An anti-virus active component of enriched bound sialic acid in edible bird's nest polysaccharide, a preparation method and application thereof

Abstract

The application relates to the field of food biotechnology, and discloses an anti-virus edible bird's nest polysaccharide active component rich in combined sialic acid, a preparation method and application thereof. The edible bird's nest polysaccharide active component has a weight-average molecular weight of 30 kDa to 36 kDa, a molecular weight distribution coefficient of 1.1 to 1.2, a combined sialic acid content of greater than or equal to 24.0%, and a total sugar content of greater than or equal to 57.0%. The preparation method comprises the following steps: after an edible bird's nest raw material is pretreated in a boiling water bath, the edible bird's nest raw material is subjected to alkaline protease and neutral protease hydrolysis in sequence; the supernatant is alcohol precipitated, redissolved, and subjected to papain hydrolysis; after enzyme inactivation, the ethanol concentration is adjusted to 75% to 85% for precipitation, and drying is performed to obtain the active component. The method can efficiently enrich the edible bird's nest polysaccharide and retain the combined sialic acid structure of the edible bird's nest polysaccharide, and the obtained component has the effects of antioxidation, pathogenic microorganism inhibition and intestinal microecosystem regulation.

Description

Technical Field

[0001] This application relates to the food field, specifically to an antiviral bird's nest polysaccharide active component enriched with bound sialic acid, its preparation method, and its application. Background Technology

[0002] Bird's nest, a traditional and precious tonic, has main active ingredients including protein (about 50%), carbohydrates (i.e., polysaccharides, about 20%), and sialic acid (about 10%).

[0003] Currently, influenced by the notion that smaller molecules have superior activity, research and development of deep-processed bird's nest products in the industry mainly focuses on bird's nest peptides. This involves converting large protein molecules into smaller peptides through protease hydrolysis, aiming to improve the product's digestibility and absorption. Larger polysaccharide components are often considered impurities and removed or not studied in depth. From the perspective of bird's nest processing technology research, traditional processes often neglect the targeted development and utilization of polysaccharide components. The carbohydrates (i.e., polysaccharides) in bird's nest mainly exist in the form of glycoproteins, with polysaccharide chains acting as bridges, connecting proteins at one end and sialic acid at the other. However, in the conventional preparation of bird's nest peptides, polysaccharide chains are often byproducts and not effectively enriched, or their structure is damaged during harsh acid-base treatments, leading to a significant loss of bound sialic acid with important biological activity.

[0004] Therefore, in order to maximize the nutritional value of bird's nest, it is urgent to study the active components of bird's nest polysaccharides and their preparation methods. Summary of the Invention

[0005] This application aims to overcome the aforementioned shortcomings of the prior art and provide an antiviral bird's nest polysaccharide active component rich in bound sialic acid. This application further provides a highly efficient preparation method for this active component, which can obtain bird's nest polysaccharides with high purity and high bound sialic acid content at a high yield. This application also provides applications of this active component in anti-oxidation, antimicrobial activity, and as a prebiotic for regulating intestinal health.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] In a first aspect, this application provides a method for preparing an antiviral bird's nest polysaccharide active component rich in bound sialic acid. According to an embodiment of this application, the method includes pretreating bird's nest raw materials by heating to obtain a pretreated material; sequentially subjecting the pretreated material to alkaline protease hydrolysis and neutral protease hydrolysis to obtain a preliminary hydrolysate; taking the supernatant of the preliminary hydrolysate, adding ethanol to achieve a final ethanol concentration of 75%–85%, allowing it to settle at low temperature, collecting the precipitate, redissolving the precipitate, and then subjecting it to papain hydrolysis; inactivating the papain hydrolysis product to obtain a secondary hydrolysate; taking the supernatant of the secondary hydrolysate, adding ethanol to achieve a final ethanol concentration of 75%–85%, allowing it to settle at low temperature, collecting the precipitate, and drying it to obtain the antiviral bird's nest polysaccharide active component rich in bound sialic acid. The method according to the embodiments of this application can efficiently prepare and completely preserve the antiviral bird's nest polysaccharide active component rich in bound sialic acid.

[0008] In some aspects of this application, the pretreatment is achieved by crushing the bird's nest raw material, soaking it in water, and subjecting it to a boiling water bath.

[0009] In some aspects of this application, the alkaline protease hydrolysis treatment is carried out under the conditions of pH 10.0~11.0 and temperature 45℃~55℃ for 3~5 hours.

[0010] In some aspects of this application, the neutral protease hydrolysis treatment is carried out under conditions of pH 6.5 to 7.5 and temperature 45°C to 55°C for 3 to 5 hours.

[0011] In some aspects of this application, the papain enzymatic hydrolysis is carried out under conditions of pH 6.0 to 7.0 and temperature 45°C to 55°C for 4 to 6 hours.

[0012] In some aspects of this application, the amount of enzyme added in the alkaline protease hydrolysis treatment, the neutral protease hydrolysis treatment, and the papain hydrolysis treatment is independently 10 × 10⁻⁶, calculated as an enzyme-to-protein ratio. 4 ~90×10 4 U / g of raw material protein.

[0013] In some aspects of this application, the temperature for the low-temperature settling is 2°C to 8°C, and the time is 8 to 16 hours.

[0014] In some aspects of this application, the boiling water bath treatment time is 1 to 3 hours.

[0015] In some aspects of this application, the ratio of the bird's nest raw material to the water is 1:30 to 1:50.

[0016] In a second aspect of this application, we provide an antiviral bird's nest polysaccharide active component rich in bound sialic acid. According to an embodiment of this application, the bird's nest polysaccharide active component is prepared by the method described in the first aspect of this application. The bird's nest polysaccharide active component according to the embodiment of this application represents the first time a bird's nest polysaccharide core active substance with a clear structure and uniform composition has been obtained.

[0017] In some aspects of this application, the weight-average molecular weight of the bird's nest polysaccharide active component is 30 kDa to 36 kDa, and the molecular weight distribution coefficient is 1.1 to 1.2; wherein, the mass percentage of bound sialic acid in the bird's nest polysaccharide active component is ≥24.0%, and the total sugar content is ≥57.0%; the total sugar content is the sum of the contents of N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose, and fucose; the monosaccharide composition of the bird's nest polysaccharide active component includes N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose, and fucose, and contains Neu5Ac-Gal-GlcNAc or Neu5Ac-Gal polysaccharide structural fragments. According to the embodiments of this application, by clearly defining its molecular weight range, highly uniform molecular weight distribution, high bound sialic acid content, and specific monosaccharide composition and structural fragments, a bird's nest polysaccharide core active substance with a clear structure and uniform composition has been obtained for the first time.

[0018] In a third aspect of this application, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid, as described in the second aspect of this application, in the preparation of products with antioxidant functions.

[0019] In a fourth aspect of this application, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid as described in the second aspect of this application in the preparation of products for inhibiting Escherichia coli, Bacillus subtilis and / or having antiviral functions.

[0020] In a fifth aspect of this application, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid, as described in the second aspect of this application, in the preparation of intestinal microecological regulators or prebiotic foods.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 The chromatogram shows the monosaccharide composition of the bird's nest polysaccharide in Example 1;

[0024] Figure 2 The image shows the GPC-MALLS analysis of bird's nest polysaccharides in Example 1.

[0025] Figure 3 The image shows the ultraviolet scan spectrum of bird's nest polysaccharides in Example 1;

[0026] Figure 4 The infrared scanning spectrum of bird's nest polysaccharide in Example 1;

[0027] Figure 5 The chromatogram of polysaccharide in bird's nest polysaccharide in Example 1 is shown below.

[0028] Figure 6 This is a graph showing the free radical scavenging rate of different concentrations of bird's nest polysaccharides in Example 2;

[0029] Figure 7 This is a diagram showing the antibacterial relationship of bird's nest polysaccharides in Example 3;

[0030] Figure 8 This is a diagram illustrating the inhibitory effect of bird's nest polysaccharide on MAL-II binding MDCK cells in Example 4.

[0031] Figure 9 This is a graph showing the pH changes during the fermentation of bird's nest polysaccharides in Example 5;

[0032] Figure 10 This is a graph showing the short-chain fatty acid content in Example 5;

[0033] Figure 11 This is a diagram showing the differences in gut microbiota composition in Example 5;

[0034] Figure 12 This is a diagram showing the alpha diversity analysis of gut microbiota in Example 5;

[0035] Figure 13 This is a diagram showing the scratch healing process of HaCaT cells in Example 6;

[0036] Figure 14 This is a graph showing the change in transmembrane resistance (TEER) of cells after DSS induction in Example 6;

[0037] Figure 15 This is a graph showing the change in alkaline phosphatase specific activity after DSS induction in Example 6.

[0038] Note: Figure 1 The above figure shows the characteristic peak of Neu5Ac determined by high performance liquid chromatography (HPLC); Figure 1The figure below shows the characteristic peaks of Fuc, Gal, Man, GlcNAc and GalNAc determined by ion chromatography (IC). The two figures together constitute the complete analytical spectrum of the monosaccharide composition of this active component 1. Figure 2 The image shows the GPC-MALLS analysis chromatogram of bird's nest polysaccharides. The first peak on the left is the main peak of the target polysaccharide. Because the content of the target peak is very high, multiple data points need to be collected during the acquisition period, resulting in a dotted pattern in the spectrum. This is a normal phenomenon in GPC-MALLS detection of high-concentration polysaccharide samples. Figure 5 In this application, the abbreviation "SA" refers to N-acetylneuraminic acid (Neu5Ac). Specifically: SAGal represents N-acetylneuraminic acid-galactose; Gal3GlcNAc represents galactose-galactose-galactose-N-acetylglucosamine; SAGalGlcNAc represents N-acetylneuraminic acid-galactose-N-acetylglucosamine; SAGalGlcNAcMan represents N-acetylneuraminic acid-galactose-N-acetylglucosamine-mannose; and ManGlcNAc represents mannose-N-acetylglucosamine. Figure 6 and Figure 8 The letters a, b, c, etc. in the table are statistical significance markers, used to visually demonstrate whether the differences between different groups are statistically significant. Different letters represent significant differences, and the same letter represents no significant differences. Detailed Implementation

[0039] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0040] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0041] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this application, all other technical and scientific terms used in this application shall have the meaning commonly understood by one of ordinary skill in the art to which this application pertains.

[0042] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0043] In this application, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0044] In this document, the term "bound sialic acid" refers to sialic acid molecules covalently linked to the ends of polysaccharides or sugar chains via glycosidic bonds (usually α-2,3 or α-2,6 bonds), primarily N-acetylneuraminic acid (Neu5Ac). It differs significantly from free sialic acid (sialic acid molecules existing alone without glycosidic bonds) in structure, stability, and biological activity. This application emphasizes the preservation of this sialic acid structure with its specific linkage.

[0045] In this paper, the term "bird's nest polysaccharide active component" refers to a class of high molecular weight polysaccharide components extracted and purified from bird's nest through a specific process. Its core characteristics are that it has the specific molecular weight range described in this application, a high content of bound sialic acid, and a clear monosaccharide composition and structural fragments.

[0046] In this document, the term "weight-average molecular weight (Mw)" refers to a commonly used parameter in polymer science for characterizing the size of polymer molecules, which is obtained by statistical averaging over molecular weight. In this application, the molecular size of the active component was determined by gel permeation chromatography-multi-angle laser light scattering (GPC-MALLS) and ranged from 30 kDa to 36 kDa.

[0047] In this document, the term "molecular weight distribution coefficient (Mw / Mn)" refers to the polydispersity index (PDI), which is the ratio of weight-average molecular weight (Mw) to number-average molecular weight (Mn). The closer this value is to 1, the more uniform the size of the polymer molecules in the sample. In this application, this coefficient is 1.1 to 1.2, indicating that the obtained polysaccharide components have a high degree of homogeneity.

[0048] In this paper, the term "Neu5Ac-Gal-GlcNAc / Neu5Ac-Gal structural fragment" refers to the characteristic oligosaccharide chain sequence present in the active components of the bird's nest polysaccharide. Neu5Ac represents N-acetylneuraminic acid (sialic acid), Gal represents galactose, and GlcNAc represents N-acetylglucosamine. These fragments indicate that sialic acid is linked to glycosyl groups such as galactose via specific glycosidic bonds, and is the basic unit constituting its bound sialylated structure.

[0049] In this document, the term "enzyme-to-substrate ratio" refers to the ratio of the enzyme activity units (U) of the added protease to the mass (g) of the substrate to be hydrolyzed (protein in the raw bird's nest) in the preparation method of this application. The unit is U / g. It is a key parameter for controlling the degree of enzymatic hydrolysis.

[0050] In this paper, the term "U (enzyme activity unit)" refers to the amount of enzyme required to catalyze the conversion of a substrate into 1 micromolar product within 1 minute under specific conditions (such as a certain temperature and pH), and is defined as 1 enzyme activity unit.

[0051] In this document, the term "prebiotic activity" refers to the property of a substance that, without being digested by the host's digestive tract, selectively stimulates the growth and / or activity of one or more beneficial bacteria in the host's gut, thereby producing a beneficial health effect on the host. This application evaluates this activity through in vitro fermentation experiments (such as promoting the production of short-chain fatty acids and regulating gut microbiota structure).

[0052] In this paper, the term "DPPH free radical scavenging assay" refers to a commonly used in vitro method for evaluating antioxidant activity. DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable free radical, deep purple in color. Antioxidants can reduce it, causing the color to lighten. The free radical scavenging rate can be calculated by measuring the change in absorbance at 517 nm.

[0053] In this article, the term "ABTS free radical scavenging assay" refers to another common in vitro method for evaluating antioxidant activity. ABTS (2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid) is oxidized to produce a blue-green ABTS. + • Free radical cations can be decolorized by antioxidants, and their scavenging ability can be evaluated by measuring the change in absorbance at 734 nm.

[0054] In this article, the term "short-chain fatty acids (SCFAs)" mainly refers to organic fatty acids containing 1-6 carbon atoms, such as acetic acid, propionic acid, and butyric acid, produced by the fermentation of carbohydrates such as dietary fiber by gut microbiota. They are important metabolites for gut health and have various physiological functions, including maintaining the intestinal barrier and anti-inflammation.

[0055] In this paper, the term "transmembrane resistance (TEER)" primarily refers to an electrophysiological indicator used to assess the integrity of the monolayer epithelial or endothelial cell barrier. A higher resistance value indicates better intercellular junctions and stronger barrier function. This application uses a Caco-2 cell model to measure TEER to evaluate the protective effect of bird's nest polysaccharides on the intestinal barrier.

[0056] In this paper, the term "16S rRNA gene sequencing" primarily refers to a molecular biology technique used to analyze the composition and diversity of microbial communities. By amplifying and determining specific regions of the 16S rRNA gene sequence of microorganisms in a sample, the types of bacteria present and their relative abundance can be identified. This application utilizes this technique to analyze the effects of bird's nest polysaccharides on the structure of the gut microbiota.

[0057] First, the understanding of the fine structure of bird's nest polysaccharides is still relatively vague. There is a lack of systematic characterization of its sugar chain composition, molecular weight uniformity, and sialylation pattern, which restricts the in-depth understanding of its structure-activity relationship and the development of high-value products.

[0058] Secondly, although existing studies have shown that bird's nest possesses various biological activities, these are mainly concentrated on bird's nest peptides and whole bird's nest extracts. In-depth research and comparison of the specific functional activities of bird's nest polysaccharides are lacking, especially regarding their mechanisms of action in combating pathogenic microorganisms, regulating gut microbiota, and protecting the intestinal barrier, which remain unclear.

[0059] Furthermore, traditional methods of hot water extraction combined with ethanol precipitation are inefficient for extracting glycoprotein matrices like bird's nest, resulting in low polysaccharide yields and difficulty in effectively removing coexisting proteins, leading to poor product purity. Enzymatic hydrolysis using a single protease often results in incomplete degradation due to the complex structure of bird's nest proteins, leading to incomplete polysaccharide release and potentially affecting the integrity of the polysaccharide's active structure. Acid hydrolysis to release sialic acid disrupts glycosidic bonds, converting the more bioactive bound sialic acid into a free state, and may also damage the overall structure of the polysaccharide chain. However, the applicant's detailed research has revealed that bound sialic acid exhibits superior bioactivity compared to its free form.

[0060] Although bird's nest as a whole is known to have antioxidant and repair functions, the unique activity and advantages of bird's nest polysaccharide components rich in bound sialic acid, extracted through specific processes, in terms of skin barrier repair, regulation of intestinal health, and inhibition of specific pathogenic microorganisms, have not yet been fully revealed and utilized.

[0061] Therefore, there is an urgent need in this field to develop a preparation method that can efficiently enrich the active components of bird's nest polysaccharides and retain their natural bound sialic acid structure to the maximum extent, while also exploring the unique bioactivity of this component that is different from bird's nest peptides, so as to maximize the utilization of the nutritional value of bird's nest resources.

[0062] Based on this, this application provides an antiviral bird's nest polysaccharide active component rich in bound sialic acid. This application further provides a highly efficient preparation method for this active component, which can obtain bird's nest polysaccharides with high purity and high bound sialic acid content at a high yield. This application also provides the applications of this active component in anti-oxidation, antimicrobial activity, and as a prebiotic for regulating intestinal health, which will be described in detail below.

[0063] Methods for preparing active components of bird's nest polysaccharides

[0064] In a first aspect, this application provides a method for preparing an antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid. According to an embodiment of this application, the method includes pretreating bird's nest raw materials by heating to obtain a pretreated product; sequentially subjecting the pretreated product to alkaline protease hydrolysis and neutral protease hydrolysis to obtain a preliminary hydrolysate; taking the supernatant of the preliminary hydrolysate, adding ethanol to achieve a final ethanol concentration of 75%~85%, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or a range between these values ​​(76%~85%, 77%~85%); allowing the mixture to settle at low temperature; and collecting the precipitate. The precipitate is reconstituted and then subjected to papain enzymatic hydrolysis. The papain hydrolysis product is inactivated to obtain a secondary hydrolysate. The supernatant of the secondary hydrolysate is taken, and ethanol is added to make the final ethanol concentration in the system reach 75%~85%, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or a range between the two, 76%~85%, 77%~85%. The mixture is allowed to stand at low temperature to precipitate, and the precipitate is collected and dried to obtain the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid. According to the method of this application embodiment, firstly, a boiling water bath is used to open the dense tertiary structure of bird's nest protein, exposing more enzyme cleavage sites, which is beneficial for alkaline protease to perform large-scale peptide chain cleavage; secondly, a neutral protease is used to specifically cleave the exposed hydrophobic regions (aromatic amino acids) to prevent protein fragment re-aggregation; finally, after removing some small molecule impurities, papain's broad substrate specificity is used to precisely modify and remove residual stubborn peptides directly linked to polysaccharide chains. This multi-enzyme synergy and site complementarity strategy overcomes the shortcomings of single enzymatic hydrolysis sites and many dead zones, significantly improving the yield of polysaccharides. After the combined treatment with alkaline and neutral protease, this intermediate alcohol precipitation step is introduced, which has key phase transfer and impurity removal functions: firstly, it realizes the cleaning and reset of the enzymatic hydrolysis system. A large number of free amino acids, short peptide fragments, inorganic salt ions and other byproducts generated in the first two enzymatic hydrolysis processes are not easily precipitated under this alcohol precipitation condition, and are thus removed with the supernatant. This provides a pure reaction environment with low background interference and low product inhibition for subsequent papain enzymatic hydrolysis, allowing papain to bind to the substrate more efficiently. Secondly, it initially enriches large glycopeptide substrates. At this stage, the precipitate mainly consists of incompletely degraded glycoprotein / glycopeptide complexes tightly bound to polysaccharides. Redissolving the precipitate increases the substrate concentration, which facilitates papain's targeted recognition of remaining sites (such as Arg, Lys, and Phe), enabling site-specific removal of stubborn protein residues.The final ethanol concentration of 75%–85% was selected, combined with low-temperature settling, to achieve the dual effects of molecular weight cutoff and structural screening: First, it precisely cuts off active components with specific molecular weights. When the ethanol concentration is controlled within the 75%–85% range, the bird's nest polysaccharide active components with a weight-average molecular weight of 30 kDa–36 kDa described in this application can undergo maximum precipitation (maximum yield); while degradation fragments or free sugars with too small a molecular weight either do not precipitate at this concentration or have already been removed in previous steps, thus ensuring a high degree of uniformity in the molecular weight distribution of the final product (PDI 1.1–1.2). Second, it solidifies and stabilizes the bound structure. The high-concentration ethanol environment rapidly reduces the water activity, promoting the formation of dense precipitates through hydrogen bonding between polysaccharide molecules. This process effectively freezes the sugar chain conformation, preventing structural collapse or hydrolysis loss of bound sialic acid during drying, ensuring that the final powder retains its natural bioactive conformation after reconstitution.

[0065] According to the method of the embodiments of this application, bird's nest polysaccharides with high purity and high bound sialic acid content can be obtained with high yield, that is, the active components of bird's nest polysaccharides are prepared efficiently and completely preserved.

[0066] According to an embodiment of this application, the heating pretreatment can be a boiling water bath or a hot water immersion at 80-100°C.

[0067] According to an embodiment of this application, the heating pretreatment is achieved by crushing the bird's nest raw material, soaking it in water, and then subjecting it to a boiling water bath treatment.

[0068] According to the embodiments of this application, the alkaline protease hydrolysis is carried out at a pH of 10.0-11.0 and a temperature of 45℃-55℃ for 3-5 hours. According to the embodiments of this application, strictly controlling the alkaline protease hydrolysis at pH 10.0-11.0 and 45℃-55℃ for 3-5 hours maximizes the activation of the enzyme activity, efficiently hydrolyzing specific peptide bonds (such as Ala, Leu, Val residues), achieving preliminary and large-scale degradation of bird's nest proteins. Subsequently, the system is adjusted to pH 6.5-7.5 for neutral protease hydrolysis, also at a mild temperature (45℃-55℃) for 3-5 hours. This condition ensures that the neutral protease specifically cleaves aromatic amino acid sites (Tyr, Phe, Trp), further breaking down the protein network, while avoiding potential damage to released polysaccharide chains and sensitive sialic acid structures caused by extreme pH. This sequential, conditionally precisely controllable complex enzymatic hydrolysis strategy synergistically covers a wider range of protein hydrolysis sites, thereby significantly improving the protein removal rate and polysaccharide release, laying a key foundation for obtaining high-purity products.

[0069] According to embodiments of this application, the neutral protease hydrolysis is performed for 3-5 hours at a pH of 6.5-7.5 and a temperature of 45°C-55°C. According to embodiments of this application, deep degradation is carried out targeting specific cleavage sites. After the preliminary treatment with boiling water bath and alkaline protease, the three-dimensional compact structure of the bird's nest protein is opened, fully exposing the previously encapsulated hydrophobic regions. The neutral protease utilizes this opportunity to precisely identify and cleave the peptide bonds of hydrophobic aromatic amino acid residues such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). This further fragmentation targeting the hydrophobic core effectively shortens large polypeptide molecules into shorter small peptide fragments, effectively preventing the re-aggregation or precipitation of proteins due to hydrophobic interactions during the pH correction from alkaline to neutral, thus ensuring the homogeneity of the hydrolysate. Compared to the strongly alkaline environment of alkaline protease, the working environment of neutral protease (pH 6.5-7.5) is more moderate. Enzymatic hydrolysis at this stage avoids both the β-elimination reaction of glycosidic bonds that may occur under prolonged strong alkaline conditions (i.e., preventing the loss of sialic acid) and the risk of hydrolysis under acidic conditions. This step maintains a high rate of proteolytic hydrolysis while preserving the acid- and base-sensitive bound sialic acid (Neu5Ac) and its core glycopeptide structural fragment (Neu5Ac-Gal-GlcNAc) to the greatest extent possible, laying the material foundation for the high bioactivity of the final product.

[0070] According to an embodiment of this application, the papain enzymatic hydrolysis is performed at a pH of 6.0–7.0 and a temperature of 45°C–55°C for 4–6 hours. Specifically, the conditions for papain hydrolysis are: pH 6.0–7.0, temperature 45°C–55°C, and time 4–6 hours. This embodiment of the application sets the papain hydrolysis at pH 6.0–7.0 and 45°C–55°C for 4–6 hours, achieving a dual optimization purpose. According to an embodiment of the application, a mild near-neutral environment and suitable temperature maintain good catalytic activity of papain, specifically hydrolyzing basic amino acids (Arg, Lys) and other sites, as detailed in Table 1 below. This process also removes protein fragments or protein residues tightly bound to polysaccharides that may remain after the first two enzymatic hydrolysis steps. Secondly, the relatively long hydrolysis time (4-6 hours) ensures the sufficiency of this hydrolysis, aiming to minimize protein residue in the final product, thereby increasing the purity of polysaccharides to a higher level, while ensuring that the entire hydrolysis process is always under mild conditions that do not break glycosidic bonds (especially the bonds connecting sialic acid).

[0071] Table 1: Enzyme cleavage sites

[0072]

[0073] It should be further explained that, as those skilled in the art will understand, in the preparation scheme of this application, heat treatment via a boiling water bath can effectively loosen the bird's nest protein from its compact state, while simultaneously dissolving some water-soluble small molecules and some inorganic residues. Preliminary enzymatic hydrolysis is performed using alkaline and neutral proteases, taking into full account the differences in enzyme cleavage sites and enzyme activities to maximize the degree of hydrolysis (enzymatic cleavage efficiency). The scheme of this application considers the enzymatic hydrolysis efficiency and synergistic effect, achieving a "1+1>2" effect in the hydrolysis results. Alcohol precipitation is also a key technical point in the preparation process. In the chemical reaction process, the role of enzymes is merely to build a bridge for the reaction; the endpoint of the reaction is not the termination of the reaction, but rather a reaction equilibrium reached during the enzymatic hydrolysis process. Alcohol precipitation can effectively remove small molecule products (byproducts) from the reaction, extracting enzymatic hydrolysis intermediates for further purity improvement. Finally, papain is selected, also based on the structural characteristics of bird's nest protein, which is conducive to further enzymatic hydrolysis. Meanwhile, this process addresses the shortcomings of using compound enzymes, with different enzymes acting on different reaction substrates, and no enzymatic competition between enzymes, thus achieving efficient preparation.

[0074] In summary, the method for preparing antiviral bird's nest polysaccharide active components rich in bound sialic acid provided in this application fully considers the types of enzymes, the sequence of enzymatic hydrolysis, the combination of enzymes, and the hydrolysis conditions. Different enzymes have different cleavage sites. Based on the structural characteristics of bird's nest, alkaline protease and neutral protease are used in combination for enzymatic hydrolysis to fully destroy the bird's nest protein while retaining the polysaccharide components. Alcohol precipitation and papain are then used to further improve the purity and content of the polysaccharides. Furthermore, the pH value remains neutral to slightly alkaline throughout the entire enzymatic hydrolysis process, ensuring mild hydrolysis conditions that do not damage the polysaccharide structure or bound sialic acid.

[0075] According to embodiments of this application, the amounts of the alkaline protease, the neutral protease, and the papain added, based on the enzyme-to-protein ratio, are each independently 10 × 10⁻⁶. 4 ~90×10 4 U / g of raw material protein, for example, can be 10×10 4 U / g, 20×10 4 U / g, 30×10 4 U / g, 40×10 4 U / g, 50×10 4 U / g, 60×10 4 U / g, 70×10 4 U / g, 80×10 4 U / g, 90×10 4 U / g or the range between the two, 20 × 10⁻⁶. 4 ~90×10 4U / g raw protein, 30×10 4 ~90×10 4 U / g of raw protein. According to the embodiments of this application, the addition amounts of alkaline protease, neutral protease, and papain are uniformly limited to 10 × 10⁻⁶ U / g raw material protein. 4 ~90×10 4 The wide and effective range of U / g raw material protein brings significant process advantages and application flexibility.

[0076] According to the embodiments of this application, the temperature for low-temperature static precipitation is 2℃~8℃, for example, it can be 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃ or a range between the two, such as 3℃~8℃, 4℃~8℃, and the time is 8~16 hours, for example, it can be 8, 9, 10, 11, 12, 13, 14, 15, 16 hours or a range between the two, such as 9~16 hours, 10~16 hours.

[0077] According to an embodiment of this application, the boiling water bath treatment time is 1 to 3 hours, for example, it can be 1 hour, 1.3 hours, 1.5 hours, 1.7 hours, 2 hours, 2.3 hours, 2.5 hours, 2.7 hours, 3 hours or a range between the two, such as 1.3 to 3 hours or 1.5 to 3 hours.

[0078] According to the embodiments of this application, the ratio of the bird's nest raw material to the water is 1:30 to 1:50, for example, it can be 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:42, 1:44, 1:46, 1:48, 1:50 or a range between the two, such as 1:32 to 1:50 or 1:34 to 1:50.

[0079] Antiviral bird's nest polysaccharide active components rich in bound bird's nest sialic acid

[0080] In a second aspect of this application, we provide an antiviral bird's nest polysaccharide active component rich in bound sialic acid. According to an embodiment of this application, the bird's nest polysaccharide active component is prepared by the method described in the first aspect of this application. The bird's nest polysaccharide active component according to the embodiment of this application represents the first time a bird's nest polysaccharide core active substance with a clear structure and uniform composition has been obtained.

[0081] According to embodiments of this application, the weight-average molecular weight of the active component of bird's nest polysaccharide is 30 kDa to 36 kDa, and the molecular weight distribution coefficient is 1.1 to 1.2; wherein, the mass percentage of bound sialic acid (N-acetylneuraminic acid) in the active component of bird's nest polysaccharide is ≥24.0%, and the total sugar content (including sialic acid) is ≥57.0%; the total sugar content is the sum of the contents of N-acetylneuraminic acid (Neu5Ac), galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), mannose (Man), and fucose (Fuc); the monosaccharide composition of the active component of bird's nest polysaccharide includes N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose, and fucose, and contains Neu5Ac-Gal-GlcNAc or Neu5Ac-Gal polysaccharide structural fragments. According to the active components of bird's nest polysaccharides in the embodiments of this application, a core active substance of bird's nest polysaccharides with a clear structure and uniform composition was obtained for the first time by clearly defining its molecular weight range (30-36 kDa), highly uniform molecular weight distribution (Mw / Mn 1.1-1.2), high bound sialic acid content (≥24.0%), and specific monosaccharide composition and structural fragments. The active components of bird's nest polysaccharides in the embodiments of this application, with a high content of bound sialic acid polysaccharides, exhibit excellent antioxidant, inhibitory, antiviral, and prebiotic activities that regulate the intestinal microecology.

[0082] It should be noted that the total sugar in the embodiments of this application refers to the sum of monosaccharides in bird's nest, including N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose and fucose.

[0083] According to embodiments of this application, the mass percentage content of the bound sialic acid is 24.0% to 37.7%, for example, it can be 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 37.7%, or a range between the two, such as 24.0% to 26%, 24.0% to 27%, 24.0% to 28%, 24.0% to 29%, 25.0% to 37.7%, or 26.0% to 37.7%.

[0084] According to embodiments of this application, the total sugar content is 57.0% to 66.1%, for example, it can be 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 66.1%, or a range between the two, such as 58.0% to 66.1% or 59.0% to 66.1%.

[0085] use

[0086] In a third aspect, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid, as described in the second aspect, in the preparation of products with antioxidant functions. According to embodiments of this application, the bird's nest polysaccharide active component exhibits significant scavenging ability against DPPH free radicals, ABTS free radicals, and hydroxyl free radicals, showing a dose-dependent effect. This indicates that the component itself possesses good antioxidant activity and can be used as a natural antioxidant ingredient in the preparation of products requiring antioxidant functions, such as additives to delay food oxidation or health foods and skincare products that help resist oxidative stress, providing new directions and data support for its application in the health industry.

[0087] use

[0088] In a fourth aspect, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound sialic acid, as described in the second aspect of this application, in the preparation of products for inhibiting *Escherichia coli*, *Bacillus subtilis*, and / or possessing antiviral functions. According to embodiments of this application, this application systematically discloses and confirms for the first time that the bird's nest polysaccharide active component has a significant inhibitory effect on specific pathogenic microorganisms (such as *Escherichia coli* and *Bacillus subtilis*) and virus mimics. Particularly important, comparative experiments revealed that its inhibitory effect is significantly better than that of similar polysaccharides without bound sialic acid and free sialic acid monomers, proving that its antimicrobial activity is inseparable from its unique and complete bound sialic acid structure. This provides a solid experimental basis and unique application value for this component in the development of novel antibacterial and antiviral functional products or adjuvant formulations.

[0089] use

[0090] In a fifth aspect, this application provides the use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid, as described in the second aspect of this application, in the preparation of intestinal microecological regulators or prebiotic foods. According to embodiments of this application, the bird's nest polysaccharide active component can be effectively utilized by the human intestinal flora, significantly promoting the growth of beneficial bacteria (such as Bacteroides), inhibiting potentially harmful bacteria (such as Proteobacteria), increasing the diversity of intestinal microorganisms, and promoting the production of short-chain fatty acids (such as acetic acid and butyric acid), lowering intestinal pH, and exhibiting excellent prebiotic properties. Simultaneously, this component can also promote the proliferation of intestinal epithelial cells and enhance their barrier function. These effects collectively indicate that this component can be used to prepare foods, health products, or special medical foods that regulate intestinal flora balance and improve intestinal health, or as a raw material for prebiotics, showing broad application prospects in the field of intestinal health.

[0091] To enable those skilled in the art to better understand the technical solutions of this application, and to ensure that the key physicochemical parameters defined in this application (including weight-average molecular weight, molecular weight distribution coefficient, bound sialic acid content, and total sugar content, etc.) have clear definition standards and reproducibility, the general determination methods and experimental conditions involved in the embodiments of this application are first described in detail below. Unless otherwise specifically noted in the specific embodiments, the structural characterization and parameter data of all samples in this application are strictly determined according to the following methods:

[0092] 1. Reagent and Instrument Instructions

[0093] The chemical reagents used in the embodiments of this application, including methanol, formic acid, acetonitrile, sulfuric acid, and ammonium sulfate, were all of analytical or chromatographic grade and purchased from conventional commercial channels such as TEDIA (USA) and Shanghai Maclean Biochemical Reagent Co., Ltd. The instruments and equipment used included an LC-30AD ultra-high performance liquid chromatography-mass spectrometry system (equipped with an LC MS-8050), a 940 ion chromatograph (Metroporte, Switzerland); an LC20A high performance liquid chromatograph (Shimadzu, Japan); a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA); a T18DS25 homogenizer (Sartorius, Germany); a 3K-15 centrifuge (Sigma, Germany); an MS3BS025 vortex mixer (Eka, Germany); and an automated Kjeldahl nitrogen analyzer (KJELTEC 8400, FOSS, Denmark), and other standard laboratory instruments, all of which were calibrated according to standard operating procedures before use.

[0094] 2. Specific measurement methods

[0095] I. Method for determining molecular weight and molecular weight distribution coefficient (GPC-MALLS method)

[0096] In this application, the weight-average molecular weight (Mw) and molecular weight distribution coefficient (Mw / Mn) of the active components of bird's nest polysaccharides were determined using gel permeation chromatography-multi-angle laser light scattering (GPC-MALLS). Specific conditions are as follows:

[0097] 1. Instrumentation System: A U3000 high-performance liquid chromatograph (Thermo Fisher Scientific) was used, coupled with a DAWN HELEOS II multi-angle laser light scattering detector (Wyatt Technology) and an Optilab T-rEX differential refractive index detector (Wyatt Technology).

[0098] 2. Chromatographic columns: guard column (6×40 mm, 6μm) and TM 1000 analytical column (7.8×300 mm, 12μm).

[0099] 3. Mobile phase: 0.2 mol / L NaNO3 solution, filtered through a 0.22 μm aqueous filter membrane and degassed by ultrasonication.

[0100] 4. Testing conditions:

[0101] (1) Flow rate: 0.9 mL / min;

[0102] (2) Column temperature: 45℃;

[0103] (3) Injection volume: 100 μL;

[0104] (4) Sample preparation: The sample was prepared into a 1 mg / mL solution and filtered through a 0.22 μm filter membrane;

[0105] (5) dn / dc value: 0.146 mL / g.

[0106] 5. Data processing: Data was collected and calculated using ASTAR 7.3.2 software.

[0107] II. Methods for determining the content of bound sialic acid and monosaccharide composition (acid hydrolysis-HPLC method)

[0108] In this application, "bound sialic acid" refers to N-acetylneuraminic acid covalently linked to the polysaccharide chain via glycosidic bonds. Since the preparation process in this application has removed most of the free small molecules through alcohol precipitation and dialysis, to ensure data rigor, the content of bound sialic acid in bird's nest polysaccharides is calculated by subtracting free sialic acid from total sialic acid, specifically referring to the Chinese National Standard (GB 31614-2023):

[0109] 1. Total sialic acid: Weigh 0.1 g of bird's nest polysaccharide sample, mix with 10 mL of 0.05 mol / L hydrochloric acid solution, incubate at 80℃ for 40 min, shaking every 5 min during the incubation period, remove and cool to room temperature, dilute to the mark with water, and mix well. Filter using a 0.22 μm syringe filter.

[0110] 2. Free sialic acid: Accurately measure 0.1 g of bird's nest polysaccharide sample, place it in a 10 mL volumetric flask, add acetonitrile-0.1% phosphoric acid (60+40) solution to make up to the mark, transfer the liquid through a 0.22 μm filter membrane, and run it on the instrument.

[0111] 3. HPLC detection conditions:

[0112] (1) Instrument: LC20A high performance liquid chromatograph.

[0113] (2) Chromatographic column: 300SAX strong anion exchange chromatography (4.6 mm × 250 mm, 5 μm).

[0114] (3) The mobile phase was 0.1% phosphoric acid:acetonitrile (40:60, volume ratio). The column temperature was 30 ℃, the flow rate was 1.0 mL / min, the injection volume was 10 μL, and the detection wavelength was 205 nm.

[0115] 4. Quantitative calculation:

[0116] The content was calculated using the external standard method with N-acetylneuraminic acid (Neu5Ac) standard as a reference.

[0117] III. Determination of Monosaccharides in Bird's Nest Polysaccharides

[0118] In this application, the monosaccharide composition of bird's nest polysaccharides was determined by acid hydrolysis and then by ion chromatography (IC).

[0119] 1. Sample hydrolysis (release of bound sialic acid):

[0120] (1) Accurately weigh 5 mg of bird's nest polysaccharide sample, add 2 mL of 2 mol / L trifluoroacetic acid (TFA), and seal with nitrogen gas.

[0121] (2) Hydrolyze at 110℃ for 4 hours.

[0122] (3) After hydrolysis, methanol is added and evaporated under reduced pressure to remove TFA. This process is repeated several times, and the residue is re-dissolved in water.

[0123] 3. HPLC detection conditions:

[0124] (1) Instrument: 940 series ion chromatograph.

[0125] (2) Chromatographic column: Metrosep Carb 2 (4.0 mm × 250 mm, 5 μm).

[0126] (3) Mobile phase: Isocratic elution with a mixed solution of 1.0 mmol / L sodium hydroxide and 1.0 mmol / L sodium acetate.

[0127] (4) Flow rate: 0.5 mL / min.

[0128] (5) Detector: Pulse Ampere Detector.

[0129] (6) Column temperature: 32℃.

[0130] IV. Method for Determination of Total Sugar Content

[0131] In this application, the total sugar content was determined using the phenol-sulfuric acid method.

[0132] (1) Prepare a standard curve using glucose (or galactose / sialic acid mixed standard) as the standard.

[0133] (2) Accurately pipette 1 mL of the sample solution, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid, shake to mix, and heat in a boiling water bath for 15 minutes. After cooling to room temperature, measure the absorbance at 490 nm and calculate the total sugar content according to the standard curve.

[0134] V. Methods for analyzing polysaccharide structures

[0135] In this application, the polysaccharide structure was analyzed by mass spectrometry. Specifically, 1 mL of a 10 mg / mL bird's nest polysaccharide solution was placed in a hydrolysis tube, 0.5 mL of 2 mol / L trifluoroacetic acid was added, and the mixture was hydrolyzed at 100℃ for 1 h. Then, 0.5 mL of water was added, and the trifluoroacetic acid was removed by rotary evaporation. This process was repeated three times. The volume was then adjusted to 5 mL with water, filtered through a 0.22 μm filter membrane, and analyzed by HPLC-MS / MS.

[0136] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0137] Example 1: Preparation of bird's nest polysaccharides rich in bound sialic acid

[0138] Take 100 g of Indonesian white bird's nest, crush it, and pass it through a 60-mesh sieve. Add 4000 mL of distilled water at a material-to-liquid ratio of 1:40, soak at room temperature for 30 minutes, and then treat in a boiling water bath for 2 hours. After cooling to room temperature, adjust the pH to 10.5 with 0.1 mol / L Na2CO3 solution, and add 160 g of alkaline protease (enzyme activity 20 × 10⁻⁶). 4 The enzyme was hydrolyzed for 4 hours with shaking in a 50℃ incubator. After hydrolysis, the enzyme was inactivated by heating in a 100℃ water bath for 10 minutes. After cooling, the pH was adjusted to 7.0 with acetic acid, and 40 g of neutral protease (enzyme activity 20 × 10⁻⁶ U / g) was added. 4 The enzyme was hydrolyzed in a 50℃ incubator with shaking for 4 hours, followed by enzyme inactivation at 100℃ for 10 minutes. The hydrolysate was centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected. Four volumes of anhydrous ethanol were added, and the mixture was allowed to stand at 4℃ for 12 hours to precipitate. The precipitate was collected by centrifugation, reconstituted with 1000 mL of distilled water, and 40 g of papain (enzyme activity 80 × 10⁻⁶ U / g) was added. 4 The enzyme was hydrolyzed at 50℃ with shaking for 5 hours, and then inactivated at 100℃ for 10 minutes. The mixture was centrifuged again at 10000 rpm for 5 minutes, and the supernatant was collected. The supernatant was precipitated with 80% ethanol, and the precipitate was collected by centrifugation. The residual ethanol was removed by water bath, and the mixture was freeze-dried to obtain 25.4 g of bird's nest polysaccharide, with a yield of 25.4%.

[0139] Product characterization results: Total sugar content 66.1% (of which sialic acid content 24.9%), protein content 20.7%, moisture 12.0%, ash content 3.9%. Relative molecular weight 33 kDa, Mw / Mn 1.125. Monosaccharide composition (percentage of total sugar): Neu5Ac 37.66%, Gal 25.28%, GlcNAc 18.54%, GalNAc 11.27%, Man 5.48%, Fuc 1.77%. The chromatogram of the monosaccharide composition of the product is shown below. Figure 1 As shown. The molecular weight distribution and parameters obtained from GPC-MALLS analysis are as follows. Figure 2 As shown in Table 2, the product molecular weight is uniform. It's important to note that the GPC-MALLS analysis chromatogram for bird's nest polysaccharides is a molecular weight determination chromatogram. The chromatogram is generated by the device collecting data points through detector responses. The first peak is the target peak for bird's nest polysaccharides. Due to the high content of the target peak, multiple data points need to be collected in one acquisition cycle, resulting in a dotted pattern in the spectrum, which is normal. Blue represents the values ​​monitored by the differential refractive index detector; the peak values ​​indicate the number of components in the polysaccharide extract. Red represents the molecular weight distribution; the concentrated color area represents the molecular weight range, which is the main function of this chromatogram. The ultraviolet scanning spectrum is shown below. Figure 3 The presence of an absorption peak at 260-280 nm indicates the presence of a small number of protein residues. Figure 4The infrared scanning spectrum shown indicates: 3435 cm⁻¹ -1 (OH stretching vibration), 2937 cm -1 (CH stretching vibration), 1639 cm -1 (C=O stretching vibration), 1071 cm -1 (CO stretching vibration), consistent with typical polysaccharide characteristic absorption peaks. For example... Figure 5 The glycan scanning chromatogram shown further confirms its unique glycan structure characteristics.

[0140] Table 2: Molecular weight parameters of bird's nest polysaccharides

[0141]

[0142] Example 2: Evaluation of the antioxidant activity of bird's nest polysaccharides rich in bound sialic acid

[0143] The bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 was prepared into solutions of 50, 100, 250, 500, 1000, and 2500 μg / mL, respectively.

[0144] (1) DPPH free radical scavenging experiment: 2 mL of polysaccharide solutions of different concentrations were taken and 2 mL of 0.04 mg / mL DPPH ethanol solution was added. After reacting at room temperature in the dark for 30 minutes, the absorbance was measured at 517 nm. The results showed that the DPPH free radical scavenging rate gradually increased with the increase of concentration. When the concentration was 2500 μg / mL, the scavenging rate reached 37.1%.

[0145] (2) ABTS free radical scavenging experiment: 0.4 mL of polysaccharide solutions of different concentrations were taken, and 4 mL of ABTS free radical working solution was added. After reacting in the dark for 6 minutes, the absorbance was measured at 734 nm. The results showed that when the concentration was 2500 μg / mL, the ABTS free radical scavenging rate was 35.8%.

[0146] (3) Hydroxyl radical scavenging experiment: 1 mL of polysaccharide solutions of different concentrations were taken, and 1 mL of ferrous sulfate, 1 mL of salicylic acid and 1 mL of hydrogen peroxide were added in sequence. After incubating in a water bath at 37℃ for 60 minutes, the absorbance was measured at 510 nm. The results showed that when the concentration was 2500 μg / mL, the hydroxyl radical scavenging rate was 15.0%.

[0147] The curves showing the change in the scavenging rates of the three free radicals as a function of concentration are as follows: Figure 6 As shown, the antioxidant activity and dose-dependent properties of the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 are demonstrated.

[0148] exist Figure 6In this context, the letters a, b, c, etc., are statistical significance markers, used to visually demonstrate whether the differences between different groups are statistically significant. Different letters represent significant differences, while the same letter represents no significant differences.

[0149] Taking the DPPH free radical scavenging rate chart as an example, there was no significant difference in DPPH free radical scavenging rates between 50 mg / L and 100 mg / L. However, significant differences were observed at concentrations of 100 mg / L, 250 mg / L, 500 mg / L, 1000 mg / L, and 2500 mg / L. Similarly, it can be seen that the ABTS free radical scavenging rate showed significant differences at each concentration.

[0150] Taking the hydroxyl radical scavenging rate chart as an example, there is no significant difference in hydroxyl radical scavenging rate below 500 mg / L (all contain the same letter 'c'), but as the concentration increases, there are significant differences at 1000 mg / L and 2500 mg / L (the difference is marked as 'b' and 'a').

[0151] Example 3: Evaluation of the antibacterial activity of bird's nest polysaccharides rich in bound sialic acid

[0152] Escherichia coli and Bacillus subtilis were activated and cultured in LB liquid medium, and the bacterial suspension concentration was adjusted to 10. 7 -10 8 CFU / mL. Using a two-fold dilution method, the 20 mg / mL bird's nest polysaccharide solution rich in bound sialic acid prepared in Example 1 was serially diluted to 0.625, 1.25, 2.5, 5, and 10 mg / mL. For each concentration, 0.2 mL of bacterial suspension and 3 mL of LB medium were added. After incubation at 35°C for 24 hours, the OD value was measured at 600 nm, and the inhibition rate was calculated.

[0153] The results showed that the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 exhibited good antibacterial activity against both *Escherichia coli* and *Bacillus subtilis*, and this activity was dose-dependent. At a concentration of 2.5 mg / mL, the inhibition rate against *Escherichia coli* was 57.5%, and against *Bacillus subtilis* was 56.3%; at a concentration of 10 mg / mL, the inhibition rates increased to 74.3% and 84.3%, respectively. The relationship between the inhibition rate and concentration is as follows: Figure 7 As shown.

[0154] Example 4: Evaluation of the antiviral activity of bird's nest polysaccharides rich in bound sialic acid

[0155] Using Huaihuai lectin MAL-II as a mimic of avian influenza virus, the inhibitory effect of bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 on its binding to MDCK cells was evaluated after Cy5 fluorescent labeling. The results showed that the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 significantly inhibited the binding of MAL-II to MDCK cells in a dose-dependent manner. At a concentration of 10 mg / mL, the inhibition efficiency reached 84.5%. After removing the terminal sialic acid from the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 by oxidation with sodium periodate, the inhibition efficiency significantly decreased to 36.4%. Simultaneously, compared with monomers of the same solubility of sialic acid, the inhibition efficiency was 13.8%, demonstrating that the bound sialylated structure of the bird's nest polysaccharide plays a key role in antiviral activity. The inhibitory effect is as follows: Figure 8 As shown.

[0156] Example 5: Evaluation of prebiotic activity of bird's nest polysaccharides rich in bound sialic acid

[0157] Fresh fecal samples were collected from healthy volunteers (BMI 18.5-25, no digestive system diseases, and no antibiotic use within the past 3 months). The samples were diluted to 25% (w / v) with pre-sterilized PBS in an anaerobic incubator and filtered before use. 50 mg of the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1, 4.8 mL of BCM culture medium, and 0.2 mL of fecal suspension were added to a 250 mL fermentation flask. The flask was anaerobically incubated at 37°C, and samples were taken for analysis at 0, 6, 12, 24, and 48 hours. A corresponding control group was also set up, consisting of 50 mg of water added to a 250 mL fermentation flask. This control group did not include the bird's nest polysaccharide rich in bound sialic acid; the remaining experimental procedures were the same as for the polysaccharide treatment. Figure 11 , Figure 12 In the diagram, letter A represents the control group, and letter B represents the polysaccharide group.

[0158] 1. pH Changes: The results showed that the pH of the bird's nest polysaccharide group rich in bound sialic acid prepared in Example 1 decreased significantly after 12 hours and stabilized at 6.5-7.0 after 48 hours, significantly lower than that of the blank control group (P<0.05). The pH changes are as follows... Figure 9 As shown.

[0159] 2. Short-chain fatty acid determination results showed that the total short-chain fatty acid content of the bird's nest polysaccharide group rich in bound sialic acid prepared in Example 1 was 362.42 μg / mL, significantly higher than the 326.18 μg / mL of the control group, with a significant increase in acetic acid and butyric acid content (P<0.05). Specific contents are as follows... Figure 10 As shown.

[0160] 3. Gut Microbiota Structure Analysis: 16S rRNA gene sequencing analysis showed that, at the phylum level, the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 significantly increased the relative abundance of Bacteroides (from 18% in the control group to 41%), and decreased the relative abundance of Proteobacteria and Enterobacteriaceae (from 37% in the control group to 28%). At the genus level, the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 increased the abundance of beneficial bacteria such as Blautia, Faecalibacterium, Phocaeicola, and Collinsella, and decreased the abundance of harmful bacteria such as Megamonas. Differences in gut microbiota composition are shown in the figure below. Figure 11 As shown.

[0161] 4. Alpha diversity analysis showed that the Shannon and Simpson indices of the bird's nest polysaccharide group rich in bound sialic acid prepared in Example 1 were significantly higher than those of the control group, indicating increased species richness and diversity of the microbial community. The analysis results are as follows: Figure 12 As shown.

[0162] Example 6: Evaluation of the cell repair activity of bird's nest polysaccharides rich in bound sialic acid

[0163] 1. HaCaT cell scratch healing assay: HaCaT immortalized human keratinocytes were seeded into 6-well plates, 6 × 10⁶ cells per well. 5 Cells were cultured for 24 hours to allow complete cell adhesion. Scratches were created on the cell monolayer using a pipette tip. After washing twice with PBS, fresh culture medium containing different concentrations of the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 (0.0625%, 0.125%, and 0.25%) was added. Photos were taken at 0, 12, and 24 hours, and the scratch area was analyzed using ImageJ software to calculate the healing rate. The results showed that the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 significantly promoted the migration and proliferation of HaCaT cells, accelerating scratch healing. When the concentration was 0.125%, the healing rate reached 77% after 24 hours, significantly higher than the control group (36%) and the bird's nest peptide group at the same concentration (52%). The scratch healing status is shown in the figure below. Figure 13 As shown.

[0164] 2. Experiment on the protection of intestinal barrier function by Caco-2 cells:

[0165] (1) Transmembrane resistance (TEER): Caco-2 cells were seeded in Transwell chambers and cultured for 14-21 days until the TEER value was stably greater than 400 Ω·cm. 2Intestinal barrier damage was induced by adding a culture medium containing bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 and adding 4% DSS. TEER values ​​and alkaline phosphatase specific activity were measured daily. Results showed that DSS treatment significantly reduced TEER values, while the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 significantly increased TEER values, promoted tight cell junctions, and thus protected intestinal barrier function. TEER value changes are shown below. Figure 14 As shown.

[0166] (2) Alkaline phosphatase (AKP) specific activity: Alkaline phosphatase specific activity was measured in the same model. The results showed that the bird's nest polysaccharide rich in bound sialic acid prepared in Example 1 could significantly increase the alkaline phosphatase specific activity of cells after DSS damage, and its effect was significantly stronger than that of the bird's nest peptide group at the same concentration. The changes in specific activity are as follows: Figure 15 As shown.

[0167] Comparative Example

[0168] Comparative Example 1: Enzymatic hydrolysis by a single protease

[0169] This comparative example used the same raw materials and pretreatment methods as Example 1. In the enzymatic hydrolysis step, only alkaline protease was added for a single hydrolysis (enzyme dosage, pH, temperature, and time were the same as in Example 1). After hydrolysis, the enzyme was directly inactivated, centrifuged, precipitated with alcohol (80% ethanol), and freeze-dried. Subsequent hydrolysis with neutral protease and papain was not performed; the remaining steps were the same as in Example 1. The results showed that the polysaccharide yield was only 17.9%, the total sugar content was 20.6%, and the protein content was 41.6%, significantly lower than the preparation effect of Example 1. This indicates that compound enzymatic hydrolysis can more thoroughly degrade proteins and improve the release rate and purity of polysaccharides.

[0170] Comparative Example 2: Traditional water extraction and alcohol precipitation method

[0171] This comparative example uses a traditional hot water extraction method without adding any proteases. The bird's nest raw material was added to water at a ratio of 1:40, and extracted three times in a 100°C water bath, each time for 2 hours. The three extracts were combined, concentrated to an appropriate volume, and then ethanol was added to a final concentration of 80% for precipitation. The precipitate was collected and freeze-dried. The remaining steps were the same as in Example 1. The results showed that the polysaccharide yield was only 9.2%, the total sugar content was 30.4%, and the protein content was as high as 52.3%, indicating that simple water extraction is difficult to effectively separate polysaccharides and proteins, and the yield and purity are far lower than the method of this application.

[0172] Comparative Example 3: Purification and Separation Methods

[0173] This comparative example uses the exact same raw materials, pretreatment, complex enzymatic hydrolysis, and enzyme inactivation steps as Example 1. Only the final "alcohol precipitation purification" step is replaced with one of the following four conventional purification methods; all other parameters remain unchanged:

[0174] Comparative Example 3-1 (Sevag method): Deproteinization was performed using a mixture of chloroform and n-butanol (volume ratio 5:1).

[0175] Comparative Example 3-2 (Salting-out Method): Salting-out precipitation was carried out using a saturated ammonium sulfate solution.

[0176] Comparative Example 3-3 (Trichloroacetic Acid Method): Trichloroacetic acid was added to a final concentration of 15% to precipitate the protein.

[0177] Comparative Examples 3-4 (Ion Exchange Method): The enzymatic hydrolysate was loaded onto a DEAE-Sepharose Fast Flow chromatography column for elution and separation.

[0178] The results are shown in Table 3. The product yield, polysaccharide content and protein residue of various alternative methods are all inferior to the alcohol precipitation method of this application.

[0179] Table 3: Comparison of Polysaccharide Purification and Separation Methods

[0180]

[0181] Comparative Example 4: Acid Hydrolysis

[0182] This comparative example uses a strong acid hydrolysis method. Bird's nest raw materials were taken and added to a 2 mol / L sulfuric acid solution, and hydrolyzed at 80°C for 2 hours. After neutralization with alkali, ethanol was added to a final concentration of 80% to precipitate. The precipitate was collected by centrifugation and freeze-dried. Other steps were the same as in Example 1. The results showed that although the polysaccharide yield was 22.8%, the total sialic acid content was only 8.3%, significantly lower than the 24.9% in Example 1. Most of the sialic acid was in free form; bound sialic acid was not detected, indicating that most sialic acid existed in free form or was destroyed.

[0183] Comparative Example 5: Enzymatic hydrolysis sequence

[0184] This comparative example aims to verify the necessity of a specific enzymatic hydrolysis sequence (alkaline protease → neutral protease → papain).

[0185] This comparative example uses the same raw materials and enzyme amounts as Example 1, but the enzymatic hydrolysis method is adjusted:

[0186] Comparative Example 5-1: The difference between Comparative Example 5-1 and Example 1 is that the enzymatic hydrolysis order is adjusted as follows: first add papain for enzymatic hydrolysis, then add neutral protease, and finally add alkaline protease. The pH and temperature at each stage are adjusted accordingly, while other steps are the same as in Example 1.

[0187] Comparative Example 5-2: The difference between Comparative Example 5-2 and Example 1 is that alkaline protease, neutral protease and papain were added simultaneously, the pH was adjusted to 8.0 (compromise pH), and enzymatic hydrolysis was performed at 50°C for 5 hours. Other steps were the same as in Example 1.

[0188] The results showed that the polysaccharide yield of Comparative Example 5-1 was 20.4%, and the molecular weight distribution coefficient (Mw / Mn) was 2.241 (broadened distribution). The polysaccharide yield of Comparative Example 5-2 was 19.1%, and the protein residue was high (>35.6%). This indicates that random or simultaneous enzymatic hydrolysis could not achieve the effect of Example 1. This may be because a specific hydrolysis sequence can gradually open the spatial structure of bird's nest glycoproteins, fully exposing the cleavage sites; while an incorrect sequence may lead to the masking of cleavage sites or interference between enzymes. The specific sequence in Example 1 ensured that the product had extremely high molecular weight uniformity (Mw / Mn≈1.1) and purity.

[0189] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0190] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for preparing antiviral bird's nest polysaccharide active components rich in bound bird's nest sialic acid, characterized in that, include: The bird's nest raw material is pretreated by heating to obtain a pretreated product. The pretreated material was subjected to alkaline protease hydrolysis and neutral protease hydrolysis in sequence to obtain a preliminary hydrolysate. Take the supernatant of the preliminary enzymatic hydrolysate, add ethanol to make the final ethanol concentration in the system reach 75%~85%, let it stand at low temperature to precipitate, collect the precipitate, and then reconstitute the precipitate for papain enzymatic hydrolysis. After inactivating the papain enzyme in the product, a secondary enzymatic hydrolysate was obtained. The supernatant of the secondary enzymatic hydrolysate was taken, and ethanol was added to make the final ethanol concentration in the system reach 75%~85%. The mixture was allowed to stand at low temperature to precipitate, and the precipitate was collected and dried to obtain the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid. The heating pretreatment is achieved by crushing the bird's nest raw material, soaking it in water, and then subjecting it to a boiling water bath.

2. The method according to claim 1, characterized in that, The alkaline protease hydrolysis treatment was carried out under the conditions of pH 10.0~11.0 and temperature 45℃~55℃ for 3~5 hours.

3. The method according to claim 1, characterized in that, The neutral protease hydrolysis treatment was carried out under the conditions of pH 6.5~7.5 and temperature 45℃~55℃ for 3~5 hours.

4. The method according to claim 1, characterized in that, The papain enzymatic hydrolysis was carried out at a pH of 6.0-7.0 and a temperature of 45℃-55℃ for 4-6 hours.

5. The method according to claim 1, characterized in that, The amounts of the alkaline protease, the neutral protease, and the papain added, based on the enzyme-to-protein ratio, are each independently 10 × 10⁻⁶. 4 ~90×10 4 U / g of raw material protein.

6. The method according to claim 1, characterized in that, The temperature for the low-temperature settling is 2℃~8℃, and the time is 8~16 hours.

7. An antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid, characterized in that, The active component of bird's nest polysaccharide is prepared by the method described in any one of claims 1 to 6.

8. The antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid according to claim 7, characterized in that, The weight-average molecular weight of the active component of the bird's nest polysaccharide is 30 kDa to 36 kDa, and the molecular weight distribution coefficient is 1.1 to 1.

2. The active component of the bird's nest polysaccharide contains ≥24.0% bound sialic acid and ≥57.0% by mass. The total sugar content is the sum of the contents of N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose, and fucose. The monosaccharide composition of the active component of the bird's nest polysaccharide includes N-acetylneuraminic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose and fucose, and contains Neu5Ac-Gal-GlcNAc or Neu5Ac-Gal polysaccharide structural fragments.

9. The antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid according to claim 8, characterized in that, The bound sialic acid has a mass percentage content of 24.0% to 37.7%.

10. The antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid according to claim 8, characterized in that, The total sugar content is 57.0%~66.1%.

11. Use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid as described in any one of claims 8 to 10 in the preparation of products with antioxidant functions.

12. The use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid as described in any one of claims 8 to 10 in the preparation of products for inhibiting Escherichia coli, Bacillus subtilis and / or having antiviral functions.

13. The use of the antiviral bird's nest polysaccharide active component rich in bound bird's nest sialic acid as described in any one of claims 8 to 10 in the preparation of intestinal microecological regulators or prebiotic foods.

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