Bird's nest sialic acid glycan peptide capable of improving lung inflammation and application of bird's nest sialic acid glycan peptide

Abstract

The invention discloses cubilose sialic acid glycan peptide capable of improving lung inflammation and application of the cubilose sialic acid glycan peptide. The ratio of fucose modified sugar chains in the cubilose sialic acid glycan peptide is 30-60%, the ratio of sialic acid modified sugar chains in the cubilose sialic acid glycan peptide is 20-40%, the core structure of the fucose type is (HexNAc) 3 (Man) 3 (Fuc) 1, the core structure of the sialic acid type is (HexNAc) 3 (Man) 3 (Gal) 1 (Fuc) 1 (NeuAc) 1, the neutral sugar dissolution rate of the cubilose sialic acid glycan peptide is high, and the glycopeptide shown in SEQ ID NO 2 has a good inflammation improving effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of glycoprotein engineering, and in particular relates to a bird's nest sialic acid polyglycopeptide capable of improving lung inflammation and an application thereof. Background Art

[0002] Bird's nest, a precious natural tonic, is primarily made from nests built by swiftlets and various other swiftlet species using secretions from their sublingual glands. In Traditional Chinese Medicine, bird's nest is believed to have numerous medicinal benefits, including nourishing yin and the lungs, enhancing beauty, and boosting brain function. The main components of bird's nest include water-soluble protein, carbohydrates, trace elements, and amino acids. Sialic acid (bird's nest acid) is its most nutritionally valuable component, comprising up to 10% of the nest, and plays a crucial role in supporting the nervous and immune systems.

[0003] Protein accounts for approximately 50% of the edible bird's nest, while carbohydrates account for approximately 30%. These are the primary components of bird's nest and play a crucial role in its nutritional value and physiological functions. The proteins and carbohydrates present in bird's nest do not exist independently; rather, they are combined through glycosylation to form bird's nest glycoproteins, including N-glycosylated and O-glycosylated proteins, with N-glycoproteins being the predominant form. The N-glycan structure in glycoproteins refers to a complex formed by the covalent attachment of a sugar chain to an asparagine residue on a protein. The structure of N-glycans is complex and diverse, significantly influencing protein stability, bioactivity, and immune recognition. N-glycans in bird's nest are composed of various monosaccharides, including sialic acid, N-acetylglucosamine, and N-acetylglucosamine. Studies have shown that the highly branched sialylated N-glycans in bird's nest help inhibit influenza virus infection. Furthermore, advances in the structural and functional research of N-glycans suggest that N-glycans play a key role in the anti-inflammatory and neuroprotective effects of bird's nest.

[0004] Sugar chains in bird's nests are mostly present in a protein-bound form. Studies have shown that bird's nests have a high degree of swelling after absorbing water and are not easily digested by pepsin and trypsin, resulting in limited bioavailability. Patent CN110257459B discloses a method for preparing bird's nest peptides by denaturing and enzymatically hydrolyzing bird's nests; patent CN107974479A discloses a method for preparing bird's nest oligopeptides using a combination of microwave and membrane technologies; and patent CN114848791A discloses a method for preparing small-molecule bird's nest glycopeptides for preventing and improving skin inflammation. However, the bird's nest peptides prepared by these methods ignore the functionality of the sugar chains in the bird's nest glycoproteins. The resulting bird's nest peptides have relatively low sugar and sialic acid content, and the sugar chain structure of the bird's nest is not characterized. Furthermore, there is currently little research on the anti-inflammatory efficacy of bird's nest sialoglycopeptides.

[0005] Therefore, the present invention uses a step-by-step enzymatic hydrolysis method combined with acetone enrichment to prepare bird's nest sialoglycopeptide. Through liquid chromatography-mass spectrometry tandem analysis, the MSfragger database is used to search for Apodiformes (Swifts) to retrieve the bird's nest sialoglycoprotein chain glycoprotein to obtain the product N-sugar chain fine structure. Animal experiments were designed to use lipopolysaccharide (LPS) to create a mouse inflammation model. It was verified that the bird's nest sialoglycopeptide has an ameliorative effect on mouse blood, nerve, liver, and lung inflammation, verifying that the product of the present invention has anti-inflammatory efficacy. Summary of the Invention

[0006] To address the current problems, this patent uses a step-by-step enzymatic hydrolysis method combined with acetone enrichment to isolate bird's nest sialoglycopeptides with anti-inflammatory effects. Liquid chromatography-mass spectrometry tandem analysis is then performed, and the MSfragger database is used to search for Apodiformes (Swifts) for bird's nest sialoglycoproteins to obtain the fine structure of the N-glycans in the bird's nest sialoglycopeptides. The present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a bird's nest glycopeptide with anti-inflammatory activity, characterized in that the amino acid sequence of the glycopeptide is shown as SEQ ID NO 2.

[0008] In some aspects of the bird's nest glycopeptide having anti-inflammatory activity as described above, the glycoform of the sugar chain to which the glycopeptide is connected is Hex(4)HexNAc(5), and the structural unit of the sugar chain is as follows:

[0009]

[0010] In a second aspect, the present invention provides a method for preparing the glycopeptide mixture according to the first aspect, comprising the following steps:

[0011] The following steps are involved:

[0012] (1) The bird's nest is soaked, crushed, and hydrolyzed with protease, the enzyme is inactivated, and the supernatant is collected by centrifugation; the mixture is concentrated and separated by liquid chromatography. Preferably, the soaking step is to add the bird's nest to water and then fully mix it.

[0013] Furthermore, the mass of the water is 20 to 50 times the dry weight of the bird's nest, for example, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, or 50 times.

[0014] Preferably, the expanded bird's nest is crushed by a colloid mill with a grinding gap of 50 to 120 μm, so that the particles in the bird's nest slurry reach the micron level.

[0015] Preferably, the grinding time of the colloid mill is 20 to 40 min, for example: 20 min, 25 min, 30 min, 35 min, 40 min.

[0016] Preferably, the protease in step (2) is selected from any one or more combinations of hydrolytic protease, papain, and alkaline protease.

[0017] Furthermore, when the protease is alkaline protease, the pH of the bird's nest slurry is adjusted to 9.5-10.5, and alkaline protease is added with an enzyme-substrate ratio of 6000-12000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is placed in an enzymatic hydrolysis reaction tank equipment and treated at 35-50°C and a rotation speed of 80-150 r / min for 10-14 hours.

[0018] Furthermore, when the protease is papain, the pH of the bird's nest slurry is adjusted to 5.5-7.5, and papain is added with an enzyme-substrate ratio of 6000-12000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is put into an enzymatic hydrolysis reaction tank equipment and treated at 37-60°C and a rotation speed of 80-150 r / min for 10-14 hours.

[0019] Furthermore, when the protease is a hydrolytic protease, the pH of the bird's nest slurry is adjusted to 7.5-10.5, and the hydrolytic protease is added at an enzyme-substrate ratio of 6000-12000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is put into the enzymatic hydrolysis reaction tank equipment and treated at 50-70°C and a rotation speed of 80-150 r / min for 10-14 hours.

[0020] Furthermore, when the protease is hydrolytic protease and papain, the pH of the bird's nest slurry is adjusted to 7.5-10.5, and the hydrolytic protease is added at an enzyme-substrate ratio of 6000-12000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is put into the enzymatic hydrolysis reaction tank equipment, and treated at 50-70°C and a rotation speed of 80-150 r / min for 10-14 hours. The enzyme is inactivated, and the pH of the bird's nest slurry is adjusted to 5.5-7.5. The papain is added at an enzyme-substrate ratio of 6000-12000 U / mg (enzyme activity / bird's nest dry weight). After mixing, the mixture is put into the enzymatic hydrolysis reaction tank equipment, and treated at 37-60°C and a rotation speed of 80-150 r / min for 1-5 hours.

[0021] Preferably, the enzyme inactivation temperature in step (2) is 90-100° C., for example, 90° C., 92° C., 95° C., 97° C., or 100° C. The enzyme inactivation time is 10-30 min, for example, 10 min, 15 min, 20 min, 25 min, or 30 min.

[0022] Preferably, the centrifugal speed in step (2) is 5000-10000 r / min, for example: 5000 r / min, 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min.

[0023] Preferably, the centrifugation time in step (2) is 15 to 30 min, for example: 15 min, 20 min, 25 min, 30 min.

[0024] Preferably, when acetone is enriched in step (3), the volume fraction of anhydrous acetone is 40% to 80% of the total system, for example, 40%, 50%, 60%, 70%, or 80%.

[0025] Preferably, the enrichment time in step (3) is 10 to 30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0026] Preferably, the centrifugal speed in step (3) is 3000-5000 r / min, for example: 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min, 5000 r / min.

[0027] Preferably, the centrifugation time in step (3) is 15 to 30 min, for example: 10 min, 15 min, 20 min, 25 min, 30 min.

[0028] Preferably, the rotary evaporation speed in step (3) is 100 to 200 r / min, for example: 100 r / min, 120 r / min, 150 r / min, 180 r / min, 200 r / min.

[0029] Preferably, the rotary evaporation temperature in step (3) is 40-60°C, for example: 40°C, 45°C, 50°C, 55°C, 60°C.

[0030] Preferably, the drying in step (3) is performed by freeze drying or low-temperature drying, more preferably freeze drying.

[0031] Furthermore, the freeze-drying step is to firstly quick-freeze the concentrated liquid after rotary evaporation and then freeze-dry it.

[0032] Furthermore, the freeze-drying temperature is -90 to -60°C, for example: -90°C, -85°C, -80°C, -75°C, -70°C, -65°C, -60°C.

[0033] Furthermore, the quick freezing time is 10 to 12 hours, for example: 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours.

[0034] Furthermore, the freeze-drying time is 12 to 24 hours, for example: 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours.

[0035] Furthermore, the protease hydrolysis comprises the following steps: adjusting the pH value to 8.0-8.5, adding hydrolyzing protease, inactivating the enzyme after enzymatic hydrolysis for 12 hours, adjusting the pH value of the enzymatic hydrolysis solution to 7.0-7.4, and adding papain for enzymatic hydrolysis for 2 hours.

[0036] Furthermore, the enrichment and concentration includes: washing the amide-bonded silica gel filler with 100% acetonitrile, activating it with an equilibrium buffer, and evenly loading it into a chromatography column; centrifuging the bird's nest hydrolysate and filtering it with a filter membrane, removing small molecule impurities through an ultrafiltration tube, replacing the solvent with an equilibrium buffer and concentrating it to less than 20 mL, and then performing a secondary filtration; loading the sample, washing it with an equilibrium buffer to remove impurities, performing gradient elution, detecting the fractions by LC-MS / MS, matching the target glycopeptide retention time and characteristic ion pairs, and combining the fractions containing the target product.

[0037] In some embodiments, the steps of the method for preparing the bird's nest glycopeptide are:

[0038] (1) Soaking the bird's nest is to fully soak the bird's nest (based on dry weight) in 20-50 times water at a water temperature of 25°C for 2 hours;

[0039] (2) The pulverization is performed by colloid milling, preferably with a colloid mill tooth gap of 80 μm, and the slurry obtained in step (1) is subjected to colloid milling for 20-40 minutes;

[0040] (3) The protease hydrolysis comprises adjusting the pH value of the feed solution obtained in step (2) to 8.0-8.5 using HCL and NaOH, adding hydrolyzing protease (enzyme amount 6000U / mg), mixing, placing in an enzymatic fermentation tank, treating at 55-60°C and a rotation speed of 150r / min for 12h, killing the enzyme after treatment, adjusting the pH value of the enzymatic hydrolyzate to 7.0-7.4 using HCL and NaOH, adding papain, mixing, placing in an enzymatic fermentation tank, and treating at 50°C and a rotation speed of 150r / min for 2h;

[0041] (4) First, the amide bonded silica gel filler was washed with 100% acetonitrile and then activated with equilibrium buffer, and evenly loaded into the chromatography column. It was rinsed at a flow rate of 2 mL / min until the column pressure was stable and there were no bubbles. The bird's nest hydrolysate was centrifuged and filtered with a 0.22 μm filter membrane. Small molecular impurities were removed by a 3 kDa ultrafiltration tube. The solvent was replaced with equilibrium buffer and concentrated to less than 20 mL and then filtered twice. The sample was loaded at a flow rate of 1 mL / min, and 5 times the column volume was rinsed with equilibrium buffer to remove impurities. Then, a gradient elution was performed, with acetonitrile decreasing from 80% to 70% in 0-10 min and 10-50 mi The acetonitrile content was reduced from 70% to 40% during n-term, and from 40% to 20% during 50-60 min, with the flow rate maintained at 2 mL / min. The fractions were detected by LC-MS / MS, and the fractions containing the target product were combined after matching the target glycopeptide retention time of 47.94 min ± 2% and characteristic ion pairs. The enriched solution was concentrated through a 10 kDa ultrafiltration tube, washed with ultrapure water three times, transferred to a 10 kDa dialysis bag, dialyzed at 4°C for 24 hours, and freeze-dried to obtain the glycopeptide powder. Finally, the column was rinsed with 10 times the column volume of 100% acetonitrile and equilibration buffer, and stored in 80% acetonitrile at 4°C.

[0042] In a fourth aspect, the present invention provides a composition comprising the bird's nest glycopeptide described in the first aspect, wherein the composition is prepared as food, health care product, cosmetic or medicine, and excipients allowed to be added thereto.

[0043] In a fifth aspect, the present invention provides use of the bird's nest sialic acid polysaccharide peptide described in the first aspect or the composition described in the fourth aspect in the preparation of an anti-inflammatory product.

[0044] In some embodiments, as described above, the anti-inflammatory product is a food, a health product, a cosmetic or a medicine.

[0045] In some embodiments, as described above, the anti-inflammatory product is used to prevent or treat inflammation-related diseases.

[0046] Furthermore, the dosage of the bird's nest glycopeptide is 100 mg / kg body weight / day to 300 mg / kg body weight / day, for example: 100 mg / kg body weight / day, 150 mg / kg body weight / day, 200 mg / kg body weight / day, 250 mg / kg body weight / day, 300 mg / kg body weight / day.

[0047] Beneficial effects of the present invention:

[0048] The present invention obtains the complete N-sugar chain structure of bird's nest sialylated polyglycopeptide and innovatively points out the core structure of bird's nest sugar chain. In addition, the prepared bird's nest sialylated polyglycopeptide has anti-inflammatory effect. The glycopeptide mixture containing bird's nest sialylated polyglycopeptide has broad application prospects in the fields of cosmetics, food, medicine, etc., promotes the further development of bird's nest products, and improves the economic value and utilization value of bird's nest. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shown is a schematic diagram of the process of preparation, separation, structure determination and functional research of bird's nest sialic acid polysaccharide peptides in the present invention.

[0050] Figure 2 Shown is the mass spectrum of the glycopeptide represented by SEQ ID NO 2.

[0051] Figure 3 Shown is the sugar chain structure of the glycopeptide represented by SEQ ID NO 2. DETAILED DESCRIPTION

[0052] The technical solutions of the present invention are described clearly and completely below in conjunction with the embodiments and accompanying drawings. The described embodiments are only a portion of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] It should be noted that, unless otherwise specified, the experimental methods and reagents used in the embodiments of the present invention are conventional experimental methods and reagents in the art.

[0054] The main reagents used in the examples are as follows:

[0055] Hydrolytic protease ( 2.4L FG, Novozymes Biotechnology Co., Ltd.), papain (S10011, Shanghai Yuanye Biotechnology Co., Ltd.), and alkaline protease (S10154, Shanghai Yuanye Biotechnology Co., Ltd.).

[0056] Example 1 Preparation of glycopeptide mixture

[0057] 1.1 Preparation and application of glycopeptide mixture

[0058] (1) Soaking the bird's nest: Soak the bird's nest (based on dry weight) in 30 times the amount of water at 25°C for 2 hours.

[0059] (2) Colloid mill grinding: The colloid mill tooth gap is set to 80 μm, and the slurry obtained in step (1) is subjected to colloid mill treatment for 30 min.

[0060] (3) Enzymatic hydrolysis in an enzymatic reaction tank: The pH value of the liquid obtained in step (2) was adjusted to 8.5 using HCL and NaOH, and hydrolyzing protease (enzyme addition amount 6000 U / mg) was added. After mixing, the mixture was placed in an enzymatic fermentation tank and treated at 60°C and a rotation speed of 150 r / min for 12 h. After treatment, the enzyme was inactivated. The pH value of the enzymatic hydrolyzate was adjusted to 7.0 using HCL and NaOH, and papain (enzyme addition amount 10000 U / mg) was added. After mixing, the mixture was placed in an enzymatic fermentation tank and treated at 50°C and a rotation speed of 150 r / min for 2 h.

[0061] (4) Inactivation of enzyme and centrifugation: The enzymatic solution obtained in step (3) was inactivated at 95°C for 20 min, cooled to room temperature, and centrifuged at 5000 rpm for 20 min to obtain the supernatant.

[0062] (5) Acetone enrichment and rotary evaporation concentration: Add anhydrous acetone to the supernatant obtained in step (4) to make the acetone volume fraction in the final system reach 60%, and stir to mix. Let it stand for more than 10 minutes, centrifuge at 3500r / min for 20 minutes to separate the precipitate and supernatant, transfer all the supernatant to a rotary evaporation bottle, set the water bath temperature to 60℃, and rotary evaporate at 100r / min to remove ethanol until the volume in the bottle remains basically unchanged. Pour out the concentrate, add a small amount of water to rinse the rotary evaporation bottle for recovery, and measure the total volume of the concentrate. According to the measured volume of the concentrate, add anhydrous acetone again to make the acetone volume fraction in the final system reach 70%, and stir to mix. Let it stand for more than 10 minutes, centrifuge at 3500r / min for 20 minutes to separate the precipitate and supernatant. Take the supernatant again and transfer it to a rotary evaporation bottle for rotary evaporation to remove acetone.

[0063] (6) Separation and preparation: The concentrated solution obtained in step (5) was dispensed into glass plates, with each plate containing about 25 to 30 mL. After quick freezing at -80°C for 12 h, the mixture was placed in a freeze dryer and lyophilized for 24 h to obtain a powdered glycopeptide mixture.

[0064] (7) Determination of sialic acid content: The enzymatic hydrolysis product powder was hydrolyzed in a boiling water bath with 1% phosphoric acid solution and centrifuged to obtain a supernatant. An equal volume of o-phenylenediamine hydrochloride solution was added and mixed with the supernatant. The mixture was derivatized in a dark water bath at 80°C for 40 min. After cooling, the mixture was filtered through a 0.45 mm filter membrane and the sialic acid content of the enzymatic hydrolysis product was determined to be 17.84 wt%.

[0065] (8) Determination of neutral sugar dissolution rate: The neutral sugar content in dry bird's nest and bird's nest sialic acid polysaccharide peptide was determined by phenol-sulfuric acid method. The dry bird's nest powder and the enzymatic hydrolysate powder were weighed, 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid were added, vortexed and mixed, and placed at room temperature for 20 min. The absorbance was measured at 490 nm, and the neutral sugar dissolution rate of the enzymatic hydrolysate was 75%.

[0066] (9) Glycopeptide ratio determination: The biuret method was used to determine the peptide content in the enzymatic hydrolysis product. After the enzymatic hydrolysis sample was prepared to an appropriate concentration, biuret (sample to be tested: biuret reagent = 3:2, v / v) was added and mixed evenly on a vortex mixer. The absorbance of the supernatant was measured at 540 nm, and the glycopeptide ratio of the enzymatic hydrolysis product was measured to be 0.78.

[0067] Example 2 Determination of N-sugar chain structure of bird's nest glycopeptide

[0068] 2.1 Bird’s Nest Glycopeptide N-Glycan Structure Determination Process

[0069] (1) Protein extraction: Add 4 times the volume of lysis buffer (1% SDS, 1% protease inhibitor) to the enzymatic hydrolyzate and sonicate. Centrifuge at 4°C, 12,000 rpm for 10 min. Transfer the supernatant to a new centrifuge tube and determine the protein concentration using a BCA kit.

[0070] (2) Enzymatic hydrolysis with pancreatic enzymes: The protein extracted in step (1) was hydrolyzed by enzymes. The volume was adjusted to a uniform volume with lysis buffer, and TCA was slowly added to a final concentration of 20%, vortexed to mix, and precipitated at 4°C for 2h. Centrifuged at 4500r / min for 5min, the supernatant was discarded, and the precipitate was washed 2-3 times with pre-cooled acetone. After drying the precipitate, TEAB was added to a final concentration of 200mM, and the precipitate was broken up by ultrasound. Trypsin was added at a ratio of 1:50 (protease: protein, m / m), and the hydrolysis was carried out overnight. Dithiothreitol (DTT) was added to a final concentration of 5mM, and the mixture was reduced at 56°C for 30min. Iodoacetamide (IAA) was then added to a final concentration of 11mM, and the mixture was incubated at room temperature in the dark for 15min.

[0071] (3) Modification and enrichment: The peptides obtained by enzymatic hydrolysis in step (2) were dissolved in 200 mL of enrichment buffer (80% acetonitrile / 5% trifluoroacetic acid), and the supernatant was transferred to a hydrophilic (HILIC) microcolumn and centrifuged at 1000 r / min for about 15 min to complete the enrichment. The hydrophilic microcolumn was then washed three times with the enrichment buffer. The glycopeptides were then eluted using 0.1% trifluoroacetic acid, 50 mM ammonium bicarbonate solution, and 50% acetonitrile, respectively. The eluates were collected and combined and vacuum-freezed. Finally, desalting was performed according to the C18 ZipTips instructions, and the samples were vacuum-freezed and then subjected to LC-MS analysis.

[0072] (4) Liquid chromatography-mass spectrometry analysis (LC-MS / MS): The peptides obtained by modification and enrichment in step (3) were dissolved in liquid chromatography mobile phase A and separated using an EASY-nLC 1200 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-2 min, 4%-7% B; 2-42 min, 7%-20% B; 42-52 min, 20%-30% B; 52-56 min, 30%-80% B; 56-60 min, 80% B, and the flow rate was maintained at 500 nl / min. After separation by the ultra-high performance liquid chromatography system, the peptides were injected into the NSI ion source for ionization and then entered the Orbitrap Exploris 480 mass spectrometer for analysis. The ion source voltage was set to 2100 V, and the FAIMS compensation voltage (CV) was set to . Both the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometer scan range was set to 700–2000 m / z, with a scan resolution of 60,000. The secondary mass spectrometer scan range was fixed at 100 m / z, with a secondary scan resolution of 30,000, and TurboTMT was set to off. Data acquisition used a data-dependent scanning (DDA) program. After the primary scan, the pre-peptide precursor ions with the highest signal intensity were selected and sequentially entered the HCD collision cell for fragmentation at 40% fragmentation energy. Secondary mass spectrometry analysis was also performed sequentially. To maximize mass spectrometry utilization, the automatic gain control (AGC) was set to 200%, the signal threshold to 50,000 ions / s, the maximum injection time to 75 ms, and the dynamic exclusion time for the tandem mass spectrometer scan to 30 s to avoid duplicate scanning of precursor ions.

[0073] 2.2 Glycopeptide N-glycan structure determination results

[0074] (1) Basic glycoform classification: According to the distribution of sugar composition in the sugar chain, sugar chains are divided into five categories: ① Paucimannose type ② High mannose type ③ Fucosylated type ④ Sialylated type ⑤ Complex / hybrid type.

[0075] (2) Complete glycopeptide identification results and glycoform statistics: The secondary mass spectrometry data were searched using MSFragger (v3.4) and the Glygen database. The search parameter settings were as follows: the database was Apodiformes (Apodiformes), a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 7 amino acid residues; the maximum number of peptide modifications was set to 3; the mass error tolerance of the primary parent ion and secondary fragment ion was set to 20ppm. Cysteine ​​alkylation Carbamidomethyl (C) was set as a fixed modification, and the variable modifications were methionine oxidation and protein N-terminal acetylation. The FDR for protein identification and PSM identification was set to 1%. In order to obtain high-quality analysis results, the search analysis results need to be further filtered.

[0076] The overall status of the complete glycopeptides identified after data filtering of the search results is shown in Table 1. A total of 92 glycopeptides, 63 glycans, and 9 glycoproteins were identified in the enzymatic hydrolysis products. In addition, 8 glycoproteins contained only one glycosylation site, and each modification site was often distributed with two or five or more glycans.

[0077] Table 1 Identification results of intact glycopeptide quantity

[0078]

[0079] As shown in Table 2, among the 92 identified glycopeptides, 36 were modified with sialylated glycoforms, 29 with fucose glycoforms, 18 with complex glycoforms, 7 with high mannose glycoforms, and 2 with oligomannose glycoforms.

[0080] Table 2 Glycoform classification statistics

[0081]

[0082]

[0083] N-acetylglucosamine and mannose constitute the basic skeleton of the sugar chain, while special monosaccharides such as fucose and sialic acid can be considered as modifications occurring on the basic skeleton of the sugar chain. The sugar chain is modified by different special monosaccharides to obtain different special sugar chain structures, which in turn enables the sugar chain to perform different functions. For example, fucose modification has been found to be related to tumor migration in studies, and terminal sialic acid modification is related to adhesion or recognition, antiviral and other functions.

[0084] The proportion of glycopeptides modified with fucose accounted for 52.38%, of which 50.79% were modified with a single fucose; the proportion modified with sialic acid was 36.51%, of which 26.98% were modified with a single sialic acid. Furthermore, statistics of the detected glycans revealed that a total of four glycosylation sites were identified, consisting of a (HexNAc)5(Man)3(Gal)3(Fuc)1(NeuAc)1 glycan. Furthermore, three glycans, (HexNAc)5(Man)3(Gal)2(Fuc)1, (HexNAc)5(Man)3(Gal)3(Fuc)1, and (HexNAc)5(Man)3(Gal)3(Fuc)1(NeuAc)2, were identified at three glycosylation sites each.

[0085] (3) Glycopeptide sugar chain structure composition: Structural images corresponding to sugar chains were extracted based on the Glygen database. Due to the complexity and diversity of sugar structures, a sugar chain may usually have multiple matches in the database. We prioritized outputting sugar structure images classified as N-linked in the database and with the highest scores. Ten sugar chain structures are listed according to the matching scores as shown in Table 3.

[0086] Table 3 Sugar chain structure composition of bird's nest sialic acid polysaccharide peptide

[0087]

[0088]

[0089] Note: Blue square: N-acetylglucosamine (HexNAc); Yellow square: N-acetylgalactosamine (GalNAc); Green circle: Mannose (Man); Yellow circle: Galactose (Gal); Red triangle: Fucose (Fuc); Purple diamond: Sialic acid (NeuAc)

[0090] Among the 63 identified glycan structures, two were oligomannose, six were high-mannose, 12 were complex, 20 were fucosylated, and 23 were sialylated. Twelve of these glycans contained both sialic acid and fucose. Based on the identified structures, the core composition of the fucosylated glycans was (HexNAc)3(Man)3(Fuc)1, while the core composition of the sialylated glycans was (HexNAc)3(Man)3(Gal)1(Fu c)1(NeuAc)1. The structures are shown in Table 4.

[0091] Table 4 Core structures of fucose and sialylated glycoforms

[0092]

[0093]

[0094] After database analysis, eight glycopeptides were found to have the potential to inhibit and eliminate inflammation. Cell tests were performed on each of them, and three glycopeptides with strong inflammation inhibition effects were screened out. Their amino acid sequences and glycoforms are as follows:

[0095] Table 5 Sequences and glycoforms of bird's nest glycopeptides with anti-inflammatory effects

[0096]

[0097] Example 3 Purification of Bird's Nest Sialyl Glycan Peptide

[0098] Materials: Amide-bonded silica gel filler (Amide-Sepharose 4B, particle size 50-100 μm, pore size The mobile phase of the laboratory glass column (inner diameter 1.6 cm, column length 20 cm, packing volume 40 mL) includes equilibration / starting buffer (80% acetonitrile (ACN) + 20% 10 mM ammonium formate solution (containing 0.1% formic acid, pH 4.5)), elution buffer (20% acetonitrile + 80% 10 mM ammonium formate solution), regeneration buffer (100% acetonitrile, all degassed by 0.22 μm filter membrane), peristaltic pump, UV detector (214 nm), automatic fraction collector, 10 kDa ultrafiltration tube, dialysis bag (molecular weight cutoff 10 kDa), and freeze dryer.

[0099] First, the amide-bonded silica gel filler was washed with 100% acetonitrile and then activated with equilibration buffer. It was evenly loaded into the chromatography column and flushed at a flow rate of 2 mL / min until the column pressure was stable (0.3-0.5 MPa) to ensure that there were no bubbles. The bird's nest enzymatic hydrolysate was centrifuged (12,000 × g, 30 min) and filtered with a 0.22 μm filter membrane. Small molecular impurities were removed by a 3 kDa ultrafiltration tube. The solvent was replaced with equilibration buffer and concentrated to less than 20 mL before secondary filtration. The sample was loaded at a flow rate of 1 mL / min (not exceeding 1 times the column volume), and 5 times the column volume was flushed with equilibration buffer to remove impurities. Then, a gradient elution was performed, with acetonitrile decreasing from 80% to 70% (discarded) in 0-10 min and acetonitrile increasing to 10% in 10-50 min. The concentration was reduced from 70% to 40% (collected at 1 mL / tube), and the acetonitrile was reduced from 40% to 20% (discarded) within 50-60 minutes, with the flow rate maintained at 2 mL / min; the fractions were detected by LC-MS / MS, and the fractions containing the target product were combined after matching the retention time of the target glycopeptide (47.94 min ± 2%) and the characteristic ion pairs; the enriched liquid was concentrated by a 10 kDa ultrafiltration tube, washed with ultrapure water three times, transferred to a dialysis bag (10 kDa), dialyzed at 4°C for 24 hours (changing the water three times), and freeze-dried (pre-frozen at -80°C, vacuum dried at -55°C for 24 hours) to obtain the glycopeptide powder; finally, the column was washed with 10 times the column volume of 100% acetonitrile and the equilibrium buffer, and stored in 80% acetonitrile at 4°C.

[0100] Example 4: Improvement of blood inflammation in mice by bird's nest sialoglycopeptide

[0101] 4.1 Animal model establishment

[0102] Fifty-six 8-week-old C57BL / 6J male mice were selected. According to the dosing design, they were divided into blank control group, model group, bird's nest group and bird's nest glycopeptide group (Groups I to III), with 8 mice in each group. The adaptation period for all mice was 1 week. From the second week on, the control group and model group were gavaged with 0.2 mL of 0.9% normal saline every day, the bird's nest group was gavaged with an equal volume of bird's nest homogenate, and the bird's nest sialic acid polyglycopeptide group was gavaged with an equal volume of glycopeptide (200 mg / kg / d) for 7 consecutive weeks. In the last week of feeding, the control group was intraperitoneally injected with 0.2 mL of 0.9% normal saline every day, and the other groups were intraperitoneally injected with an equal volume of LPS solution (2 mg / kg) for 1 week. The mice were fasted for 12 hours and then killed.

[0103] 4.2 Blood index determination

[0104] 50mL of fresh whole blood was used to count blood cells using a fully automatic blood cell analyzer, including inflammatory cells such as white blood cells, neutrophils, and lymphocytes. An appropriate amount of fresh blood was taken, allowed to stand at room temperature for 30 minutes, centrifuged at 4°C and 3000r / min for 15 minutes, and the supernatant was prepared to obtain serum. The levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum were determined using a commercial Elisa kit. The operating steps were guided by the instructions of the kit, and the absorbance of the 96-well plate was measured at 490nm. The inflammatory factors were quantified according to the standard curve. The effects of bird's nest peptides on mouse blood indicators are shown in Table 6, and the effects on mouse blood inflammatory factors are shown in Table 3.

[0105] Table 6 Effects of bird's nest sialic acid polysaccharide peptides on the blood count indicators of mice

[0106]

[0107]

[0108] Note: Groups I to III represent the bird's nest glycopeptides with sequence numbers 1, 2, and 3 in Table 5, respectively.

[0109] As can be seen from Table 7, there are significant differences in the content of white blood cells, neutrophils, monocytes, and eosinophils in the model group compared with the normal group (p < 0.05), indicating that the modeling is successful. After intervention in mice, each intervention group (bird's nest group, bird's nest glycopeptide group) has a significant callback effect on the content of white blood cells, neutrophils, monocytes, and eosinophils in the mouse blood compared with the model group. There is no significant difference in the intervention effect of the bird's nest group on inflammatory cells in the blood, while the bird's nest glycopeptide group I to III, which is obtained by step-by-step enzymatic hydrolysis, is significantly different from the bird's nest group, indicating that the bird's nest glycopeptide prepared by the present invention has a significant improvement effect on mouse blood inflammation.

[0110] Table 7 Effects of bird's nest sialic acid polysaccharide peptides on inflammatory factors in mouse blood

[0111]

[0112] Note: Groups I to III represent the bird's nest glycopeptides with sequence numbers 1, 2, and 3 in Table 5, respectively.

[0113] As can be seen from Table 7, there are significant differences (p < 0.05) in the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group, indicating that the modeling is successful. After intervention in mice, each intervention group (bird's nest group, bird's nest glycopeptide group) has a significant callback effect on the levels of TNF-α, IL-1β, and IL-6 in the mouse blood compared with the model group. There is no significant difference in the intervention effect on inflammatory factors in the blood compared with the bird's nest group, while the bird's nest glycopeptide I to III groups obtained by step-by-step enzymatic hydrolysis are significantly different from the other groups. This shows that the bird's nest glycopeptide prepared by the present invention has a significant improvement effect on mouse blood inflammation.

[0114] Example 5: Improvement of neuroinflammation in mice by bird's nest peptide

[0115] 5.1 Animal model establishment: The animal model establishment method is the same as that in 3.1 of Example 3.

[0116] 5.2 Neuroinflammatory Factor Assay: After obtaining the mouse cerebral cortex, the cerebral cortex was homogenized and the serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were measured using commercial ELISA kits. The procedures were as instructed by the kits. The absorbance of a 96-well plate was measured at 490 nm, and the inflammatory factors were quantified according to the standard curve. The results are shown in Table 8.

[0117] Table 8 Effects of bird's nest glycopeptides on neuroinflammatory factors in mice

[0118]

[0119] Note: Groups I to III represent the bird's nest glycopeptides with sequence numbers 1, 2, and 3 in Table 5, respectively.

[0120] As can be seen from Table 8, there are significant differences (p<0.05) in the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group, indicating that the modeling is successful. After intervention in mice, the intervention groups (bird's nest group, bird's nest glycopeptide group) have a significant callback effect on the content of TNF-α, IL-1β, and IL-6 in the mouse cerebral cortex compared with the model group. There is no significant difference in the intervention effect of the bird's nest group on the inflammatory factors in the mouse cerebral cortex. The bird's nest glycopeptide group I and group II have a certain improvement effect on neuroinflammatory factors, and the product bird's nest glycopeptide group III obtained by step-by-step enzymatic hydrolysis is significantly different from the other groups. This shows that the bird's nest glycopeptide prepared by the present invention has a significant improvement effect on mouse neuroinflammation.

[0121] Example 6: Improvement of liver inflammation in mice by bird's nest glycopeptide

[0122] 6.1 Animal model establishment: The animal model establishment method is the same as that in 4.1 of Example 4.

[0123] 6.2 Assay of Liver Inflammatory Factors: After harvesting mouse livers, the livers were homogenized and serum levels of the inflammatory factors TNF-α, IL-1β, and IL-6 were measured using commercial ELISA kits. The procedures were performed according to the kit instructions. The absorbance was measured at 490 nm in a 96-well plate, and the inflammatory factors were quantified using a standard curve. The results are shown in Table 9.

[0124] Table 9 Effects of bird's nest glycopeptides on inflammatory factors in mouse liver

[0125]

[0126]

[0127] Note: Groups I to III represent the bird's nest glycopeptides with sequence numbers 1, 2, and 3 in Table 5, respectively.

[0128] As can be seen from Table 9, there are significant differences (p<0.05) in the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group, indicating that the modeling is successful. After intervention in mice, each intervention group (bird's nest group, bird's nest peptide group) has a significant callback effect on the content of TNF-α, IL-1β, and IL-6 in the mouse liver compared with the model group. Bird's nest glycopeptide group I-II has a certain improvement effect on liver inflammatory factors, while group III is more significantly different from the other groups. This shows that the bird's nest glycopeptide prepared by the present invention has a significant improvement effect on mouse liver inflammation.

[0129] Example 7: Improvement of lung inflammation in mice by bird's nest peptide

[0130] 7.1 Animal model establishment: The animal model establishment method is the same as that in Example 3, Section 3.1.

[0131] 7.2 Pulmonary Inflammatory Factor Assay: After harvesting mouse lungs, the lungs were homogenized and serum levels of the inflammatory factors TNF-α, IL-1β, and IL-6 were measured using commercial ELISA kits. The procedures were as instructed by the kits. The absorbance of a 96-well plate was measured at 490 nm, and the inflammatory factors were quantified using a standard curve. The results are shown in Table 10.

[0132] Table 10 Effects of bird's nest peptides on inflammatory factors in the lungs of mice

[0133]

[0134] Note: Groups I to III represent the bird's nest glycopeptides with sequence numbers 1, 2, and 3 in Table 5, respectively.

[0135] As can be seen from Table 10, there are significant differences (p<0.05) in the three inflammatory factors (TNF-α, IL-1β, and IL-6) between the model group and the normal group, indicating that the modeling is successful. After intervention in mice, each intervention group (bird's nest group, bird's nest peptide group) has a significant callback effect on the content of TNF-α, IL-1β, and IL-6 in the mouse lung tissue compared with the model group. There is no significant difference in the intervention effect on the inflammatory factors of the mouse lung compared with the bird's nest group. The bird's nest glycopeptide group I and group II have a certain improvement effect on the inflammatory factors of the lung, while the difference between group III and other groups is more significant. This shows that the bird's nest glycopeptide prepared by the present invention has a significant improvement effect on mouse lung inflammation.

[0136] The above experiments show that the bird's nest sialic acid polysaccharide peptide prepared by the method of step-by-step enzymatic hydrolysis combined with acetone enrichment in the present invention has a significant improvement effect on inflammation in the blood, nerves, liver, and lungs of mice, and the improvement effect is higher than that of the group taking bird's nest, indicating that decomposing bird's nest into glycopeptides with a higher sugar content can better exert its anti-inflammatory effect, and it can be applied to the production of food, health products, and medicines.

[0137] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A bird's nest glycopeptide capable of improving lung inflammation, characterized in that: The amino acid sequence of the glycopeptide is shown as SEQ ID NO 2.

2. The bird's nest glycopeptide according to claim 1, characterized in that The glycoform of the sugar chain to which the glycopeptide is connected is Hex(4)HexNAc(5), and the structural unit of the sugar chain is as follows:

3. A method for preparing the bird's nest glycopeptide according to claim 1, characterized in that: The following steps are included in sequence: The bird's nest is soaked, crushed, hydrolyzed with protease, the enzyme is inactivated, centrifuged to obtain the supernatant, enriched, concentrated, separated and purified.

4. The method for preparing bird's nest glycopeptide according to claim 3, characterized in that: The protease hydrolysis comprises the following steps: adjusting the pH value to 8.0-8.5, adding hydrolyzing protease, performing enzymatic hydrolysis for 12 hours and then killing the enzyme, adjusting the pH value of the enzymatic hydrolysis solution to 7.0-7.4, and adding papain for enzymatic hydrolysis for 2 hours.

5. The preparation method according to claim 3, characterized in that The enrichment and concentration method includes: washing an amide-bonded silica gel filler with 100% acetonitrile, activating it with an equilibrium buffer, and uniformly loading it into a chromatography column; centrifuging the bird's nest hydrolysate, filtering it with a filter membrane, removing small molecular impurities through an ultrafiltration tube, replacing the solvent with an equilibrium buffer, concentrating it to less than 20 mL, and then performing a secondary filtration; loading the sample, washing it with an equilibrium buffer to remove impurities, performing gradient elution, detecting the fractions by LC-MS / MS, matching the retention time and characteristic ion pairs of the target glycopeptide, and combining the fractions containing the target product.

6. The preparation method according to claim 3, characterized in that The steps of the method include: (1) Soaking the bird's nest is to fully soak the bird's nest (based on dry weight) in 20-50 times water at a water temperature of 25°C for 2 hours; (2) The pulverization is performed by colloid milling, preferably with a colloid mill tooth gap of 80 μm, and the slurry obtained in step (1) is subjected to colloid milling for 20-40 minutes; (3) The protease hydrolysis comprises adjusting the pH value of the feed solution obtained in step (2) to 8.0-8.5 using HCL and NaOH, adding hydrolyzing protease at an enzyme dosage of 6000 U / mg, mixing, placing in an enzymatic fermentation tank, treating at 55-60°C and a rotation speed of 150 r / min for 12 hours, killing the enzyme after treatment, adjusting the pH value of the enzymatic hydrolyzate to 7.0-7.4 using HCL and NaOH, adding papain, mixing, placing in an enzymatic fermentation tank, and treating at 50°C and a rotation speed of 150 r / min for 2 hours; (4) The enrichment and concentration is as follows: first, the amide bonded silica gel filler is washed with 100% acetonitrile and then activated with an equilibrium buffer, and then evenly loaded into the chromatography column, and flushed at a flow rate of 2 mL / min until the column pressure is stable to ensure that there are no bubbles; the bird's nest hydrolysate is centrifuged and filtered with a 0.22 μm filter membrane, and then small molecular impurities are removed by a 3 kDa ultrafiltration tube, the solvent is replaced with an equilibrium buffer and concentrated to less than 20 mL, and then filtered twice; the sample is loaded at a flow rate of 1 mL / min, and 5 times the column volume is flushed with the equilibrium buffer to remove impurities, followed by gradient elution, 0-10 min acetonitrile is reduced from 80% to 70%, 10-50 min acetonitrile is reduced from 70% to 40%, 50-60 min acetonitrile is reduced from 40% to 20%, and the flow rate is maintained at 2 mL / min; the fractions are detected by LC-MS / MS, and the fractions containing the target product are combined after matching the target glycopeptide retention time of 47.94 min ± 2% and the characteristic ion pair; (5) The separation and purification process is as follows: the enriched liquid is concentrated through a 10 kDa ultrafiltration tube, washed with ultrapure water three times, transferred to a 10 kDa dialysis bag, dialyzed at 4°C for 24 hours, and freeze-dried to obtain glycopeptide powder; finally, the column is washed with 10 times the column volume of 100% acetonitrile and equilibrium buffer in sequence, and sealed and stored in 80% acetonitrile at 4°C.

7. A composition comprising the bird's nest glycopeptide according to claim 1 or 2, wherein the composition is prepared as food, health care product, cosmetic or medicine, and excipients allowed to be added thereto.

8. Use of the bird's nest glycopeptide according to claim 1 or 2 or the composition according to claim 7 in the preparation of an anti-inflammatory product.

9. The use according to claim 8, characterized in that The anti-inflammatory product is food, health product, cosmetic or medicine.

10. The use according to claim 8, characterized in that The application includes at least one of the following: (1) Application in the preparation of products capable of improving neuroinflammation; (2) Application in the preparation of products capable of improving lung inflammation; (3) Application in the preparation of products capable of improving liver inflammation.

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