Radix astragali glycosylated polypeptide prepared by coupling of enzyme and fermentation and preparation method and application thereof

Abstract

The application discloses a kind of enzyme fermentation coupling in-situ glycosylation modification preparation radix astragali sugar polypeptide and its preparation method and application, belong to the field of bioengineering technology.The preparation process of radix astragali sugar polypeptide provided by the present application is mainly with deep enzymolysis and fermentation, enzymatic reaction and physical membrane separation in whole process, without chemical organic solvent addition, production waste liquid is easy to handle;Extraction-reaction-separation link is constructed as integrated preparation system, realizes high-efficiency cooperation and has continuity, adaptability;Compound broken wall enzyme, polysaccharide degrading enzyme and protease, endogenous polysaccharide and protein are simultaneously realized to small molecule, combined with compound strain fermentation, optimize substrate structure and reaction environment, improve the output efficiency of polypeptide and glycosyl donor;Radix astragali sugar polypeptide dry powder prepared has the functions of antioxidant, antifatigue, relieving chemical liver injury, etc., can be widely applied in biological medicine and health field.

Description

Technical Field

[0001] This invention relates to a glycopeptide, its preparation method and application, specifically to an astragalus glycopeptide prepared by enzyme-ferment coupling synergistic in situ glycosylation modification, its preparation method and application, belonging to the field of bioengineering technology. Background Technology

[0002] Astragalus polysaccharide (APS) has antioxidant, NF-κB inhibition, and Nrf2 pathway activation effects, and can alleviate CCl4 and acetaminophen-induced chemical liver damage.

[0003] Astragalus small molecule polypeptides and crude extracts have liver-protective and anti-inflammatory activities.

[0004] While physically mixing Astragalus polysaccharides with Astragalus small molecule peptides (or crude extracts) can be used to protect against chemical liver injury, the mixture dissociates rapidly after entering the digestive tract, resulting in only a simple additive effect of antioxidant and anti-inflammatory properties. It also has multiple inherent drawbacks, such as peptides being easily degraded by digestive enzymes, unstable formulations that are prone to stratification, low oral bioavailability, weak repair effect on severe chemical liver injury, and high raw material costs.

[0005] Glycopeptides are complex active substances formed by the covalent bonding of carbohydrate units and polypeptide chains. They possess the dual structural and physicochemical properties of both polypeptides and polysaccharides, exhibiting stable molecular structures and abundant active sites. Existing research and applications have confirmed that glycopeptides possess excellent physiological activities such as antioxidant, immunomodulatory, and glycemic regulation, and demonstrate good biocompatibility and high human absorption rate. As a novel green material, they have extremely high development value and broad market application prospects in the fields of biomedicine and health.

[0006] Existing studies have shown that Ganoderma lucidum glycopeptides and Lycium barbarum glycopeptides have protective effects against alcohol-induced and metabolic liver injury. However, there are no reports on whether Astragalus glycopeptides derived from Astragalus membranaceus have protective effects against acute chemical liver injury induced by chemical drugs and toxins.

[0007] The current mainstream preparation processes for glycopeptides have many technical shortcomings: (1) Chemical synthesis preparation method: The process steps are complicated, the preparation cost is high, and the production process is prone to generating harmful by-products, which is contrary to the concept of green biomanufacturing development.

[0008] (2) Single enzymatic hydrolysis preparation process: The process is difficult to control, the resulting product has poor bioactivity stability, a wide molecular weight distribution range, and poor product uniformity.

[0009] (3) Pure microbial fermentation preparation process: The utilization rate of raw material substrate is low, the production cycle is long, and the overall conversion and preparation efficiency is difficult to meet the industrialization requirements.

[0010] (4) Traditional enzyme-fermentation coupling process: It generally adopts a step-by-step sequential processing mode, which only realizes the simple combination of enzymatic hydrolysis and fermentation processes, and cannot achieve the synergistic adaptation and optimization of reaction system, process parameters and mechanism of action.

[0011] (5) Traditional chemical glycosylation: The reaction conditions are demanding, the site-directed modification ability is weak, the by-product output is large, which greatly increases the difficulty of subsequent separation and purification; moreover, this process is mostly carried out in the later stage, making it difficult to coordinate with the front-end biotransformation process, and the overall integration of the process is low.

[0012] Therefore, there is an urgent need to develop a comprehensive and efficient green process for preparing glycopeptides, break through existing technological bottlenecks, and realize the large-scale, industrialized production and widespread application of glycopeptides. Summary of the Invention

[0013] To address the shortcomings of existing technologies, the first objective of this invention is to provide an astragalus glycopeptide that offers better protection against acute chemical liver injury induced by chemical drugs and toxins. The second objective of this invention is to provide a comprehensive and efficient method for preparing the aforementioned astragalus glycopeptide through synergistic in-situ glycosylation modification of enzyme-ferment coupling.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing astragalus glycopeptides through enzyme-ferment coupling synergistic in-situ glycosylation modification includes the following steps: (1) Homogenization: Pulverize Astragalus membranaceus, add it to 40℃ warm water at a material-liquid ratio of 1:1 and soak for 12 hours to obtain pretreated liquid. Then grind the pretreated liquid with a colloid mill to obtain homogenized liquid. (2) Warm water extraction: Add water to the homogenate at a volume ratio of 1:5 and extract at 60℃ for 3 hours to obtain the water extract; (3) Enzyme-fermentation coupling reaction: Add compound biological enzyme and compound microbial fermentation agent to the aqueous extract, adjust the system temperature to 37℃ and pH value to 6.5, and carry out the enzyme-fermentation reaction simultaneously for 24 hours to obtain enzyme-fermentation products; (4) In-situ glycosylation modification: After the enzyme-fermentation coupling reaction is completed, a complex glycosyltransferase is immediately added to the enzyme-fermentation product system. The reaction temperature is set to 60℃ and the pH value to 8.0. The enzymatic grafting reaction is carried out for 8 hours. Then the temperature is rapidly increased to 80℃ and the cross-linking and ripening is stirred for 3 hours. After the reaction is completed, the product is naturally cooled to room temperature to obtain the glycosylated modified product. (5) Separation and purification: The glycosylation modified product system was purified and filtered stepwise using a butterfly centrifuge, a tubular centrifuge, a microfiltration ceramic membrane, an ultrafiltration ceramic membrane, and a nanofiltration ceramic membrane to obtain a concentrated glycopeptide solution. (6) Spray dry or vacuum freeze dry the concentrated glycopeptide solution to obtain glycopeptide powder.

[0015] Preferably, in step (1), the grinding process using a colloid mill is as follows: the conveying pump feeds material at a uniform speed of 300L / h, first sets the grinding gap to 0.2-0.3mm and the operating speed to 3000r / min, and performs one fine grinding, then adjusts the grinding gap to 0.10-0.15mm and the operating speed to 4000r / min, and performs two fine grindings.

[0016] Preferably, in step (3), the compound bio-enzyme is composed of cellulase, hemicellulase, pectinase, neutral protease and papain, and the amount added is 0.5% of the mass of the water extract; the compound ratio of cellulase, hemicellulase, pectinase, neutral protease and papain by mass is 2:1:2:2:1 or 1:2:3:3:2.

[0017] Preferably, in step (3), the compound microbial fermentation agent is obtained by fermentation of a compound fermentation strain in MRS medium, with an inoculum amount of 3% of the water extract volume. The compound fermentation strain is composed of *Lactobacillus plantarum* LHP710, *Lactobacillus paracasei* SQ01, *Lactobacillus rhamnosus* SQ02, *Candida tropicalis* CICC31854, and *Pichia pastoris* CICC33372. *Lactobacillus plantarum* LHP710 was deposited on June 4, 2021, at the China Center for Type Culture Collection (CCTCC), located in Wuhan, with accession number CCTCC NO: M 2021675, and classified as *Lactobacillus plantarum* LHP710. *Lactobacillus paracasei* SQ01 was deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC), located in Wuhan, with accession number CCTCC NO: M Lactobacillus paracasei SQ01 (accession number 2024934) and Lactobacillus rhamnosus SQ02 (accession number CCTCC NO: M 2024933) were deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan, China. The compounding ratio of Lactobacillus plantarum LHP710, Lactobacillus paracasei SQ01, Lactobacillus rhamnosus SQ02, Candida tropicalis CICC31854, and Pichia pastoris CICC33372, by weight, was 2:2:2:1:1 or 1:1:4:2:3.

[0018] Preferably, in step (4), the complex glycosyltransferase is composed of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase, and xylosyltransferase; by weight, the ratio of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase, and xylosyltransferase is 2:2:1:3:3 or 1:3:3:2:1.

[0019] Preferably, in step (5), the pore size of the microfiltration ceramic membrane is 0.2 μm, the molecular weight cutoff of the ultrafiltration ceramic membrane and the nanofiltration ceramic membrane is 10 kDa and 300 Da, respectively, and the temperature of the membrane separation system is controlled at 40℃ and the pH value is maintained in the range of 6.5-8.5.

[0020] An astragalus glycopeptide was prepared by the aforementioned method.

[0021] The aforementioned applications of astragalus glycopeptides include: (1) Preparation of antioxidant drugs; (2) Preparation of antihypertensive drugs; (3) Preparation of drugs to protect against chemical liver injury.

[0022] The advantages of this invention are: (1) The preparation process of Astragalus glycopeptide mainly involves deep enzymatic hydrolysis and fermentation, enzymatic reaction and physical membrane separation. No chemical organic solvents are added, and the production waste liquid is easy to treat, which meets the green production standards.

[0023] (2) This invention integrates the extraction-reaction-separation process into a unified preparation system, achieving high efficiency, synergy, continuity, and adaptability. It effectively solves the defects of traditional processes such as disconnection, high intermediate product loss, and cumbersome production process, realizing integrated green and efficient preparation from medicinal and edible raw materials to glycopeptides.

[0024] (3) This invention combines cell wall-breaking enzymes, polysaccharide-degrading enzymes, and proteases to simultaneously achieve the miniaturization of endogenous polysaccharides and proteins. Combined with synergistic fermentation of complex strains, it optimizes the substrate structure and reaction environment, thereby improving the yield efficiency of peptides and glycosyl donors. The use of low-temperature enzymatic grafting combined with high-temperature cross-linking and ripening segmented glycosylation strengthens the covalent binding of glycopeptides and effectively improves the structural stability of the product.

[0025] (4) The present invention uses ceramic membrane gradient separation and purification. Through multi-stage ceramic membrane cross-flow separation of microfiltration-ultrafiltration-nanofiltration, it accurately classifies, removes impurities, desalinates and concentrates according to molecular weight. The whole process is carried out at low temperature, which maximizes the preservation of biological activity and improves the purity and yield of the product.

[0026] (5) The astragalus glycopeptide prepared by the present invention has significantly improved water solubility, dispersibility and thermal stability after dual modification by enzyme-fermentation coupling and glycosylation. Compared with ordinary plant polypeptides and polysaccharides, it has a higher in vivo absorption and utilization rate. The preparation system retains natural active substances to the greatest extent. After microbial fermentation, new active substances (such as small molecule organic acids, microbial extracellular polysaccharides, functional active oligopeptides, etc.) are added. Moreover, the moisture content, powder flowability and particle size of the finished product can be precisely controlled.

[0027] (6) The Astragalus polypeptide dry powder prepared by the present invention has functions such as anti-oxidation, anti-fatigue and relief of chemical liver damage, and can be widely used in the fields of biomedicine and health. Attached Figure Description

[0028] Figure 1 This is a graph showing the concentration of ALT in the serum of mice in each group. Figure 2 This is a graph showing the concentration of AST in the serum of mice in each group; Figure 3 This is a graph showing the concentration of SOD in the serum of mice in each group. Figure 4 This is a graph showing the concentration of MDA in the serum of mice in each group. Figure 5 These are images showing the HE staining results of liver tissue from each group of mice; Figure 6 These are images showing the Masson staining results of liver tissue from each group of mice. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0030] I. Preparation of Astragalus Glycopeptides Astragalus, Astragalus polysaccharide, Astragalus polypeptide, cellulase, hemicellulase, pectinase, neutral protease, papain, β-galactosyltransferase, transglutaminase, α-mannosyltransferase, α-glucosyltransferase, and xylosyltransferase were all purchased from the online store.

[0031] Lactobacillus plantarum LHP710 was deposited on June 4, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan, with accession number CCTCC NO: M 2021675, and classified as Lactobacillus plantarum LHP710.

[0032] Lactobacillus paracasei SQ01: deposited on May 15, 2024 at the China Center for Type Culture Collection (CCTCC), Wuhan, with accession number CCTCC NO: M 2024934, and classified as Lactobacillus paracasei SQ01.

[0033] Lactobacillus rhamnosus SQ02 was deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2024933, and classified as Lactobacillus rhamnosus SQ02.

[0034] Candida tropicalis CICC 31854, Pichia pastoris CICC 33372, Lactobacillus plantarum CICC 20265, and Bacillus subtilis ATCC 6633 were purchased from the China Industrial Microbial Culture Collection Center.

[0035] Example 1

[0036] 1. Homogenize Pulverize the astragalus powder and pass it through a 24-mesh sieve. Then, add 40°C warm water to the astragalus powder at a material-to-liquid ratio of 1 kg: 1 L and soak it at a constant temperature for 12 hours to obtain a pretreated liquid.

[0037] The pretreated liquid is fed into a colloid mill for grinding. The feed pump delivers the liquid at a constant speed of 300 L / h. First, the grinding gap is set to 0.3 mm and the operating speed is 3000 r / min for one fine grinding. Then, the grinding gap is adjusted to 0.15 mm and the operating speed is 4000 r / min for two fine grindings to obtain a homogenized slurry.

[0038] 2. Warm water extraction Water was added to the homogenate at a volume ratio of 1:5 to dilute it, and then the mixture was heated to 60°C and extracted for 3 hours to obtain an aqueous extract.

[0039] 3. Enzyme-fermentation coupling reaction Add a compound biological enzyme to the aqueous extract at a concentration of 0.5% of the extract's mass, then inoculate with a compound microbial fermentation agent at a concentration of 3% of the extract's volume. Adjust the system temperature to 37°C and pH to 6.5, and allow the enzyme fermentation reaction to proceed simultaneously for 24 hours to obtain the enzyme fermentation product.

[0040] The compound bio-enzyme is composed of cellulase, hemicellulase, pectinase, neutral protease, and papain in a mass ratio of 2:1:2:2:1. Among them, cellulase, hemicellulase, and pectinase are mainly used to break down the cell walls of Astragalus membranaceus and release active substances (such as Astragalus polysaccharides and Astragalus polypeptides), while neutral protease and papain are mainly used to hydrolyze the large protein molecules in Astragalus membranaceus into water-soluble polypeptides or amino acids.

[0041] Preparation method of compound microbial fermentation agent: Lactobacillus plantarum LHP710, Lactobacillus paracasei SQ01, Lactobacillus rhamnosus SQ02, Candida tropicalis CICC31854, and Pichia pastoris CICC33372 were respectively prepared into bacterial powders according to conventional methods. Then, Lactobacillus plantarum LHP710 (bacterial powder), Lactobacillus paracasei SQ01 (bacterial powder), Lactobacillus rhamnosus SQ02 (bacterial powder), Candida tropicalis CICC31854 (bacterial powder), and Pichia pastoris CICC33372 (bacterial powder) were compounded in a mass ratio of 2:2:2:1:1 to obtain a compound fermentation strain. The compound fermentation strain was activated in 37℃ warm water for 5±1h, and then inoculated into MRS medium at an inoculation amount of 2g (dry powder) / 100mL. The system temperature was adjusted to 37℃ and pH 5.5, and the culture was allowed to ferment statically for 24h.

[0042] 4. In-situ glycosylation modification Immediately after the enzyme-fermentation coupling reaction is completed, a complex glycosyltransferase is added to the enzyme-fermentation product system at a concentration of 0.1% of the product's mass. The reaction temperature is set at 60℃ and the pH at 8.0. The enzymatic grafting reaction is carried out for 8 hours (directed enzymatic glycosylation to achieve synergy between glycosylation and enzyme fermentation, completing the directional grafting modification of glycosyl groups onto polypeptide molecular sites). Subsequently, the temperature is rapidly increased to 80℃, and the mixture is stirred and cross-linked for 3 hours. After completion, the mixture is allowed to cool naturally to room temperature (to further enhance the stability of the binding between glycosyl groups and polypeptides and improve the molecular modification structure), thus obtaining the glycosylated modified product.

[0043] The complex glycosyltransferase is composed of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase, and xylosyltransferase in a mass ratio of 2:2:1:3:3.

[0044] 5. Separation and purification First, a primary solid-liquid separation of the glycosylation-modified product system was performed using a disc centrifuge at 8000 r / min, collecting the continuously flowing clear liquid phase A (light liquid and heavy liquid). Then, a secondary solid-liquid separation was performed using a tubular centrifuge at 16000 r / min, collecting the continuously flowing clear liquid phase B. Subsequently, clear liquid phase B was sequentially filtered through a 0.2 μm microfiltration ceramic membrane at 0.2 MPa and 200 L / (m²·h) to remove suspended solids and colloidal impurities, and then at 0.3 MPa and 200 L / (m²·h). Under conditions of m²·h, the solution is filtered through an ultrafiltration ceramic membrane with a molecular weight cutoff of 10 kDa (retaining large molecular impurities and achieving the separation of effective glycopeptide components), and then through a nanofiltration ceramic membrane with a molecular weight cutoff of 300 Da (concentrating glycopeptides while removing salts and free monosaccharides) at 0.4 MPa and 100 L / (m²·h) to complete the stepwise purification and filtration. The temperature of the membrane separation system is controlled at 40℃ and the pH value is maintained within the range of 6.5-8.5 throughout the process, resulting in a concentrated glycopeptide solution (with a soluble solids content of 20-40%).

[0045] 6. Dry into powder Maltodextrin (carrier) was added to the concentrated glycopeptide solution at a concentration of 5% of the solution's mass, and dissolved by stirring at 45°C. The solution was then homogenized under high pressure (20 MPa in the first stage and 10 MPa in the second stage) to obtain a uniform and stable mixture. The mixture was then spray-dried with the spray drying tower set at an inlet temperature of 170°C, an outlet temperature of 85°C, and a slight negative pressure of -100 Pa to obtain glycopeptide powder (moisture content ≤ 5.0%).

[0046] Example 2

[0047] The process is basically the same as Example 1, except that the ratio of the compound bio-enzyme and the compound fermentation strain is different in the enzyme-fermentation coupling reaction process. The compound bio-enzyme is composed of cellulase, hemicellulase, pectinase, neutral protease and papain in a mass ratio of 1:2:3:3:2. The compound fermentation strain is composed of Lactobacillus plantarum LHP710, Lactobacillus paracasei SQ01, Lactobacillus rhamnosus SQ02, Candida tropicalis CICC31854 and Pichia pastoris CICC33372 in a mass ratio of 1:1:4:2:3.

[0048] Example 3

[0049] The process is basically the same as in Example 1, except that the ratio of the complex glycosyltransferase is different in the in situ glycosylation modification process. The complex glycosyltransferase is composed of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase and xylosyltransferase in a mass ratio of 1:3:3:2:1.

[0050] Example 4

[0051] The process is essentially the same as in Example 1, except that in the powdering process, vacuum freeze-drying is used. Specifically, the concentrated glycopeptide solution is spread evenly on a tray and pre-frozen at -45°C for 5 hours. It is then placed in a vacuum freeze-drying chamber, with the vacuum level controlled at 20 Pa, the cold trap temperature ≤ -40°C, and the heating plate temperature at 30°C. A first sublimation drying is performed for 12 hours. The vacuum level is then adjusted to 15 Pa, and the heating plate temperature is raised to 45°C for a second desorption drying for 12 hours. After drying, the material is discharged at low temperature and continuously ultra-finely pulverized at 5000 r / min using an ultra-micro pulverizer to obtain glycopeptide dry powder (300 mesh).

[0052] Comparative Example 1 The process is basically the same as in Example 1, except that the enzyme-fermentation reaction steps are different. This method adopts a step-by-step process of enzymatic hydrolysis and fermentation. Specifically, a compound biological enzyme (0.5% of the water extract mass) is first added to the water extract, and the system temperature is controlled at 37°C and pH at 6.5. The enzymatic hydrolysis reaction is carried out alone for 12 hours. After the enzymatic hydrolysis reaction is completed, the enzyme is first inactivated at 70°C for 20 minutes, and then cooled to 37°C. Then, a compound microbial fermentation agent (3% of the water extract volume) is added, and the system temperature is controlled at 37°C and pH at 6.5. Fermentation is carried out alone for 12 hours.

[0053] Comparative Example 2 The process is basically the same as in Example 1, except that the enzyme-fermentation reaction process is different. This method only performs the enzymatic hydrolysis reaction and does not carry out microbial fermentation treatment. Specifically, a compound biological enzyme (0.5% of the mass of the water extract) is added to the water extract, the system temperature is controlled at 37°C and the pH value is 6.5, and the enzymatic hydrolysis reaction is carried out for 24 hours.

[0054] Comparative Example 3 The process is basically the same as Example 1, except that the types and ratios of the compound fermentation strains are different in the enzyme-fermentation coupling reaction process. The compound fermentation strains in this scheme are composed of Lactobacillus plantarum CICC 20265 and Bacillus subtilis ATCC 6633 in a mass ratio of 1:1.

[0055] Comparative Example 4 The process is basically the same as in Example 1, except that the glycosylation modification process is different. This method adopts a stepwise glycosylation modification process. First, the enzyme fermentation product system is subjected to solid-liquid separation treatment, and then the glycosylation modification reaction is carried out. Specifically, the enzyme fermentation product system is first subjected to primary solid-liquid separation using a disc centrifuge at 8000 r / min, and the continuously flowing clear liquid phase A is collected. Then, the clear liquid phase A is subjected to secondary solid-liquid separation using a tubular centrifuge at 16000 r / min, and the continuously flowing clear liquid phase B is collected. Then, a complex glycosyltransferase is added to the clear liquid phase B (the amount added is 0.1% of the mass of the clear liquid phase B). After that, the enzymatic grafting reaction (temperature 60℃, pH 8.0, reaction time 8h) and cross-linking ripening reaction (temperature 80℃, pH 8.0, reaction time 3h) are carried out in stages.

[0056] Comparative Example 5 The process is basically the same as in Example 1, except that the glycosylation modification process is different. This method only performs the first stage of enzymatic grafting reaction and does not carry out the second stage of temperature-induced cross-linking and ripening reaction. Specifically, a complex glycosyltransferase is added to the enzyme fermentation product system at a rate of 0.1% of the mass of the enzyme fermentation product system. The reaction temperature is set at 60°C and the pH value at 8.0. The enzymatic grafting reaction is carried out for 8 hours and then naturally cooled to room temperature.

[0057] Comparative Example 6 The process is basically the same as in Example 1, except that the type and ratio of the complex glycosyltransferase are different in the in situ glycosylation modification process. In this scheme, the complex glycosyltransferase is composed of β-galactosyltransferase and transglutaminase in a mass ratio of 1:1.

[0058] Comparative Example 7 The process is basically the same as in Example 1, except that the glycosylation modification process is different. This method only performs the second stage of heating and cross-linking ripening reaction and does not carry out the first stage of enzymatic grafting reaction. Specifically, a complex glycosyltransferase is added to the enzyme fermentation product system at a rate of 0.1% of the mass of the enzyme fermentation product system. The reaction temperature is set at 80°C and the pH value is set at 8.0. The cross-linking ripening is carried out by stirring for 3 hours and then naturally cooled to room temperature.

[0059] Comparative Example 8 The process is basically the same as in Example 1, except that the separation and purification steps are different. This method uses only a single device, a butterfly centrifuge, for primary solid-liquid separation, without secondary solid-liquid separation. It also uses only a single membrane, a microfiltration ceramic membrane, for membrane separation, without ultrafiltration ceramic membrane or nanofiltration ceramic membrane separation. Finally, it undergoes vacuum low-temperature concentration to obtain a concentrated glycopeptide solution.

[0060] II. Detect the grafting degree, browning degree, and purity of glycopeptides, and calculate the extraction rate and yield of glycopeptides. Degree of grafting (DG): The degree to which a polypeptide (peptide chain) is covalently bonded (glycosidic bond, etc.) to glycosyl groups (monosaccharides / oligosaccharides / polysaccharides). It is a core indicator for evaluating the effect of glycosylation modification and determining the functional properties (solubility, stability, antioxidant properties, etc.) of glycopeptides. It is calculated by the change in free amino group content before and after the glycosylation reaction, and can directly reflect the grafting efficiency and molecular modification effect of glycopeptides.

[0061] Evaluation criteria: Currently, the degree of glycopeptide grafting is used as the criterion for evaluating the effect of covalent modification. A grafting degree of not less than 10% is required to achieve effective covalent binding and possess practical modification value. A grafting degree of 20-40% is the ideal modification range, within which the glycopeptide binding efficiency is excellent, which can effectively improve the antioxidant and stability properties of the product, while effectively inhibiting browning side reactions. A grafting degree higher than 40% is considered a high-quality modification level, suitable for the biopharmaceutical and health fields. When the grafting degree exceeds 50%, the browning degree of the material will be significantly aggravated.

[0062] The glycopeptide powders prepared in each example and comparative example were dissolved in pure water and stirred evenly (the rehydration time was about 30 seconds). A 50 g / L Astragalus glycopeptide solution was prepared. The solution was brown and free of flocculent matter. After standing for 30 minutes, the supernatant was taken for testing.

[0063] 1. Grafting degree Add 0.5 mL of astragalus glycopeptide supernatant to 10 mL of o-phthalaldehyde (OPA), mix thoroughly, and measure the absorbance of the mixture at 340 nm (A0). Then react in a 35°C water bath for 2 min. After the reaction, measure the absorbance of the mixture again at 340 nm (A1). Use deionized water as a blank control and calculate the grafting degree (DG) according to the following formula: DG = (A1 - A0) / A1 × 100% 2. Degree of browning Add 5 mL of 20% sodium dodecyl sulfate (SDS) solution and 1 mL of astragalus glycopeptide supernatant to a test tube, mix well, and measure the absorbance of the mixture at 420 nm. Use 10% SDS solution as a blank control. The absorbance reflects the degree of browning of Maillard glycosylation reaction.

[0064] 3. Purity The purity of Astragalus glycopeptides is determined by the purity of the polysaccharides within the Astragalus glycopeptides, which is tested using the sulfuric acid-phenol method.

[0065] 4. Extraction rate The extraction rate of Astragalus glycopeptides (hereinafter referred to as extraction rate) is calculated using the following formula: Extraction rate = (Purity of polysaccharides in astragalus glycopeptides × Mass of astragalus glycopeptides) / (Purity of polysaccharides in dried astragalus × Mass of dried astragalus) × 100% The purity of polysaccharides in Astragalus polysaccharide polypeptides and the purity of polysaccharides in dried Astragalus were determined by the sulfuric acid-phenol method.

[0066] 5. Yield The formula for calculating the yield of astragalus glycopeptides (hereinafter referred to as yield) is as follows: Yield = (Mass of astragalus glycopeptides / Mass of dried astragalus) × 100% The results of the detection of grafting degree, browning degree and purity of the glycopeptide dry powders prepared in each embodiment and each comparative example, as well as the calculation results of extraction rate and yield, are shown in Table 1.

[0067] Table 1. Grafting degree, browning degree, purity, extraction rate, and yield of glycopeptide dry powder

[0068] The grafting degree test results show that the grafting degree of the samples prepared in Examples 1 to 4 and Comparative Examples 1 to 8 all reached over 20% (23.64-43.98%). This index is a high and ideal standard in the polysaccharide-peptide covalent modification system, fully demonstrating the good covalent binding efficiency between glycosyl groups and peptide molecules. The astragalus glycopeptides prepared by the method provided in this invention not only significantly improve the comprehensive utilization of polysaccharide and peptide components in the raw materials, but also effectively optimize the various functional properties of the peptides themselves.

[0069] Based on the comprehensive test data, the samples prepared in Examples 1 to 4 were generally superior to those prepared in Comparative Examples 1 to 8 in terms of glycopeptide grafting degree, product purity, raw material extraction rate, and finished product yield, and also generally exhibited lower browning degree. The integrated enzyme-fermentation coupling reaction, the appropriate ratio of compound bio-enzymes and compound fermentation strains, and the in-situ glycosylation modification process combining low-temperature enzymatic grafting with heated cross-linking and ripening effectively enhanced the glycosylation modification binding effect. The vacuum freeze-drying mode significantly inhibited material browning, ensuring product activity and appearance quality (Example 4). The purification method combining two-stage centrifugation (butterfly and tubular) with multi-stage membrane separation efficiently removed impurities and significantly improved product purity (Examples 1 to 4, Comparative Example 8). In contrast, the groups that underwent step-by-step enzymatic hydrolysis and fermentation (Comparative Example 1), eliminated fermentation steps (Comparative Example 2), replaced fermentation strains (Comparative Example 3), simplified glycosylation reaction process, and single-stage separation treatment (Comparative Examples 4 to 7) showed a significant decrease in glycosylation grafting efficiency, increased browning of materials, insufficient dissolution of effective components, and poor impurity removal. Ultimately, both the raw material extraction rate and the product yield decreased to varying degrees.

[0070] This demonstrates that the complete set of optimized preparation methods provided by the present invention can synergistically improve the physicochemical quality (grafting degree, browning degree, purity, extraction rate, and yield) of Astragalus polypeptides, while effectively increasing production output efficiency.

[0071] III. Sensory evaluation of glycopeptides, DPPH free radical scavenging rate and ACE inhibition rate 1. Sensory evaluation A 10-member evaluation panel conducted a sensory evaluation of astragalus polypeptides based on four dimensions: color, odor, taste, and texture, in accordance with the general standards for sensory evaluation of food.

[0072] Scoring criteria: A 100-point scale is used, with 25 points for color, 25 points for aroma, 30 points for taste, and 20 points for texture. Each item is graded according to a gradient of quality, and the score is the average of 10 scores. The sensory scoring criteria are shown in Table 2.

[0073] Table 2 Sensory Rating Criteria

[0074] 2. DPPH free radical scavenging rate An alcoholic solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) is purple-red. The color of the DPPH alcoholic solution changes when solutions with different antioxidant capacities are added. The different absorbance values ​​can reflect the antioxidant capacity of the sample.

[0075] The supernatant of Astragalus glycopeptide was added to a methanol solution of DPPH (0.025 g / L), while distilled water was used as the control group. The mixture was shaken in the dark at room temperature for 30 min, and the absorbance was read at a wavelength of 515 nm. The instrument was zeroed with 0.025 g / L DPPH methanol solution before measurement.

[0076] The formula for calculating DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = (A 对照组 -A 样品组 ) / A 对照组 ×100% 3. ACE inhibition rate N-[3-(2-furanoyl)acryloyl]-phenylaminoyl-glutamyl-glutamic acid (FAPGG) can be enzymatically hydrolyzed by acetone (ACE) into phenylalanine (FAP) and diglycine peptide (GG), and can serve as an ACE mimic substrate.

[0077] The absorbance of 1 mM FAPGG completely converted into FAP and GG is 0.758. The ACE inhibition rate can be calculated based on the change in absorbance at 340 nm. Specifically: Add 50 μL of 0.1 U / mL ACE solution and 100 μL of 50 g / L Astragalus polypeptide supernatant to a centrifuge tube, react in a 37°C water bath for 1 h, and then quickly add 200 μL of 1 mol / L hydrochloric acid to terminate the reaction. Use ultrapure water as a blank control group, and calculate the ACE inhibition rate using the absorbance measured at 340 nm.

[0078] The statistical results of DPPH free radical scavenging rate, ACE inhibition rate and sensory evaluation data of the astragalus glycopeptides prepared in each example and comparative example are shown in Tables 3 and 4.

[0079] Table 3. Sensory evaluation, DPPH radical scavenging rate, and ACE inhibition rate of the astragalus glycopeptides prepared in each example.

[0080] Table 4. Sensory evaluation, DPPH radical scavenging rate and ACE inhibition rate of the astragalus glycopeptides prepared in each comparative example.

[0081] Based on the comprehensive sensory evaluation, DPPH free radical scavenging rate and ACE inhibitory activity test results, it can be seen that the overall quality of the samples prepared in Examples 1 to 4 is superior to that of the samples prepared in Comparative Examples 1 to 8.

[0082] In terms of sensory characteristics, the methods provided in Examples 1 to 4 effectively inhibit browning of materials, resulting in products with uniform color, pure Astragalus membranaceus aroma, delicate and smooth taste, and loose powder without obvious agglomeration. Among them, Example 4, which uses vacuum freeze-drying, exhibits the most significant advantage in low-temperature environment, resulting in the lowest degree of browning and the best overall sensory score. However, process modifications such as stepwise enzymatic hydrolysis and fermentation (Comparative Example 1), elimination of microbial fermentation (Comparative Example 2), replacement of fermentation strains (Comparative Example 3), and simplification of glycosylation reaction and single-stage separation and purification (Comparative Examples 4 to 6) all cause problems such as darkening of sample color, weakening of flavor, increased bitterness, and easy agglomeration of powder, resulting in a significant decline in sensory quality. Comparative Example 7, which only undergoes temperature-induced cross-linking and ripening, shows the most severe degree of deterioration.

[0083] Regarding bioactivity, the astragalus glycopeptides prepared in Examples 1 to 4 exhibited high DPPH free radical scavenging and ACE inhibition rates, indicating significant antioxidant and antihypertensive effects. In the comparative examples, due to insufficient covalent binding between glycosyl groups and peptides (Comparative Examples 4 to 7), significant loss of active components (Comparative Example 1), and incomplete removal of impurities (Comparative Example 8), both functional activities showed varying degrees of reduction. The groups lacking enzymatic glycosylation (Comparative Example 7) or the microbial fermentation process (Comparative Example 2) were unable to form stable glycopeptide active structures, resulting in the most significant decline in bioactivity.

[0084] The above results indicate that the integrated enzyme-fermentation coupling, multi-enzyme synergistic in-situ glycosylation, two-stage separation combined with multi-stage membrane separation and low-temperature freeze drying process provided by this invention can significantly improve the sensory quality of the product, such as appearance, flavor, and taste, and can also maximize the preservation and enhancement of the physiological activity of Astragalus glycopeptides. It has a significant ability to scavenge DPPH free radicals and a significant ability to inhibit ACE, indicating that it has a relatively significant antioxidant effect and blood pressure lowering effect.

[0085] IV. Protective effect of Astragalus glycopeptides on acute chemical liver injury One hundred and eighteen Kunming mice were randomly divided into six groups of 18 each, as follows: Control group: Olive oil was administered by gavage once every 3 days, 10 mL / kg each time, for 21 days.

[0086] Model group: Gavage with 0.2% CCl4 olive oil solution every 3 days, 10 mL / kg each time, for 21 days.

[0087] Astragalus polysaccharide group: Astragalus polysaccharide was administered by gavage at a dose of 100 mg / kg once daily for 7 days. Subsequently, a 0.2% CCl4 olive oil solution was administered by gavage at a dose of 10 mL / kg every 3 days, while Astragalus polysaccharide was administered by gavage at a dose of 100 mg / kg once daily for 21 days.

[0088] Astragalus polypeptide group: Astragalus polypeptide was administered orally once daily at a dose of 100 mg / kg for 7 days. Subsequently, a 0.2% CCl4 olive oil solution was administered orally every 3 days at a dose of 10 mL / kg, while simultaneously administering astragalus polypeptide orally once daily at a dose of 100 mg / kg for 21 days.

[0089] Glycopeptide mixture group: Astragalus polysaccharide and Astragalus polypeptide were mixed evenly by mass to obtain a glycopeptide mixture. The glycopeptide mixture was administered by gavage at a dose of 100 mg / kg once daily for 7 days. Subsequently, a 0.2% CCl4 olive oil solution was administered by gavage at a dose of 10 mL / kg every 3 days, while the glycopeptide mixture was administered by gavage at a dose of 100 mg / kg once daily for 21 days.

[0090] Astragalus glycopeptide group: The astragalus glycopeptide prepared in Example 1 was administered by gavage at a dose of 100 mg / kg once daily for 7 days. Afterwards, a 0.2% CCl4 olive oil solution was administered by gavage at a dose of 10 mL / kg every 3 days, while simultaneously, the astragalus glycopeptide was administered by gavage at a dose of 100 mg / kg once daily for 21 days.

[0091] Astragalus polysaccharides, astragalus polypeptides, mixtures of astragalus polysaccharides and astragalus polypeptides, and astragalus glycopeptides were all dissolved in physiological saline.

[0092] After the administration of the drug via gavage, serum samples were collected from each group of mice to measure the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA).

[0093] The concentrations of ALT, AST, SOD, and MDA in the serum of mice in each group are shown in Table 5. Figures 1 to 4 .

[0094] Table 5. Detection results of ALT, AST, SOD and MDA concentrations in mouse serum.

[0095] From Table 5 and Figures 1 to 4 It can be known that: (1) Compared with the model group, the concentrations of ALT and AST in the serum of mice in the Astragalus glycopeptide group were significantly reduced. The concentrations of ALT and AST in the serum of mice in the Astragalus polysaccharide group, Astragalus polypeptide group, and glycopeptide mixture group also decreased, but the decrease was less than that in the low Astragalus glycopeptide group. This indicates that Astragalus glycopeptide has a significant protective effect against hepatocyte damage; at the same dosage, the protective effect of free polysaccharide, free polypeptide, and glycopeptide mixture against hepatocyte damage is lower than that of Astragalus glycopeptide.

[0096] (2) Compared with the model group, the serum SOD concentration of mice in the Astragalus glycopeptide group was significantly increased, and the MDA concentration was significantly decreased. The increase in SOD concentration and the decrease in MDA concentration in the Astragalus polysaccharide group, Astragalus polypeptide group, and glycopeptide mixture group were all lower than those in the Astragalus glycopeptide group. This indicates that Astragalus glycopeptide can effectively maintain or enhance SOD activity, thereby enhancing the body's antioxidant defense capacity. Astragalus glycopeptide may exert a hepatoprotective effect by inhibiting lipid peroxidation and reducing hepatocyte membrane damage. At the same dosage, the ability of free polysaccharide, free polypeptide, and glycopeptide mixture to enhance the body's antioxidant defense capacity and reduce hepatocyte membrane damage was lower than that of Astragalus glycopeptide.

[0097] Based on the above test results, liver tissues from mice in the blank group, model group, and astragalus glycopeptide group were taken and subjected to HE staining and Masson staining, respectively.

[0098] The HE staining and Masson staining results of the liver tissues of each group of mice are shown in the figures below. Figure 5 and Figure 6 .

[0099] HE staining results showed that the liver tissue of mice in the Astragalus glycopeptide group had an intact liver capsule structure, unclear lobule boundaries, and radially arranged hepatocytes and hepatic sinusoids around the central vein with basically intact structure. Only a small amount of venous congestion (black arrow) and local hepatocyte edema (loose cytoplasm and light staining, red arrow) were observed.

[0100] Masson staining results showed that only a small amount of collagen fiber deposition was observed between liver cells and around blood vessels in mice in the Astragalus glycopeptide group (blue area).

[0101] Compared with the model group, the liver tissue pathology of mice in the Astragalus glycopeptide group was significantly improved, indicating that Astragalus glycopeptide has a clear protective effect against chemical liver injury and can inhibit early liver fibrosis.

[0102] In summary, at the same dosage, animal experimental results show that: (1) The astragalus glycopeptide prepared in this invention has a significantly better ability to reduce the concentration of ALT and AST in serum than single components and mixed components; (2) The antioxidant (SOD) capacity and the ability to clear MDA from liver tissue obtained by the present invention of the astragalus glycopeptide are significantly better than those of single component and mixed component.

[0103] In summary, the pharmacodynamic control experiment confirmed that, compared with equal doses of Astragalus polysaccharide alone, Astragalus polypeptide alone, and a mixture of Astragalus polysaccharide and polypeptide, the Astragalus glycopeptide prepared in this invention has a stronger protective effect against CCl4-induced chemical liver injury. Specifically, it can more significantly reduce the concentrations of ALT and AST in serum, increase the activity of antioxidant enzymes in liver tissue, and inhibit liver tissue inflammatory pathways and hepatocyte apoptosis. Moreover, the covalent glycopeptide is resistant to gastric acid degradation and has higher oral bioavailability, achieving the same hepatoprotective effect at low doses, thus overcoming the shortcomings of poor absorption of free polysaccharides and easy degradation of free polypeptides.

[0104] The astragalus glycopeptides prepared by this invention possess excellent antioxidant activity, blood pressure-lowering effects, and significant protective effects against chemically induced liver damage. Furthermore, the product exhibits high purity, superior sensory quality, and excellent bioavailability. The preparation process is also green, mild, and highly controllable, making it suitable for large-scale industrial production. In addition, leveraging the natural medicinal and edible properties of astragalus, these astragalus glycopeptides are highly safe and can effectively improve oxidative stress, protect liver cells, and precisely meet the health needs of contemporary individuals for liver protection, health maintenance, and antioxidant regulation. Therefore, they possess excellent development value and broad market industrial application prospects in the fields of biomedicine and the broader health industry.

[0105] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for preparing astragalus glycopeptides through enzyme-ferment coupling synergistic in-situ glycosylation modification, characterized in that, Includes the following steps: (1) Homogenization: Pulverize Astragalus membranaceus, add it to 40℃ warm water at a material-liquid ratio of 1:1 and soak for 12 hours to obtain pretreated liquid. Then grind the pretreated liquid with a colloid mill to obtain homogenized liquid. (2) Warm water extraction: Add water to the homogenate at a volume ratio of 1:5 and extract at 60℃ for 3 hours to obtain the water extract; (3) Enzyme-fermentation coupling reaction: Add compound biological enzyme and compound microbial fermentation agent to the aqueous extract, adjust the system temperature to 37℃ and pH value to 6.5, and carry out the enzyme-fermentation reaction simultaneously for 24 hours to obtain enzyme-fermentation products; (4) In-situ glycosylation modification: After the enzyme-fermentation coupling reaction is completed, a complex glycosyltransferase is immediately added to the enzyme-fermentation product system. The reaction temperature is set to 60℃ and the pH value to 8.

0. The enzymatic grafting reaction is carried out for 8 hours. Then the temperature is rapidly increased to 80℃ and the cross-linking and ripening is stirred for 3 hours. After the reaction is completed, the product is naturally cooled to room temperature to obtain the glycosylated modified product. (5) Separation and purification: The glycosylation modified product system was purified and filtered stepwise using a butterfly centrifuge, a tubular centrifuge, a microfiltration ceramic membrane, an ultrafiltration ceramic membrane, and a nanofiltration ceramic membrane to obtain a concentrated glycopeptide solution. (6) Spray dry or vacuum freeze dry the concentrated glycopeptide solution to obtain glycopeptide powder.

2. The method for preparing astragalus glycopeptides by enzyme-ferment coupling synergistic in situ glycosylation modification according to claim 1, characterized in that, In step (1), the grinding process using a colloid mill is as follows: The conveying pump feeds material at a uniform speed of 300L / h. First, set the grinding gap to 0.2-0.3mm and the operating speed to 3000r / min for one fine grinding. Then, adjust the grinding gap to 0.10-0.15mm and the operating speed to 4000r / min for two fine grindings.

3. The method for preparing astragalus glycopeptides by enzyme-ferment coupling synergistic in situ glycosylation modification according to claim 1, characterized in that, In step (3), the compound bio-enzyme is composed of cellulase, hemicellulase, pectinase, neutral protease and papain, and the amount added is 0.5% of the mass of the water extract; by mass, the compound ratio of cellulase, hemicellulase, pectinase, neutral protease and papain is 2:1:2:2:1 or 1:2:3:3:

2.

4. The method for preparing astragalus glycopeptides by enzyme-ferment coupling synergistic in situ glycosylation modification according to claim 1, characterized in that, In step (3), the compound microbial fermentation agent is obtained by fermentation of the compound fermentation strain in MRS medium. The inoculum amount is 3% of the volume of the water extract. The compound fermentation strain is composed of Lactobacillus plantarum LHP710, Lactobacillus paracasei SQ01, Lactobacillus rhamnosus SQ02, Candida tropicalis CICC31854, and Pichia pastoris CICC33372. Among them, Lactobacillus plantarum LHP710 was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan on June 4, 2021, with accession number CCTCC NO: M 2021675 and classified as Lactobacillus plantarum LHP710. Lactobacillus paracasei SQ01 was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan on May 15, 2024, with accession number CCTCC NO: M 2024934 and classified as Lactobacillus paracasei SQ01. Lactobacillus paracasei SQ01 and Lactobacillus rhamnosus SQ02 were deposited on May 15, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan, with accession number CCTCC NO: M 2024933. The classification name is Lactobacillus rhamnosus SQ02. The compounding ratio of Lactobacillus plantarum LHP710, Lactobacillus paracasei SQ01, Lactobacillus rhamnosus SQ02, Candida tropicalis CICC31854, and Pichia pastoris CICC33372, by weight, is 2:2:2:1:1 or 1:1:4:2:

3.

5. The method for preparing astragalus glycopeptides by enzyme-ferment coupling synergistic in situ glycosylation modification according to claim 1, characterized in that, In step (4), the complex glycosyltransferase is composed of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase, and xylosyltransferase; by weight, the ratio of β-galactosyltransferase, transglutaminase, α-mannosidase, α-glucosyltransferase, and xylosyltransferase is 2:2:1:3:3 or 1:3:3:2:

1.

6. The method for preparing astragalus glycopeptides by enzyme-ferment coupling synergistic in situ glycosylation modification according to claim 1, characterized in that, In step (5), the pore size of the microfiltration ceramic membrane is 0.2 μm, the molecular weight cutoff of the ultrafiltration ceramic membrane and the nanofiltration ceramic membrane is 10 kDa and 300 Da, respectively, and the temperature of the membrane separation system is controlled at 40℃ and the pH value is maintained in the range of 6.5-8.

5.

7. An astragalus polypeptide, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

8. The application of the astragalus polypeptide according to claim 7, characterized in that, The applications include: (1) Preparation of antioxidant drugs; (2) Preparation of antihypertensive drugs; (3) Preparation of drugs to protect against chemical liver injury.

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