Preparation and application of cubilose glycopeptide for prevention and adjuvant therapy of gynecological inflammation and gastrointestinal inflammation

Abstract

The invention relates to the field of cubilose peptides, in particular to preparation and application of cubilose glycopeptides capable of preventing and assisting in treating gynecological inflammation and gastrointestinal inflammation. The invention provides cubilose glycopeptide which comprises cubilose peptide, sialylated O-glycan and sialylated N-glycan, and the cubilose peptide is connected with the sialylated O-glycan and / or the sialylated N-glycan. The cubilose glycopeptide provided by the invention has unique sialylated O-glycans and N-glycans, has unique biological activity and functions and synergistic effect compared with the cubilose glycopeptide without N-glycans or O-glycans and non-sialylated cubilose peptides, and forms and enhances the inhibition effect on pathogens and the antiviral effect; therefore, the compound has unique advantages in the aspect of preventing and treating related problems. The sialic acid binding type cubilose glycopeptide has stronger antibacterial activity, especially has an inhibition effect on pathogens such as candida albicans and helicobacter pylori, and also has an antiviral effect.

Description

Technical Field

[0001] The present invention relates to the field of bird's nest peptides, and in particular to the preparation and application of bird's nest glycopeptides for preventing and assisting in the treatment of gynecological inflammation and gastrointestinal inflammation. Background Art

[0002] Sialic acid (SA) is a general term for acylation of nine-carbon sugar neuraminic acid, which is widely present in various biological tissues and is an important component of glycoproteins, oligosaccharides and glycolipids. It gives negative charge and hydrophilicity to the cell surface and plays an important role in physiological processes such as immune regulation and cell recognition. Sialic acid plays an important role in neural development, immune regulation, pathogen binding, tumor metastasis, etc. Edible bird's nest is generally defined as an edible biological product made by picking and other processes secreted by the salivary glands of several swiftlets of the genera Collocalia and Aerodramus living in Southeast Asia and neighboring islands. It has attracted attention due to its high nutritional value and market value. According to traditional Chinese medicine, bird's nest has multiple functions such as expectoration and cough, replenishing qi and nourishing the middle, nourishing yin, and moistening the lungs. Recent studies have found that bird's nest has the effects of improving neurodegenerative diseases, promoting intestinal health, whitening and anti-aging.

[0003] As people's pursuit of health and quality of life continues to improve, the demand for drugs to prevent and treat microbial and viral infections is also increasing. As a natural product with potential biological activity, sialic acid-bound bird's nest glycopeptide has broad research and application prospects, but there is a lack of relevant efficacy and mechanism analysis. Therefore, the research on its new efficacy value still needs further exploration.

[0004] N-Acetylneuraminic acid (Neu5Ac) is a type of negatively charged α-keto acid containing nine carbon atoms. It has many biological functions, such as promoting brain development, enhancing immunity, resisting and preventing hypertension, and is the most abundant type of sialic acid in mammals. Neu5Ac is widely distributed in nature, especially in dietary bird's nests. Bird's nests usually refer to nests made of a mixture of salivary gland secretions and feathers of swiftlets. They have attracted widespread attention due to their high nutritional value and market application value. According to Chinese medicine records, bird's nests, as a tonic, have many functions, such as resolving phlegm and relieving cough, replenishing qi and nourishing the middle, and nourishing yin and moistening the lungs. In recent years, many studies have found that bird's nests play an important role in improving neurodegenerative diseases, promoting intestinal health, whitening and anti-aging. The various biological functions of bird's nests are closely related to their main active ingredient, sialic acid glycoprotein, among which bound Neu5Ac plays a key role. Enzymatic hydrolysis can convert large molecular weight, water-insoluble bird's nest sialic acid glycoprotein into small molecular weight, water-soluble sialic acid glycopeptide, while also improving the bioavailability and bioaccessibility of its active ingredients.

[0005] Sialyl polysaccharides can inhibit pathogens such as Candida albicans, Helicobacter pylori and influenza virus by binding to receptors on the surface of pathogenic microorganisms. Candida albicans is a common fungus belonging to the genus Candida. Under normal circumstances, it exists in the human skin, mouth, digestive tract and vagina of women. Among them, fungal gynecological inflammation, also known as candidal vaginitis, is a common gynecological disease caused by excessive proliferation of Candida albicans. When the microbial balance in the vagina is broken, such as decreased immunity, changes in hormone levels (such as pregnancy or use of oral contraceptives), and abuse of antibiotics, Candida albicans may overgrow, thereby causing fungal gynecological inflammation. Helicobacter pylori infection is one of the common bacterial infections in the world. As a common gastric pathogen, its infection can cause inflammation of the gastric mucosa and then cause gastroenteritis.

[0006] Bird's nest glycopeptide, as a small molecule active peptide extracted from high-quality bird's nest through specific enzymatic hydrolysis technology, has shown significant potential in the prevention and adjuvant treatment of gynecological inflammation and gastrointestinal inflammation due to its excellent bioavailability and unique anti-inflammatory and antibacterial properties. Through scientific screening and refining processes, it is ensured that bird's nest glycopeptide not only retains the original rich nutrients of the bird's nest, but also significantly improves its absorption efficiency in the human body, providing a solid scientific foundation for the development of a new generation of efficient and safe nutritional health products. With the improvement of health awareness and the deepening of scientific research and technology, bird's nest glycopeptide is gradually becoming an innovative solution to the problems of gynecological inflammation and gastrointestinal inflammation, and its preparation and application technology is ushering in a new chapter of rapid development. Summary of the invention

[0007] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one object of the present invention is to provide a bird's nest glycopeptide and its use in inhibiting the growth of microorganisms. The bird's nest glycopeptide provided by the present invention has unique sialylated O-glycans and N-glycans. Compared with bird's nest peptides that do not contain N-glycans or O-glycans and are not sialylated, it has unique biological activity and function as well as synergistic effects, forming an enhanced inhibitory effect on pathogens and antiviral effects, thereby showing unique advantages in preventing and treating related problems. Sialic acid-bound bird's nest glycopeptides have stronger antibacterial activity, especially inhibitory effects on pathogens such as Candida albicans and Helicobacter pylori, as well as antiviral effects.

[0008] To this end, the first aspect of the present invention provides a bird's nest glycopeptide. In some embodiments of the present invention, the bird's nest glycopeptide comprises a bird's nest peptide, sialylated O-glycans and sialylated N-glycans, and the bird's nest peptide is connected to the sialylated O-glycans and / or sialylated N-glycans.

[0009] The bird's nest glycopeptide provided by the present invention has unique sialylated O-glycans and N-glycans, and has unique biological activity and function as well as synergistic effect compared to bird's nest peptides without N-glycans or O-glycans and non-sialylated bird's nest peptides, thereby enhancing the inhibitory effect on pathogens and the antiviral effect.

[0010] In some embodiments of the present invention, the bird's nest glycopeptide is obtained by the following method:

[0011] S1: Obtaining the pre-treated bird's nest;

[0012] S2: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1,

[0013] The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

[0014] In some embodiments of the present invention, the pre-treatment includes removing impurities from the bird's nest raw material. In some embodiments of the present invention, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin. Thus, the water-soluble protein content can be further increased.

[0015] In some embodiments of the present invention, the method for obtaining bird's nest glycopeptide further comprises:

[0016] S3: performing a second stage enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step S2 to obtain bird's nest glycopeptide E2,

[0017] The enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin, and papain. The second stage of enzymatic hydrolysis is performed to obtain more low molecular weight glycopeptides.

[0018] In some embodiments of the present invention, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease.

[0019] In some embodiments of the present invention, the bird's nest glycopeptide E2 includes bird's nest glycopeptide E2-LW less than 10 kD and bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

[0020] The second aspect of the present invention provides the use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in the bird's nest glycopeptide of the first aspect in inhibiting the growth of microorganisms. In some embodiments of the present invention, the microorganisms include Candida albicans, Helicobacter pylori and viruses containing sialic acid binding receptors on their surfaces.

[0021] At present, there are relatively few studies on the prevention and treatment of specific pathogens and antiviral effects of sialic acid-bound bird's nest glycopeptides. This limits the comprehensive evaluation of its efficacy and safety. Sialic acid and its derivatives have a certain effect on anti-microbial infections such as Candida albicans and Helicobacter pylori. This may be related to the ability of sialic acid to interfere with the growth, attachment and infection process of microorganisms. The sialic acid component in bird's nest glycopeptides further enhances the inhibitory effect on these microorganisms through its special chemical structure and biological activity. The antiviral effect of bird's nest is closely related to the bound sialic acid in its glycoprotein. Sialic acid can play an antiviral role by binding to receptors on the surface of the virus to prevent the virus from entering the cell. In addition, sialic acid may also enhance the defense against viruses by regulating the immune response of host cells.

[0022] In some embodiments of the invention, the virus containing a sialic acid binding receptor on its surface comprises an influenza virus.

[0023] The third aspect of the present invention provides the use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in the bird's nest glycopeptide described in the first aspect in the preparation of products for preventing or assisting in the treatment of gynecological inflammation and gastrointestinal inflammation.

[0024] In some embodiments of the present invention, the gynecological inflammation is caused by an overgrowth of Candida albicans.

[0025] In some embodiments of the present invention, the gastrointestinal inflammation is caused by Helicobacter pylori infection.

[0026] The inventors have found that the bird's nest glycopeptide with a special structure provided by the present invention can be used to prevent or assist in the treatment of gynecological inflammation and gastrointestinal inflammation, especially gynecological inflammation caused by excessive proliferation of Candida albicans and gastrointestinal inflammation caused by Helicobacter pylori infection.

[0027] The fourth aspect of the present invention provides a food, medicine or skin care product. In some embodiments of the present invention, the food, medicine or skin care product comprises the bird's nest glycopeptide described in the first aspect.

[0028] The various biological functions of bird's nest are related to its main bioactive component, salivary glycoprotein, among which the bound Neu5Ac plays a key role. Enzymatic hydrolysis can convert the large molecular weight, water-insoluble bird's nest salivary glycoprotein into small molecular weight, water-soluble salivary glycopeptides, which are easy to use in the fields of skin care products and food.

[0029] The fifth aspect of the present invention provides a method for preparing the bird's nest glycopeptide described in the first aspect. In some embodiments of the present invention, the preparation method comprises:

[0030] A: Get the bird's nest after pre-processing;

[0031] B: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1,

[0032] The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

[0033] In some embodiments of the present invention, the pre-treatment includes removing impurities from the bird's nest raw materials.

[0034] In some embodiments of the present invention, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin.

[0035] In some embodiments of the present invention, the method for preparing the bird's nest glycopeptide further comprises:

[0036] C: Performing a second stage of enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step B to obtain bird's nest glycopeptide E2,

[0037] Wherein, the enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin and papain.

[0038] In some embodiments of the present invention, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease.

[0039] In some embodiments of the present invention, the method for preparing the bird's nest glycopeptide further comprises:

[0040] D: Separate the bird's nest glycopeptide E2 obtained in step C to obtain bird's nest glycopeptide E2-LW less than 10 kD and bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

[0041] The present invention provides a sialic acid-bound bird's nest glycopeptide for use in the prevention and treatment of Candida albicans, Helicobacter pylori and antiviral treatment. The sialic acid-bound bird's nest glycopeptide is a bird's nest product obtained after two-stage enzymatic hydrolysis. The sialic acid-bound bird's nest glycopeptide is different from sialic acid and bird's nest peptide in efficacy. Sialic acid mainly acts on the brain and nervous system, a special polysaccharide with multiple biological activities, such as moisturizing, scavenging free radicals, promoting collagen regeneration, etc.; bird's nest peptides focus on beauty and skin care, etc.; sialic acid-bound bird's nest glycopeptide is different from the two, forming unique sialylated O-glycans and N-glycans, and relative to bird's nest peptides without N-glycans or O-glycans and non-sialylated bird's nest peptides, it has unique biological activity and function and synergistic effects, forming an enhanced inhibitory effect on pathogens and antiviral effects, thereby showing unique advantages in the prevention and treatment of related problems. Sialic acid-bound bird's nest glycopeptide has stronger antibacterial activity, especially the inhibitory effect on pathogens such as Candida albicans and Helicobacter pylori, as well as antiviral effect. This is due to the synergistic effect of sialic acid and bird's nest glycopeptide, which makes sialic acid-bound bird's nest glycopeptide have a large number of sialylated O-glycans and N-glycans at the same time, which can more effectively inhibit the growth and reproduction of Candida albicans and Helicobacter pylori and viral infection. Sialic acid-bound bird's nest glycopeptide provides a new way to prevent and treat Candida albicans, Helicobacter pylori infection and viral infection. Administration by oral, external or injection may achieve the effect of preventing the occurrence of diseases, alleviating symptoms and promoting recovery. Based on the unique biological activity and function of sialic acid-bound bird's nest glycopeptide, foods and medicines with multiple health functions can be developed. For example, corresponding foods and health foods with functions such as enhancing immunity can be developed; drugs with antibacterial and antiviral effects can also be developed for the prevention and treatment of related diseases. The sialic acid-binding bird's nest glycopeptide provided by the present invention can be used to prevent or assist in the treatment of gynecological inflammation by effectively inhibiting the growth and reproduction of Candida albicans; the sialic acid-binding bird's nest glycopeptide provided by the present invention can be used to prevent or assist in the treatment of gastrointestinal inflammation by effectively inhibiting the growth and reproduction of Helicobacter pylori.

[0042] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0044] Figure 1 The flow chart of the preparation of sialic acid-binding bird's nest glycopeptide is shown;

[0045] Figure 2The effects of different enzymes on the hydrolysis of bird's nests are shown, including: (a) the soluble protein content of bird's nests degraded by different proteases or protease combinations when treated with a single protease and two proteases; (b) the sialic acid content of each sample group; (c) and (d) the molecular weight distribution of glycoproteins of SGP-E1 after the second-stage hydrolase treatment by high-performance gel exclusion chromatography (GPC); (e) the fluorescence spectra of SGP-E1 and Desia-E1; (f) the fluorescence spectra of SGP-E2 and Desia-E2, and the significance analysis was performed by Waller-Duncan test (P<0.05, n=3);

[0046] Figure 3 The inhibitory effect of sialic acid-binding bird's nest glycopeptide on the growth of Candida albicans is shown, including: (a) the inhibitory effect of different treatment groups on Candida albicans; (b) the inhibition of Candida albicans growth by different concentrations of SGP-E2;

[0047] Figure 4 The inhibitory effect of sialic acid-binding bird's nest glycopeptide on Helicobacter pylori is shown, including: (a) the inhibitory effect of different concentrations of SGP-E1 and SGP-E2 on Helicobacter pylori; (b) the inhibitory effect of different treatment groups on Helicobacter pylori;

[0048] Figure 5 The inhibitory effect of sialic acid-binding bird's nest glycopeptide on the binding of MAL-Ⅱ to MDCK cells is shown, including: (a) The fluorescence image of the binding of Cy5 fluorescently labeled MAL-Ⅱ to MDCK cells under the action of bird's nest hydrolyzate; (b) The relative fluorescence intensity of the binding of MAL-Ⅱ to MDCK cells under the action of SGP-E1 and Desia-E1, E1-1, E1-2, E1-3, and E1-4 are 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL of E1 respectively; (c) The relative fluorescence intensity of the binding of MAL-Ⅱ to MDCK cells under the action of SGP-E2 and Desia-E2, E2-1, E2-2, E2-3, and E2-4 are 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL of E2 respectively; (d) The relative fluorescence intensity of the binding of MAL-Ⅱ to MDCK cells under the action of different substances;

[0049] Figure 6a The N-glycan profile of sialic acid-bound bird's nest glycopeptide is shown;

[0050] Figure 6b The partial sugar chain structure of sialic acid-binding bird's nest glycopeptide is shown. DETAILED DESCRIPTION

[0051] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0053] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0054] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0055] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0056] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0057] Sialyl glycans can inhibit pathogens such as Candida albicans, Helicobacter pylori and influenza viruses (such as avian influenza virus) by binding to receptors on the surface of pathogenic microorganisms. Candida albicans is an important conditional pathogen in the human gastrointestinal tract and oral cavity. It is reported that mucin, the main component of mucus, can downregulate genes related to infection in Candida albicans, inhibit its attachment to cells and biofilm formation. In addition, sugar molecules at the O-glycosylation site of mucin can bind to the surface protein of Candida albicans and interfere with its attachment to and invasion of host cells. Helicobacter pylori infection is one of the common bacterial infections in the world, which can cause peptic ulcers, gastritis and MaLT lymphoma. Helicobacter pylori can bind to mucin on the surface of oral and gastric mucosa and colonize, in which sialic acid binding adhesin (SabA) plays an important role.

[0058] The present invention obtains sialic acid-bound bird's nest glycopeptide, which is different from single sialic acid monomer and bird's nest peptide. Sialic acid monomer is obtained by biological fermentation and other ways, and is the monomer form of sialic acid; bird's nest peptide does not distinguish whether there is bound sialic acid and sugar chain, and is a peptide chain composed of amino acids obtained after protease hydrolysis of bird's nest; and sialic acid-bound bird's nest glycopeptide is a sialylated glycopeptide that links O-glycan and N-glycan at the end of bird's nest peptide and connects sialic acid at the end of sugar chain. Because of its unique structure of sialic acid-O-glycan and N-glycan-bird's nest peptide that is different from monomeric sialic acid and bird's nest peptide, it has an inhibitory effect on the growth of Candida albicans, Helicobacter pylori and avian influenza virus. Corresponding foods and health foods with functions such as enhancing immunity can be developed; medicines with antibacterial and antiviral effects can also be developed for the prevention and treatment of related diseases.

[0059] According to a specific embodiment of the present invention, the present invention provides a bird's nest glycopeptide, which comprises a bird's nest peptide, sialylated O-glycan and sialylated N-glycan, wherein the bird's nest peptide is connected to the sialylated O-glycan and / or the sialylated N-glycan.

[0060] According to a specific embodiment of the present invention, the bird's nest glycopeptide provided by the present invention, wherein the sialylated O-glycan and / or sialylated N-glycan contained therein can be connected to an amino acid at the end of the bird's nest peptide or in the middle of the bird's nest peptide chain, and the sialic acid-binding bird's nest glycopeptides of such structure are all covered within the protection scope of the present invention, and all have unique biological activities and functions as well as synergistic effects, thereby forming an enhanced inhibitory effect on pathogens and an antiviral effect.

[0061] According to a specific embodiment of the present invention, the bird's nest glycopeptide provided by the present invention contains a large amount of sialylated O-glycans and N-glycans, wherein a plurality of N-glycans contain bifurcated GlcNAc on the core pentasaccharide.

[0062] According to a specific embodiment of the present invention, the bird's nest glycopeptide is obtained by the following method:

[0063] S1: Obtaining the pre-treated bird's nest;

[0064] S2: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1,

[0065] The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

[0066] It should be noted that the pre-treatment includes not only removing impurities from the bird's nest raw materials, but also processing the bird's nest after impurities removal into different forms, such as forming a liquid bird's nest or a solid bird's nest after processing. The solid bird's nest can be in the form of freeze-dried powder, for example.

[0067] According to a preferred embodiment of the present invention, the bird's nest after pretreatment is in the form of freeze-dried powder. It should be noted that there is no particular limitation on the preparation process of the bird's nest freeze-dried powder. For example, the bird's nest freeze-dried powder can be obtained by soaking, standing, boiling, and freeze-drying the bird's nest.

[0068] According to a specific embodiment of the present invention, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin, thereby further increasing the water-soluble protein content.

[0069] According to a specific embodiment of the present invention, the method for obtaining bird's nest glycopeptide further comprises:

[0070] S3: performing a second stage enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step S2 to obtain bird's nest glycopeptide E2,

[0071] The enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin, and papain. The second stage of enzymatic hydrolysis is performed to obtain more low molecular weight glycopeptides.

[0072] The N-glycans of SGP-E1 provided by the present invention contain more sialylated N-glycans, while SGP-E2 contains relatively more N-glycans without sialic acid at the terminal.

[0073] According to a specific embodiment of the present invention, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease.

[0074] According to a specific embodiment of the present invention, the bird's nest glycopeptide E2 includes a bird's nest glycopeptide E2-LW less than 10 kD and a bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

[0075] According to a specific embodiment of the present invention, the present invention provides the use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in the aforementioned bird's nest glycopeptide in inhibiting the growth of microorganisms, including Candida albicans, Helicobacter pylori and viruses containing sialic acid binding receptors on the surface.

[0076] According to a specific embodiment of the present invention, the present invention provides the use of sialic acid-binding bird's nest glycopeptide in the prevention and treatment of Candida albicans, Helicobacter pylori and antiviral.

[0077] According to a specific embodiment of the present invention, the virus containing sialic acid binding receptors on its surface includes influenza virus.

[0078] It should be noted that the viruses containing sialic acid binding receptors on their surfaces described in the present invention include but are not limited to influenza viruses, and may also be other viruses containing sialic acid binding receptors on their surfaces, and these types of viruses are all within the protection scope of the present invention.

[0079] According to a preferred embodiment of the present invention, the virus containing sialic acid binding receptors on its surface is an avian influenza virus.

[0080] According to a specific embodiment of the present invention, the present invention provides the use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in the aforementioned bird's nest glycopeptide in the preparation of products for preventing or assisting in the treatment of gynecological inflammation and gastrointestinal inflammation.

[0081] According to a specific embodiment of the present invention, the gynecological inflammation is caused by excessive proliferation of Candida albicans.

[0082] According to a specific embodiment of the present invention, the gastrointestinal inflammation is caused by Helicobacter pylori infection.

[0083] According to a specific embodiment of the present invention, the present invention provides a food, medicine or skin care product, comprising the above-mentioned bird's nest glycopeptide.

[0084] According to a specific embodiment of the present invention, the present invention provides a method for preparing the aforementioned bird's nest glycopeptide, the preparation method comprising:

[0085] A: Get the bird's nest after pre-processing;

[0086] B: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1,

[0087] The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

[0088] According to a specific embodiment of the present invention, the pre-treatment includes removing impurities from the bird's nest raw materials.

[0089] According to a preferred embodiment of the present invention, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin.

[0090] According to a specific embodiment of the present invention, the method for preparing the bird's nest glycopeptide further comprises:

[0091] C: Performing a second stage of enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step B to obtain bird's nest glycopeptide E2,

[0092] Wherein, the enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin and papain.

[0093] According to a preferred embodiment of the present invention, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease.

[0094] According to a specific embodiment of the present invention, the method for preparing the bird's nest glycopeptide further comprises:

[0095] D: Separate the bird's nest glycopeptide E2 obtained in step C (for example, by centrifugation) to obtain bird's nest glycopeptide E2-LW less than 10 kD and bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

[0096] According to a specific embodiment of the present invention, the present invention uses sialic acid-bound bird's nest glycopeptide obtained by two-stage protease degradation as the research object, removes its terminal sialic acid by sodium periodate oxidation, and uses bird's nest desialoglycoprotein / glycopeptide (Desia-E), sialic acid monomer (Neu5Ac), 3'-sialyllactose (3'-SL) and casein glycomacropeptide (CGMP) as controls to compare its inhibitory effect on the growth of pathogenic microorganisms Candida albicans and Helicobacter pylori, as well as its inhibitory effect on influenza virus, and finally analyzes the N-sugar chain map of sialic acid-bound bird's nest glycopeptide by DSA-FACE technology to further illustrate the relationship between its structure and function. Based on the unique biological activity and function of sialic acid-bound bird's nest glycopeptide, foods and medicines with multiple health functions can be developed. For example, corresponding foods and health foods with functions such as enhancing immunity can be developed; medicines with antibacterial, antiviral and other effects can also be developed; even external skin care products with corresponding effects can be developed to prevent and treat related problems. It has broad market prospects in food, health products, skin care products, medical care and other aspects.

[0097] The scheme of the present disclosure will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present disclosure and should not be considered as limiting the scope of the present disclosure. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0098] Example 1 Preparation of sialic acid-bound bird's nest glycopeptide

[0099] The ready-to-eat dry bird's nest was rinsed with deionized water for three times and then soaked in deionized water (5 g bird's nest / 300 mL water), placed at 4°C overnight for 16 h, boiled for 30 min, freeze-dried to obtain bird's nest freeze-dried powder, and stored in a desiccating dish at room temperature.

[0100] The enzymatic hydrolysis of bird's nest freeze-dried powder is divided into two stages (reference Figure 1 ). In order to obtain a higher content of soluble protein, the first stage of enzymatic hydrolysis was carried out, including double enzyme enzymatic hydrolysis and single enzyme enzymatic hydrolysis, and the sample after enzymatic hydrolysis was SGP-E1. The double enzyme enzymatic hydrolysis combination includes pepsin + trypsin, trypsin + alkaline protease, and pronase + trypsin, and the five enzymes involved are the enzymes used for single enzyme enzymatic hydrolysis. When pepsin + trypsin enzymatic hydrolysis, bird's nest freeze-dried powder (10 mg / mL) is dissolved in 0.1M potassium chloride-hydrochloric acid buffer (pH2), pepsin (1%, w / w) is added and continuously shaken at 37°C for 2h; then the pH of the above solution is adjusted to 8 with 1M NaOH, trypsin (1%, w / w) is added and reacted at 37°C for another 2h. After the reaction is completed, it is heated in a boiling water bath for 15min to inactivate the enzyme. After cooling to room temperature, the enzymatic solution is centrifuged at 4°C, 8000rpm for 15min, the supernatant is collected, the soluble protein content is measured, and then freeze-dried. When the pancreatin + alkaline protease hydrolysis, the bird's nest freeze-dried powder (10 mg / mL) was added with pancreatin (1%, w / w, 38 ° C) and alkaline protease (1%, w / w, 60 ° C) in 0.1M potassium phosphate buffer (pH 8), and each was shaken for 2 hours. The post-treatment method was the same as described above. When the pronase + trypsin hydrolysis, the process was the same as described above, and the pronase (1%, w / w, 39 ° C, pH 8) and trypsin (1%, w / w, 39 ° C, pH 8) were reacted for 2 hours each.

[0101] The selection of hydrolase in the first stage, the results are as follows Figure 2As shown in (a), when treated with a single protease, different proteases have significant differences in the degradation effect of bird's nests. Using pepsin, trypsin, and pancreatin to degrade bird's nests can obtain more water-soluble proteins; when the bird's nest is treated with a mixture of pronase and trypsin, the water-soluble protein content is the highest. Therefore, the combination of pronase + trypsin was selected for the first stage of enzymatic hydrolysis, and the product was named SGP-E1. The first stage enzymatic hydrolysis rate was calculated to be 90.25%. The sialic acid content of SGP-E1 is 11.57%, and the total sugar content is 16.57%.

[0102] In order to obtain more bird's nest glycopeptides with smaller molecular weight, the second stage of enzymatic hydrolysis was carried out. The second stage of enzymatic hydrolysis is to add another enzyme under the conditions of the first stage. The enzymatic hydrolysis sample is SGP-E2, and the added enzymes are papain (1%, w / w), alkaline protease, pancreatin and pepsin. The optimal reaction conditions of papain are pH 6, 60°C, and the reaction time is 2h. By ultrafiltration and centrifugation of SGP-E2, the sample with a molecular weight less than 10kD is obtained as E2-LW, and the remaining part is E2-HW.

[0103] The second stage of hydrolase selection was to determine the molecular weight distribution of glycoproteins in SGP-E1 by high performance gel exclusion chromatography (GPC). Figure 2 As shown in (c) and (d), the content of glycopeptides with molecular weight less than 10 kD in SGP-E1 is relatively low, about 20%. In order to obtain more low molecular weight glycopeptides, SGP-E1 was subjected to a second stage of enzymatic hydrolysis. Figure 2 As shown in (c), after SGP-E1 was treated with alkaline protease, the content of low molecular weight glycopeptides was higher. Figure 2 From the molecular weight composition in (d), it can be seen that the proportion of glycopeptides with a molecular weight less than 10 kD in the alkaline protease treatment group was 64.25%, followed by the pepsin treatment group (59.14%), the pancreatin treatment group (40.23%), and the papain treatment group (21.52%). Therefore, alkaline protease was selected to perform the second stage enzymatic hydrolysis of SGP-E1, and the obtained product was named SGP-E2.

[0104] Example 2 Preparation of desialylated bird's nest glycopeptide

[0105] In order to destroy the terminal sialic acid of bird's nest glycopeptide, sodium periodate oxidation method was used. The specific method was to take 900 μL SGP-E1 and SGP-E2 (10 mg / mL), add 100 μL NaIO4 solution (100 mM), and shake the reaction at 4 ° C for 30 minutes. After the reaction, the SGP-E1 and SGP-E2 solutions were transferred to a 1000Da dialysis bag and dialyzed for 3 days to remove the unreacted NaIO4 in the solution. The dialyzed solution was collected and rotary evaporated to obtain the desialylated samples (Desia-E1, Desia-E2) and freeze-dried.

[0106] Example 3: Investigating whether sodium periodate has an effect on the structure of bird's nest glycoprotein

[0107] SGP-E1, SGP-E2 and Desia-E1, Desia-E2 were prepared into 10 mg / mL solutions, 1.5 mL was added to a four-way quartz cuvette, and the fluorescence spectrum of the sample solution was scanned in the range of 300-450 nm by a fluorescence spectrophotometer with an excitation wavelength of 290 nm and a voltage of 700 V ( Figure 2 (e), (f)) investigate the effect of sodium periodate on the structure of bird's nest glycoprotein.

[0108] like Figure 2 In (b), the sialic acid content of the desialylated sample Desia-E1 is 0.0917%, while that of SGP-E1 is 11.57%. Therefore, sodium periodate almost completely destroys sialic acid. Figure 2 As shown in (e) and (f), the maximum fluorescence emission wavelengths of SGP-E1 and SGP-E2 as well as desialylated Desia-E1 and Desia-E2 are all around 343 nm, and there is no obvious fluorescence peak shift, all within the range of 340-350 nm, indicating that sodium periodate only destroys the sialic acid at the terminal of bird's nest glycoprotein and does not affect its higher-order structure.

[0109] Example 4 Sialic acid-binding bird's nest glycopeptide inhibits the growth of Candida albicans

[0110] The lyophilized sample SGP-E2 was weighed and prepared into 1 mg / mL, 2 mg / mL, 10 mg / mL and 20 mg / mL with distilled water. In addition, SGP-E1, Desia-E1, Desia-E2, E2-LW and E2-HW, as well as sialic acid monomer (Neu5Ac), 3'-sialyllactose (3'-SL) and casein glycomacropeptide (CGMP) were weighed and prepared into 20 mg / mL for later use.

[0111] Take a sterile 96-well plate, first add 200 μL of sterile water to each well on the outermost edge of the 96-well plate to prevent the loss of water in the culture medium of each well during the culture process, then add 100 μL of the prepared bacterial suspension to each well in turn, and then add 100 μL of SGP-E2 of different concentrations to make the final concentrations of 0.5 mg / mL, 1 mg / mL, 5 mg / mL and 10 mg / mL respectively; add 100 μL of 20 mg / mL of SGP-E1, Desia-E1, Desia-E2, E2-LW, E2-HW, Neu5Ac (sialic acid), 3'-SL (3'-sialyllactose) and CGMP (casein glycomacropeptide) (prepared with liquid Sabouraud culture medium) to make the final concentration of 10 mg / mL; add 100 μL of sterile water to the remaining wells as a blank control, and set 3 replicates for each sample. After adding the sample to each well and mixing, the 96-well plate was placed in a constant temperature shaker at 37°C and 120 rpm for incubation. After 24 hours, the OD value of each well was measured at 600 nm using a multifunctional microplate reader.

[0112] The OD600 value at 24h reflects the growth status of Candida albicans. Figure 3 As can be seen in (a), under the same concentration conditions, the growth of Candida albicans treated with the 3'-SL group was almost the same as that of the blank control group (P>0.05), with no significant difference. The growth curve of Candida albicans treated with the Neu5Ac group was significantly different from that of the blank control group (P<0.001); at the same time, the SGP-E1 and E2-HW groups were significantly different from the Neu5Ac group; the E2-LW group was significantly different from the SGP-E1 group (P<0.001). When the terminal sialic acid of the bird's nest hydrolyzate was removed (Desia-E1, Desia-E2), its inhibitory effect was significantly weakened (P<0.001). Figure 3 (b) shows that within the concentration range of 10 mg / mL, SGP-E2 has a concentration-dependent inhibitory effect on the growth of Candida albicans, and 0.5 mg / mL, 1 mg / mL, 5 mg / mL and 10 mg / mL of SGP E2 have significant inhibitory effects on Candida albicans (P<0.001). It can be seen that within a certain concentration range (about 10 mg / mL), sialic acid-binding bird's nest glycopeptide has a significant inhibitory effect on the growth of Candida albicans, and its terminal sialic acid plays a decisive role. This may be because sialic acid in sialic acid-binding bird's nest glycopeptide can bind to the receptor on Candida albicans. However, Neu5Ac, 3'-SL and CGMP, which also contain sialic acid, have significantly weakened inhibitory effects on the growth of Candida albicans.

[0113] Example 5 Sialic acid-binding bird's nest glycopeptide inhibits the growth of Helicobacter pylori

[0114] SGP-E1, SGP-E2, Desia-E1, and Desia-E2 samples were added to Brucella broth medium and prepared to a concentration of 20 mg / mL, wherein SGP-E1 and SGP-E2 were further diluted to 1 mg / mL, 2 mg / mL, and 10 mg / mL, respectively. In addition, E2-LW, E2-HW, Neu5Ac, 3'-SL, and CGMP were added to Brucella broth medium and prepared to a concentration of 20 mg / mL for standby use.

[0115] Take a sterile 96-well plate, first add 200 μL of sterile water to the outermost wells, then add 100 μL of the prepared Helicobacter pylori suspension to the inner wells, and then add 100 μL of Brucella broth medium with and without samples, where the medium group without samples is the blank control, and each sample is set up with 3 replicates. The 96-well plate is placed at 37°C, microaerobic, 160rpm and cultured for 72 hours. After the culture is completed, the OD value of each well is measured at 600nm with an ELISA instrument, and the antibacterial rate (%) is calculated as follows.

[0116]

[0117] The results of sialic acid-binding bird's nest glycopeptide against influenza virus and Helicobacter pylori growth inhibition are as follows Figure 4 As shown. It can be seen that 0.5mg / mL, 1mg / mL, 5mg / mL and 10mg / mL of SGP-E1 and SGP-E2 all have an inhibitory effect on the growth of Helicobacter pylori, and the inhibitory effect shows a concentration-dependent increasing trend. Figure 4 As shown in (a), compared with SGP-E1, under the same concentration conditions, SGP-E2 has a more significant inhibitory effect on Helicobacter pylori (P<0.001). Figure 4 As shown in (b), under the same concentration (10 mg / mL), E2-LW (molecular weight <10kD) had the strongest inhibitory effect on Helicobacter pylori, with an inhibition rate of 37.6%, and the inhibition was significantly different from other groups (P<0.001), followed by SGP-E2, with an inhibition rate of 29.13%, followed by SGP-E1 and E2-HW, with inhibition rates of 23.35% and 14%, respectively, and there were significant differences compared with other groups (P<0.001). In the control group, the inhibition rates of Neu5Ac and CGMP were only 8.04% and 9.45%, respectively, with no significant difference between the two (P>0.05). When the sialic acid was removed from the bird's nest hydrolyzate (Desia-E1, Desia-E2), its inhibitory effect on Helicobacter pylori almost disappeared. Therefore, similarly, sialic acid-binding bird's nest glycopeptide has a concentration-dependent inhibitory effect on Helicobacter pylori within a certain concentration range, and the terminal sialic acid plays an important role in its inhibition of the growth of Helicobacter pylori.

[0118] Example 6 Sialic acid-binding bird's nest glycopeptide against influenza virus function

[0119] Add 1 mL of DMSO to 1 mg of sulfonated Cy5-NHS activated ester fluorescent powder, shake and mix to fully dissolve it, and keep it away from light at room temperature for 1 hour. Take 0.5 mL of MAL-Ⅱ (2 mg / mL) to a 5 mL centrifuge tube, add 0.06 mL of activated Cy5-NHS fluorescent dye, mix well, keep it away from light at room temperature, and shake for 3 hours. After the reaction is completed, use 10 mM HEPES buffer solution to ultrafilter and centrifuge in a 2 mL (10 kDa) ultrafiltration centrifuge tube to remove the unreacted fluorescent dye. The purified dye solution is stored at 4°C away from light. MDCK cells are cultured in MEM complete culture medium at 37°C and a 5% CO2 incubator. When the MDCK cells are cultured to a confluence of 80%, they are digested, centrifuged, resuspended and counted, and their density is adjusted to 10 4 / mL, inoculated into confocal culture dishes, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence is cultured to 60%-70%, wash with PBS, add 1mL 4% paraformaldehyde to fix, and add PBS containing 5% BSA to block the cells for 1h at room temperature. Cy5-labeled MAL-Ⅱ and samples of different concentrations were added to the culture dish and incubated overnight at 4°C in the dark with shaking. Then DAPI staining solution was added to stain the MDCK cells, and photos were taken under the DAPI and Cy5 channels with a laser confocal microscope, and the fluorescence intensity was analyzed with Image J software. The results are shown in Figure 5 As shown in (a). Note: MAL-Ⅱ is the full name of Maackia amurensis lectin II, which is a plant lectin that can be extracted from Maackia amurensis. MAL II has a high specific binding ability to sialic acid-containing sugar chains. MAL-Ⅱ can bind to MDCK cells and is used as a mimic of avian influenza virus.

[0120] The research results of sialic acid-binding bird's nest glycopeptide against influenza virus are as follows Figure 5 As shown, MAL-Ⅱ in the blank control group can obviously bind to MDCK cells; when 0.5 mg / mL SGP-E1 is added, the binding of MAL-Ⅱ to MDCK cells is significantly inhibited, and when 0.5 mg / mL SGP-E2 is added, the binding of MAL-Ⅱ to MDCK cells is also inhibited, but the inhibitory effect is not as significant as Desia-E1. Figure 5As shown in (b) and (c), within the concentration range of 10 mg / mL, as the concentration of SGP-E1 and SGP-E2 increases, the inhibitory effect on the binding of MAL-Ⅱ to MDCK cells increases. After the terminal sialic acid is removed, the inhibitory effect on the binding of MAL-Ⅱ to MDCK cells is significantly weakened. Figure 5 As shown in (d), under the same concentration (10 mg / mL), SGP-E1 had the most significant inhibitory effect on the binding of MAL-Ⅱ to MDCK cells (P<0.001), with an inhibition efficiency of 86.28%, followed by SGP-E2, with an inhibition efficiency of 75.45%, and the inhibition efficiencies of Desia-E1 and Desia-E2 were only 45.83% and 25.62%. In the control group, the inhibition efficiency of CGMP derived from milk reached 68.95%, which was close to that of SGP-E2; the inhibition efficiency of sialic acid monomer Neu5Ac was only 53.31%, and that of 3'-SL was only 26.92%. The above results show that the sialic acid-binding bird's nest glycopeptide has the strongest inhibitory effect on the binding of MAL-Ⅱ to MDCK cells, and the sialic acid glycan structure plays a key role.

[0121] Example 7 Analysis of N-glycan spectrum of sialic acid-bound bird's nest glycopeptide

[0122] The N-sugar chain structure in sialic acid-bound bird's nest glycopeptide was analyzed by DSA-FACE technology. First, the sugar chains on the EBN hydrolysate were hydrolyzed with peptide-N-glycosidase F (PNGase F), then fluorescently labeled with APTS (8-amino-1,3,6-pyrenetrisulfonic acid), and then the labeled samples were purified by magnetic beads. Finally, capillary electrophoresis was performed by DNA sequencer to obtain the N-sugar chain electrophoresis spectrum.

[0123] The results of the N-glycan spectrum of sialic acid-bound bird's nest glycopeptide are as follows Figure 6a , 6bAs shown in the figure, there are 10 identifiable sugar peaks in the N-sugar chain spectrum of sialic acid-bound bird's nest glycopeptide, most of which have sialic acid structures, and some known sugar chain structures have been marked in the figure. Among them, sugar peak No. 5 is speculated to be A2BG2S1, sugar peak No. 6 is speculated to be FA2BG2S1 or FA2G2S1, and sugar peak No. 7 is speculated to be A2G1. Among the sugar peaks of SGP-E1 and E2-HW, the highest peak is No. 2 FA2BG2S2, and the highest peak in E2 and E2-LW is No. 7 A2G1. The content of N-sugar chain peaks with sialic acid structure in SGP-E1 is higher, while the content of No. 7 and No. 8 sugar peaks near the back in SGP-E2 is higher. It may be that an additional enzyme is added to SGP-E2 to make the sugar chain unfold more thoroughly, so its antibacterial effect is better. SGP-E1 has a better antiviral effect, which may be related to its containing more N-sugar chains with sialic acid.

[0124] As can be seen from the above examples, the sialic acid-bound bird's nest glycopeptide derived from bird's nest shows good anti-pathogen and anti-avian influenza virus capabilities, and the sialylated structure plays an important role. Within a certain concentration range, SGP-E1 and SGP-E2 show stronger inhibitory effects on the growth of Candida albicans than Neu5Ac, CGMP and 3'-SL, and sialic acid glycopeptide E2-LW. For Helicobacter pylori, both SGP-E1 and SGP-E2 show concentration-dependent inhibitory effects, and E2-LW has a stronger inhibitory effect on Helicobacter pylori than Ne5Ac, CGMP and 3'-SL. In the inhibitory effect on influenza virus, within a certain concentration range, SGP-E1 and SGP-E2 both show concentration-dependent inhibitory effects on avian influenza virus. Compared with the control group Neu5Ac, CGMP and 3'-SL, SGP-E1 has a stronger inhibitory effect, which may be closely related to the more α-2,3-linked sialic acid structures on the bird's nest sialic acid glycoprotein. The N-glycan map of sialic acid-bound bird's nest glycopeptide was analyzed by DSA-FACE technology, and 10 different sugar peaks were obtained, most of which were accompanied by sialic acid.

[0125] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0126] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A bird's nest glycopeptide, characterized in that: The invention comprises a bird's nest peptide, sialylated O-glycan and sialylated N-glycan, wherein the bird's nest peptide is connected with the sialylated O-glycan and / or the sialylated N-glycan.

2. The bird's nest glycopeptide according to claim 1, characterized in that The bird's nest glycopeptide is obtained by the following method: S1: Obtaining the pre-treated bird's nest; S2: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1, The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

3. The bird's nest glycopeptide according to claim 2, characterized in that The pre-treatment includes removing impurities from the bird's nest raw materials; Optionally, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin.

4. The bird's nest glycopeptide according to claim 2 or 3, characterized in that The method for obtaining the bird's nest glycopeptide further comprises: S3: performing a second stage enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step S2 to obtain bird's nest glycopeptide E2, Wherein, the enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin, and papain; Optionally, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease.

5. The bird's nest glycopeptide according to claim 4, characterized in that The bird's nest glycopeptide E2 includes a bird's nest glycopeptide E2-LW less than 10 kD and a bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

6. Use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in any one of claims 1-5 in inhibiting the growth of microorganisms, characterized in that: The microorganisms include Candida albicans, Helicobacter pylori, and viruses containing sialic acid binding receptors on their surfaces.

7. The use according to claim 6, characterized in that The viruses containing sialic acid binding receptors on their surfaces include influenza viruses.

8. Use of bird's nest glycopeptide E1, bird's nest glycopeptide E2, bird's nest glycopeptide E2-LW and bird's nest glycopeptide E2-HW in any one of claims 1 to 5 in the preparation of products for preventing or assisting in the treatment of gynecological inflammation and gastrointestinal inflammation.

9. The use according to claim 8, characterized in that The gynecological inflammation is caused by excessive proliferation of Candida albicans; Optionally, the gastrointestinal inflammation is caused by Helicobacter pylori infection.

10. A food, medicine or skin care product, characterized in that: The invention comprises the bird's nest glycopeptide according to any one of claims 1 to 5.

11. The method for preparing the bird's nest glycopeptide according to any one of claims 1 to 5, characterized in that: include: A: Get the bird's nest after pre-processing; B: performing a first-stage enzymatic hydrolysis on the pre-treated bird's nest to obtain bird's nest glycopeptide E1, The enzymes used in the first stage of enzymatic hydrolysis include at least two selected from pepsin, trypsin, pancreatin, alkaline protease, and pronase.

12. The preparation method according to claim 11, characterized in that: The pre-treatment includes removing impurities from the bird's nest raw materials; Optionally, the enzymes used in the first stage of enzymatic hydrolysis are pronase and trypsin; Optionally, the preparation method of the bird's nest glycopeptide further comprises: C: Performing a second stage of enzymatic hydrolysis on the bird's nest glycopeptide E1 obtained in step B to obtain bird's nest glycopeptide E2, Wherein, the enzyme used in the second stage of enzymatic hydrolysis is any one of alkaline protease, pepsin, pancreatin, and papain; Optionally, the enzyme used in the second stage of enzymatic hydrolysis is alkaline protease; Optionally, the preparation method of the bird's nest glycopeptide further comprises: D: Separate the bird's nest glycopeptide E2 obtained in step C to obtain bird's nest glycopeptide E2-LW less than 10 kD and bird's nest glycopeptide E2-HW greater than or equal to 10 kD.

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