitosan. This modification ensures the uniform coating of the covalent organic framework by chitosan, providing a basis for the efficient adsorption performance of the modified covalent organic framework. Furthermore, after proline is grafted onto the chitosan molecular chain, its carboxyl and imino groups can form multiple hydrogen and ionic bonds with the amino and hydroxyl groups of the bird's nest glycopeptide, enhancing the interaction between the modified chitosan and the glycopeptide. At the same time, the pyrrolidine group of proline is a hydrophobic group, which can adsorb the hydrophobic amino acid residues of the glycopeptide through hydrophobic interactions, further improving the adsorption specificity and capacity of the modified covalent organic framework for the glycopeptide. This synergistic effect can effectively enrich the glycopeptide in the enzymatic hydrolysate, reduce impurity interference, and ultimately improve the purity of the small molecule glycopeptide composition, thus providing a material basis for its biological function. Attached Figure Description

[0020] Figure 1 This is a process flow diagram of the preparation method of the bird's nest-derived small molecule glycopeptide composition used in the embodiments of the present invention. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this invention.

[0022] Example 1: A method for preparing a small molecule glycopeptide composition derived from bird's nest, referring to... Figure 1 ,include: S1: After washing the bird's nest raw material with deionized water, soak it in 10 times its volume of deionized water for 2 hours, then stir and extract it at 50-60℃ for 1 hour, centrifuge at 4000rpm for 10 minutes, take the supernatant, and then concentrate it under reduced pressure to obtain a crude extract solution of bird's nest glycoprotein. S2: Adjust the pH of the crude extract of bird's nest glycoprotein to 2.0 with 1M HCl, then add pepsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90, and react at 35℃ for 1 h. Then adjust the pH to 7.0, add trypsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90, and continue to react at 45℃ for 3 h. After the enzymatic hydrolysis is completed, heat in an 85℃ water bath for 5 min, then centrifuge to obtain a clear enzymatic hydrolysate. S3: Mix the enzymatic hydrolysate and the loading solution at a volume ratio of 1:9. The loading solution consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 85:14.9:0.1 to obtain a mixed solution. Then, add 50 mg / mL of modified covalent organic framework to the mixed solution and react in a constant temperature shaker at 37℃ and 300 rpm for 60 min. After the reaction is complete, centrifuge at 10000 rpm for 5 min. Then, wash the precipitate with the eluent, which consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 35:64.5:0.5. Wash at 37℃ for 20 min and then centrifuge at 10000 rpm for 5 min to obtain the pretreated enzymatic hydrolysate. S4: The pretreated enzymatic hydrolysate was ultrafiltered through an ultrafiltration membrane system with a molecular weight cutoff of 2000 Da under a nitrogen pressure of 0.3 MPa to obtain an ultrafiltration permeate. The ultrafiltration permeate was purified by Sephadex G-15 gel chromatography. The purified target component solution was concentrated and freeze-dried to obtain a small molecule glycopeptide composition derived from bird's nest.

[0023] The preparation of the above-mentioned modified covalent organic framework includes: A1: 20 parts by weight of 2,4,6-tricarboxypyrogallol, 60 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 10 parts by weight of 4-aminophenylboronic acid were added to 200 parts by weight of anhydrous ethanol. The mixture was then reacted at 80°C and 300 rpm for 4 h. After the reaction was completed, the mixture was cooled to room temperature and then centrifuged at 10,000 rpm for 10 min. The precipitate was then washed three times by centrifugation with N,N-dimethylformamide and anhydrous ethanol, and finally dried under vacuum to obtain a covalent organic framework. A2: Add 5 parts by weight of modified chitosan to 30 parts by weight of deionized water and 1 part by weight of glacial acetic acid, stir and mix, then add 100 parts by weight of deionized water and mix evenly to obtain a modified chitosan solution. Disperse 3 parts by weight of covalent organic framework in 100 parts by weight of deionized water to obtain a covalent organic framework dispersion. A3: The covalent organic framework dispersion and the modified chitosan solution were mixed at a volume ratio of 1:2, and then stirred for 2 hours. After the reaction was completed, the mixture was filtered and freeze-dried to obtain the modified covalent organic framework.

[0024] The preparation of the above-mentioned modified chitosan includes: a1: Add 2 parts by weight of sodium hydroxide to 40 parts by weight of deionized water, then add 8 parts by weight of proline, stir and mix at 200 rpm for 20 min, then add 40 parts by weight of tert-butanol, stir at 500 rpm for 15 min, then add 10 parts by weight of ditert-butyl dicarbonate dropwise at 25 °C for 20 min, then react for 50 min, then raise the temperature to 33 °C and react for 20 h to obtain the reaction mixture; a2: Extract the reaction mixture twice with petroleum ether, combine the aqueous phases, then adjust the pH to 1 with dilute sulfuric acid in an ice-water bath, then extract three times with diethyl ether, combine the diethyl ether layers, then wash the diethyl ether layer with deionized water and dry it with anhydrous sodium sulfate for 10 h, filter, concentrate, and obtain the pretreated amino acids. a3: Add 5 parts by weight of chitosan to 25 parts by weight of DMF, stir and mix for 2 hours to obtain a swollen chitosan mixture. Add 8 parts by weight of pretreated amino acids to 25 parts by weight of DMF, then add 0.01 parts by weight of 4-dimethylaminopyridine, and then add 10 parts by weight of N,N-dicyclohexylcarbodiimide under ice bath conditions. Stir and mix to obtain a mixed solution. a4: Mix the mixed solution and the swollen chitosan mixture, then react in an ice bath for 2 hours, and then at 25°C for 20 hours. After filtration, wash three times with anhydrous ethanol. Place the washed product in a DCM system of 25 wt% trifluoroacetic acid and react for 2 hours. After the reaction is complete, wash with ethanol, then dialyze with deionized water, and freeze dry to obtain modified chitosan.

[0025] Example 2, a method for preparing a small molecule glycopeptide composition derived from bird's nest, see [link to example]. Figure 1 ,include: S1: After washing the bird's nest raw material with deionized water, it was soaked in 15 times its volume of deionized water for 4 hours, then stirred and extracted at 60°C for 2 hours, centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, and then concentrated under reduced pressure to obtain a crude extract solution of bird's nest glycoprotein. S2: Adjust the pH of the crude extract of bird's nest glycoprotein to 3.0 with 1M HCl, then add pepsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:110, and react at 40℃ for 3 hours. Then adjust the pH to 8.0, add trypsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:110, and continue to react at 55℃ for 6 hours. After the enzymatic hydrolysis is completed, heat in a water bath at 100℃ for 15 minutes, and then centrifuge to obtain a clear enzymatic hydrolysate. S3: Mix the enzymatic hydrolysate and the loading solution at a volume ratio of 1:9. The loading solution consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 85:14.9:0.1 to obtain a mixed solution. Then, add 60 mg / mL of modified covalent organic framework to the mixed solution and react in a constant temperature shaker at 37℃ and 400 rpm for 70 min. After the reaction is complete, centrifuge at 12000 rpm for 10 min. Then, wash the precipitate with the eluent, which consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 35:64.5:0.5. Wash at 37℃ for 30 min and then centrifuge at 12000 rpm for 10 min to obtain the pretreated enzymatic hydrolysate. S4: The pretreated enzymatic hydrolysate was ultrafiltered through an ultrafiltration membrane system with a molecular weight cutoff of 2000 Da under a nitrogen pressure of 0.5 MPa to obtain an ultrafiltration permeate. The ultrafiltration permeate was purified by Sephadex G-15 gel chromatography. The purified target component solution was concentrated and freeze-dried to obtain a small molecule glycopeptide composition derived from bird's nest.

[0026] The preparation of the above-mentioned modified covalent organic framework includes: A1: 30 parts by weight of 2,4,6-tricarboxypyrogallol, 70 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 15 parts by weight of 4-aminophenylboronic acid were added to 300 parts by weight of anhydrous ethanol. The mixture was then reacted at 90°C and 500 rpm for 5 h. After the reaction was completed, the mixture was cooled to room temperature and then centrifuged at 12,000 rpm for 20 min. The precipitate was then washed 5 times by centrifugation with N,N-dimethylformamide and anhydrous ethanol, and finally dried under vacuum to obtain a covalent organic framework. A2: Add 8 parts by weight of modified chitosan to 40 parts by weight of deionized water, add 2 parts by weight of glacial acetic acid, stir and mix, then add 120 parts by weight of deionized water and mix evenly to obtain a modified chitosan solution. Disperse 5 parts by weight of covalent organic framework in 100 parts by weight of deionized water to obtain a covalent organic framework dispersion. A3: The covalent organic framework dispersion and the modified chitosan solution were mixed at a volume ratio of 1:3, and then stirred for 3 hours. After the reaction was completed, the mixture was filtered and freeze-dried to obtain the modified covalent organic framework.

[0027] The preparation of the above-mentioned modified chitosan includes: a1: Add 3 parts by weight of sodium hydroxide to 50 parts by weight of deionized water, then add 10 parts by weight of proline, stir and mix at 300 rpm for 30 min, then add 50 parts by weight of tert-butanol, stir at 800 rpm for 20 min, then add 12 parts by weight of ditert-butyl dicarbonate dropwise at 28℃ for 30 min, then react for 60 min, then raise the temperature to 35℃ and react for 24 h to obtain the reaction mixture; a2: The reaction mixture was extracted three times with petroleum ether, the aqueous phases were combined, and then the pH was adjusted to 1.2 with dilute sulfuric acid under an ice-water bath. After extraction with diethyl ether five times, the diethyl ether layers were combined. The diethyl ether layers were then washed with deionized water and dried with anhydrous sodium sulfate for 12 hours. After filtration and concentration, the pretreated amino acids were obtained. a3: Add 8 parts by weight of chitosan to 30 parts by weight of DMF, stir and mix for 3 hours to obtain a swollen chitosan mixture. Add 10 parts by weight of pretreated amino acids to 30 parts by weight of DMF, then add 0.03 parts by weight of 4-dimethylaminopyridine, and then add 12 parts by weight of N,N-dicyclohexylcarbodiimide under ice bath conditions. Stir and mix to obtain a mixed solution. a4: Mix the mixed solution and the swollen chitosan mixture, then react in an ice bath for 3 hours, and then at 28°C for 24 hours. After filtration, wash five times with anhydrous ethanol. Place the washed product in a DCM system of 25 wt% trifluoroacetic acid and react for 3 hours. After the reaction is complete, wash with ethanol, then dialyze with deionized water, and freeze dry to obtain modified chitosan.

[0028] Example 3, a method for preparing a small molecule glycopeptide composition derived from bird's nest, see [link to example]. Figure 1 ,include: S1: After washing the bird's nest raw material with deionized water, it was soaked in 12.5 times its volume of deionized water for 3 hours, and then stirred and extracted at 55°C for 1.5 hours. After centrifugation at 6000 rpm for 15 minutes, the supernatant was collected and then concentrated under reduced pressure to obtain a crude extract solution of bird's nest glycoprotein. S2: Adjust the pH of the crude extract of bird's nest glycoprotein to 2.5 with 1M HCl, then add pepsin (5000 U / mg activity, enzyme-to-protein ratio 1:100), react at 37.5℃ for 2 hours, then adjust the pH to 7.5, add trypsin (5000 U / mg activity, enzyme-to-protein ratio 1:100), and continue reacting at 50℃ for 4.5 hours. After enzymatic hydrolysis, heat in a 92.5℃ water bath for 10 minutes, then centrifuge to obtain a clear enzymatic hydrolysate. S3: Mix the enzymatic hydrolysate and the loading solution at a volume ratio of 1:9. The loading solution consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 85:14.9:0.1 to obtain a mixed solution. Then, add 55 mg / mL of modified covalent organic framework to the mixed solution and react in a constant temperature shaker at 37℃ and 350 rpm for 65 min. After the reaction is complete, centrifuge at 11000 rpm for 7.5 min. Then, wash the precipitate with the eluent, which consists of acetonitrile:deionized water:trifluoroacetic acid at a volume ratio of 35:64.5:0.5. Wash at 37℃ for 25 min and then centrifuge at 11000 rpm for 7.5 min to obtain the pretreated enzymatic hydrolysate. S4: The pretreated enzymatic hydrolysate was ultrafiltered through an ultrafiltration membrane system with a molecular weight cutoff of 2000 Da under a nitrogen pressure of 0.4 MPa to obtain an ultrafiltration permeate. The ultrafiltration permeate was purified by Sephadex G-15 gel chromatography. The purified target component solution was concentrated and freeze-dried to obtain a small molecule glycopeptide composition derived from bird's nest.

[0029] The preparation of the above-mentioned modified covalent organic framework includes: A1: 25 parts by weight of 2,4,6-tricarboxypyrogallol, 65 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 12.5 parts by weight of 4-aminophenylboronic acid were added to 250 parts by weight of anhydrous ethanol. The mixture was then reacted at 85°C and 400 rpm for 4.5 h. After the reaction was completed, the mixture was cooled to room temperature and then centrifuged at 11,000 rpm for 15 min. The precipitate was then washed four times by centrifugation with N,N-dimethylformamide and anhydrous ethanol, and finally dried under vacuum to obtain a covalent organic framework. A2: Add 6.5 parts by weight of modified chitosan to 35 parts by weight of deionized water and 1.5 parts by weight of glacial acetic acid, stir and mix, then add 110 parts by weight of deionized water and mix evenly to obtain a modified chitosan solution. Disperse 4 parts by weight of covalent organic framework in 100 parts by weight of deionized water to obtain a covalent organic framework dispersion. A3: The covalent organic framework dispersion and the modified chitosan solution were mixed at a volume ratio of 1:2.5, and then stirred for 2.5 hours. After the reaction was completed, the mixture was filtered and freeze-dried to obtain the modified covalent organic framework.

[0030] The preparation of the above-mentioned modified chitosan includes: a1: Add 2.5 parts by weight of sodium hydroxide to 45 parts by weight of deionized water, then add 9 parts by weight of proline, stir and mix at 250 rpm for 25 min, then add 45 parts by weight of tert-butanol, stir at 650 rpm for 17.5 min, then add 11 parts by weight of ditert-butyl dicarbonate dropwise at 26.5℃ for 25 min, then react for 55 min, then raise the temperature to 34℃ and react for 22 h to obtain the reaction mixture; a2: The reaction mixture was extracted three times with petroleum ether, the aqueous phases were combined, and then the pH was adjusted to 1.1 with dilute sulfuric acid under an ice-water bath. After extraction with diethyl ether four times, the diethyl ether layers were combined. The diethyl ether layers were then washed with deionized water and dried with anhydrous sodium sulfate for 11 hours. After filtration and concentration, the pretreated amino acids were obtained. a3: Add 6.5 parts by weight of chitosan to 26.5 parts by weight of DMF, stir and mix for 2.5 hours to obtain a swollen chitosan mixture. Add 9 parts by weight of pretreated amino acids to 26.5 parts by weight of DMF, then add 0.02 parts by weight of 4-dimethylaminopyridine, and then add 11 parts by weight of N,N-dicyclohexylcarbodiimide under ice bath conditions. Stir and mix to obtain a mixed solution. a4: Mix the mixed solution and the swollen chitosan mixture, then react in an ice bath for 2.5 h, and then react at 26.5 °C for 22 h. After filtration, wash four times with anhydrous ethanol. Place the washed product in a DCM system of 25 wt% trifluoroacetic acid and react for 2.5 h. After the reaction is complete, wash with ethanol, then dialyze with deionized water, and freeze dry to obtain modified chitosan.

[0031] Comparative Example 1 differs from Example 1 in that step S3 is removed, and the pretreatment enzymatic hydrolysate in step S4 is replaced with an equal amount of enzymatic hydrolysate, while the remaining steps remain unchanged to prepare the bird's nest source small molecule glycopeptide composition, which is referred to as Comparative Example 1.

[0032] Comparative Example 2 differs from Example 1 in that 4-aminophenylboronic acid in step A1 is removed, while the remaining steps remain unchanged to prepare the bird's nest-derived small molecule glycopeptide composition. This is referred to as Comparative Example 2.

[0033] Comparative Example 3 differs from Example 1 in that steps A2-A3 and a1-a4 are removed, and the modified covalent organic framework in step S3 is replaced with an equal amount of covalent organic framework prepared by A1, while the remaining steps remain unchanged to prepare the bird's nest source small molecule glycopeptide composition, which is referred to as Comparative Example 3.

[0034] Comparative Example 4 differs from Example 1 in that steps a1-a4 are removed, and the modified chitosan in step A2 is replaced with an equal amount of chitosan, while the remaining steps remain unchanged to prepare the bird's nest-derived small molecule glycopeptide composition. This is referred to as Comparative Example 4.

[0035] The yield and total sialic acid content of the bird's nest-derived small molecule glycopeptide compositions prepared in Examples 1-3 and Comparative Examples 1-4 were determined. The determination was performed three times, and the average value was taken. The results are shown in Table 1.

[0036] Product yield (%) = (mass of bird's nest source small molecule glycopeptide composition / dry weight of starting bird's nest raw material) × 100%.

[0037] The total sialic acid content in each group of 10mg bird's nest-derived small molecule glycopeptide compositions was determined by high performance liquid chromatography (HPLC).

[0038] Table 1. Results of product yield and total sialic acid content determination Product yield (%) Total sialic acid content (%) Example 1 8.5 11.2 Example 2 9.2 12.3 Example 3 8.9 11.8 Comparative Example 1 2.4 3.9 Comparative Example 2 4.9 6.2 Comparative Example 3 5.5 7.8 Comparative Example 4 6.8 8.9 As can be seen from the data in Table 1, Comparative Example 1 shows that the present invention introduces a modified covalent organic framework as an adsorbent during the purification stage, which can significantly improve the product yield and total sialic acid content compared to direct ultrafiltration and gel chromatography. Comparative Examples 2 and 3 show that the introduction of functional monomers 4-aminophenylboronic acid and chitosan modification into the covalent organic framework can significantly improve the selectivity and enrichment efficiency of the covalent organic framework, thereby effectively improving the product yield and total sialic acid content. Comparative Example 4 shows that the modification of the covalent organic framework using proline-modified chitosan has a better modification effect than chitosan itself, and is more conducive to the enrichment of glycopeptides.

[0039] The DPPH free radical scavenging rate of the bird's nest-derived small molecule glycopeptide compositions prepared in Examples 1-3 was determined. The determination was performed three times, and the average value was taken. The results are shown in Table 2.

[0040] Table 2. DPPH free radical scavenging rate of bird's nest-derived small molecule glycopeptide compositions DPPH radical scavenging rate (%) Example 1 73.2 Example 2 74.4 Example 3 73.7 As can be seen from the data in Table 2, the small molecule glycopeptide composition of bird's nest extracted by the present invention has a good DPPH free radical scavenging rate, indicating that it has high antioxidant activity.

[0041] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims. Parts not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A method for preparing a small molecule glycopeptide composition derived from bird's nest, characterized in that, include: S1: The bird's nest raw material is pretreated by washing and soaking in water, then extracted and centrifuged to obtain a crude extract solution of bird's nest glycoprotein; S2: The crude extract of bird's nest glycoprotein was first hydrolyzed with pepsin at a pH of 2.0-3.0, an enzyme-to-protein ratio of 1:90-110, a hydrolysis temperature of 35-40℃, and a hydrolysis time of 1-3 hours. Then, it was hydrolyzed with trypsin at a pH of 7.0-8.0, an enzyme-to-protein ratio of 1:90-110, a hydrolysis temperature of 45-55℃, and a hydrolysis time of 3-6 hours. Finally, the enzyme was inactivated and centrifuged to obtain the hydrolysate. S3: The enzymatic hydrolysate is first enriched and impurities removed by a modified covalent organic framework to obtain a pretreated enzymatic hydrolysate; S4: The pretreated enzymatic hydrolysate is passed through an ultrafiltration membrane system with a molecular weight cutoff of 1000-3000 Da, and then purified by gel chromatography. The purified target component solution is concentrated and freeze-dried to obtain a small molecule glycopeptide composition from bird's nest. The preparation of the above-mentioned modified covalent organic framework includes: adding 20-30 parts by weight of 2,4,6-tricarboxypyrogallol, 60-70 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 10-15 parts by weight of 4-aminophenylboronic acid to anhydrous ethanol for reaction to obtain a covalent organic framework; then modifying the covalent organic framework with modified chitosan to obtain a modified covalent organic framework. The preparation of the modified chitosan includes: first, dissolving sodium hydroxide and proline in water and stirring, adding tert-butanol and stirring at high speed, and then adding di-tert-butyl dicarbonate dropwise under controlled temperature to obtain a mixed solution; after extraction, pH adjustment, washing and drying, pretreated amino acids are obtained; then, a swollen chitosan mixture and an amino acid mixture are prepared, and the two are mixed and reacted, followed by washing, system reaction, dialysis and freeze drying to obtain modified chitosan.

2. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 1, characterized in that, Specifically, the steps include the following: S1: After washing the bird's nest raw material with deionized water, soak it in 10-15 times its volume of deionized water for 2-4 hours. Then, stir and extract it at 50-60℃ for 1-2 hours. Centrifuge at 4000-8000rpm for 10-20 minutes, take the supernatant, and then concentrate it under reduced pressure to obtain a crude extract solution of bird's nest glycoprotein. S2: Adjust the pH of the crude extract of bird's nest glycoprotein to 2.0-3.0 with 1M HCl, then add pepsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90-110, and react at 35-40℃ for 1-3 hours. Then adjust the pH to 7.0-8.0, add trypsin with an activity of 5000 U / mg and an enzyme-to-protein ratio of 1:90-110, and continue to react at 45-55℃ for 3-6 hours. After the enzymatic hydrolysis is completed, heat in a water bath at 85-100℃ for 5-15 minutes, then centrifuge to obtain a clear enzymatic hydrolysate. S3: Mix the enzymatic hydrolysate and the loading solution at a volume ratio of 1:9 to obtain a mixed solution. Then, add 50-60 mg / mL of modified covalent organic framework to the mixed solution and react in a constant temperature shaker at 37℃ and 300-400 rpm for 60-70 min. After the reaction is complete, centrifuge at 10000-12000 rpm for 5-10 min. Then, wash the precipitate with the elution buffer at 37℃ for 20-30 min and centrifuge at 10000-12000 rpm for 5-10 min to obtain the pretreated enzymatic hydrolysate. S4: The pretreated enzymatic hydrolysate is passed through an ultrafiltration membrane system with a molecular weight cutoff of 1000-3000 Da and ultrafiltration is performed under a nitrogen pressure of 0.3-0.5 MPa to obtain an ultrafiltration permeate. The ultrafiltration permeate is purified by gel chromatography, and the purified target component solution is concentrated and freeze-dried to obtain a bird's nest-derived small molecule glycopeptide composition.

3. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 2, characterized in that, The preparation of the modified covalent organic framework in step S3 specifically includes the following steps: A1: Add 20-30 parts by weight of 2,4,6-tricarboxypyrogallol, 60-70 parts by weight of 2,5-diamino-1,4-benzenedisulfonic acid and 10-15 parts by weight of 4-aminophenylboronic acid to 200-300 parts by weight of anhydrous ethanol, and then react at 80-90℃ and 300-500 rpm for 4-5 h. After the reaction is completed, cool to room temperature, then centrifuge at 10000-12000 rpm for 10-20 min. Then wash the precipitate 3-5 times by centrifugation with N,N-dimethylformamide and anhydrous ethanol, and finally vacuum dry to obtain a covalent organic framework. A2: Add 5-8 parts by weight of modified chitosan to 30-40 parts by weight of deionized water, and add 1-2 parts by weight of glacial acetic acid. Stir and mix, then add 100-120 parts by weight of deionized water and mix evenly to obtain a modified chitosan solution. Disperse 3-5 parts by weight of covalent organic framework in 100 parts by weight of deionized water to obtain a covalent organic framework dispersion. A3: Mix the covalent organic framework dispersion and the modified chitosan solution at a volume ratio of 1:2-3, stir and react for 2-3 hours, filter after the reaction is complete, and freeze dry to obtain the modified covalent organic framework.

4. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 3, characterized in that, The preparation of the above-mentioned modified chitosan specifically includes the following steps: a1: Add 2-3 parts by weight of sodium hydroxide to 40-50 parts by weight of deionized water, then add 8-10 parts by weight of proline, stir and mix at 200-300 rpm for 20-30 min, then add 40-50 parts by weight of tert-butanol, stir at 500-800 rpm for 15-20 min, then add 10-12 parts by weight of ditert-butyl dicarbonate dropwise at 25-28℃ for 20-30 min, then react for 50-60 min, then raise the temperature to 33-35℃ and react for 20-24 h to obtain the reaction mixture; a2: Extract the reaction mixture with petroleum ether 2-3 times, combine the aqueous phases, then adjust the pH to 1-1.2 with dilute sulfuric acid in an ice-water bath, then extract with diethyl ether 3-5 times, combine the diethyl ether layers, then wash the diethyl ether layer with deionized water and dry with anhydrous sodium sulfate for 10-12 hours, filter, concentrate, and obtain the pretreated amino acids. a3: Add 5-8 parts by weight of chitosan to 25-30 parts by weight of DMF, stir and mix for 2-3 hours to obtain a swollen chitosan mixture. Add 8-10 parts by weight of pretreated amino acids to 25-30 parts by weight of DMF, then add 0.01-0.03 parts by weight of 4-dimethylaminopyridine. Then, under ice bath conditions, add 10-12 parts by weight of N,N-dicyclohexylcarbodiimide, stir and mix to obtain a mixed solution. a4: Mix the mixed solution and the swollen chitosan mixture, then react in an ice bath for 2-3 hours, and then react at 25-28℃ for 20-24 hours. After filtration, wash with anhydrous ethanol 3-5 times. Place the washed product in a 25wt% trifluoroacetic acid DCM system and react for 2-3 hours. After the reaction is complete, wash with ethanol, then dialyze with deionized water, and freeze-dry to obtain modified chitosan.

5. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 2, characterized in that, The sample loading solution in step S3 consists of acetonitrile:deionized water:trifluoroacetic acid in a volume ratio of 85:14.9:0.

1.

6. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 2, characterized in that, The eluent in step S3 consists of acetonitrile:deionized water:trifluoroacetic acid in a volume ratio of 35:64.5:0.

5.

7. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 2, characterized in that, In step S4, an ultrafiltration membrane system with a molecular weight cutoff of 2000 Da is preferred.

8. The method for preparing a small molecule glycopeptide composition derived from bird's nest according to claim 2, characterized in that, In step S4, gel chromatography was performed using a Sephadex G-15 column.

9. A small molecule glycopeptide composition derived from bird's nest, characterized in that, It is prepared by the method for preparing a small molecule glycopeptide composition derived from bird's nest as described in any one of claims 1-8.

10. The use of the bird's nest-derived small molecule glycopeptide composition as described in claim 9 in the preparation of drugs, health foods, and food additives for promoting nerve cell development, improving memory, treating insomnia, and delaying neurodegenerative diseases.

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