Preparation method of rhizoma polygonati peptide for resisting inflammation and protecting kidney

Abstract

The invention relates to the technical field of bioactive peptide preparation, and discloses a preparation method of rhizoma polygonati peptide for resisting inflammation and protecting kidney, which comprises the following steps: (1) crushing rhizoma polygonati rhizome and then pretreating with an ethanol solution to obtain a water extract concentrated solution; (2) adding the water extraction concentrated solution into a buffer solution to extract total protein of rhizoma polygonati; (3) carrying out enzymolysis on the total protein solution and enzyme under the enzymolysis conditions that the enzyme concentration is 2%-5% of the mass of the substrate, the pH range is 7.5-8.5, the temperature is 45-55 DEG C, and the time is 35 hours; and (4) carrying out ultrafiltration on the enzymatic hydrolysate to remove macromolecular impurities, carrying out dynamic coordination separation on a target peptide fragment by using a metal modified medium, purifying the target peptide fragment through countercurrent partition chromatography, and carrying out freeze drying to obtain the polygonatum sibiricum peptide. According to the present invention, the compound enzyme synergistic enzymolysis, the multi-site dynamic regulation and the dynamic coordination separation technology are adopted, such that the compound enzyme can be well adopted as the anti-inflammatory kidney protection raw material, and can be used for prevention and auxiliary treatment of kidney-related diseases.

Description

Technical Field

[0001] The invention relates to the technical field of bioactive peptide preparation, in particular to a method for preparing polyanthocyanidin for anti-inflammatory and kidney-protecting. Background Art

[0002] Huangjing peptide has attracted much attention in the field of functional foods and drugs in recent years due to its significant anti-inflammatory and kidney-protecting functions. As an active peptide with a clear molecular weight range (800~1,200Da), Huangjing peptide protects kidney cells by regulating inflammatory factors and anti-oxidative stress. At present, the preparation of Huangjing peptide mainly includes raw material pretreatment, protein extraction, enzymatic hydrolysis, separation and purification. However, these processes still have many limitations in efficiency, target peptide generation and final product purity in actual production, which limits the large-scale application of Huangjing peptide.

[0003] In the prior art, a single enzyme is used in the enzymatic hydrolysis stage to degrade proteins under fixed conditions, which fails to fully utilize the synergy of the compound enzyme, resulting in a low target peptide generation rate. At the same time, the separation and purification stages mostly use ultrafiltration combined with conventional chromatographic separation technology, which mainly relies on molecular weight or physical properties for coarse separation, lacks specific selection for the target peptide, and the final product has low purity. In addition, the lack of dynamic control means severely limits the enzymatic hydrolysis efficiency, separation effect, and the stability and efficiency of the overall process. The prior art has deficiencies in the synergistic mechanism of enzymatic hydrolysis, separation accuracy, process stability, etc., making it difficult to achieve efficient preparation and high-purity separation of target peptides. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for preparing Polygonatum sibiricum peptide for anti-inflammatory and kidney-protecting purposes, which solves the problems of low efficiency in generating target peptide segments of Polygonatum sibiricum peptide, insufficient separation purity and lack of dynamic regulation of the process in the prior art.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for preparing Huangjing peptide for anti-inflammatory and kidney protection, comprising the following steps: (1) crushing the rhizome of Polygonatum sibiricum and pre-treating it with an ethanol solution to obtain a water extract concentrate; (2) adding the water extract concentrate to a buffer solution to extract total protein from Polygonatum cyrtonema; (3) The total protein solution is enzymatically hydrolyzed with enzymes under the following conditions: enzyme concentration is 2% to 5% of substrate mass, pH range is 7.5-8.5, temperature is 45-55°C, and time is 35 hours; (4) After ultrafiltration of the enzymatic hydrolysate to remove macromolecular impurities, the target peptides were separated using metal-modified media for dynamic coordination. The separation conditions were a metal ion concentration of 0.1-1 mmol / L and a solution pH of 6.8-7.2. (5) The target peptide was purified by countercurrent distribution chromatography and freeze-dried to obtain polysaccharide peptide.

[0006] Preferably, the particle size of the crushed Polygonatum rhizome is 80 mesh, the concentration of the ethanol solution used is 70%, the pretreatment temperature is 40-50°C, the time is 24 hours, the solid-liquid ratio is 1:8-1:10, and the filtrate obtained after ethanol treatment needs to be concentrated to 1 / 51 / 10 of the original volume, and the precipitate is removed for standby use.

[0007] Preferably, the buffer used for extracting total protein from Polygonatum sibiricum is Tris-HCl buffer, the concentration of the buffer is 20-50 mmol / L, the solid-liquid ratio during the extraction process is 1:5, the extraction temperature is 4°C, the extraction time is 23 hours, and the extracted liquid needs to be centrifuged at 4,000-6,000 rpm for 1015 minutes to separate the supernatant and remove impurities.

[0008] Preferably, the enzyme used in the enzymatic hydrolysis reaction is a composite enzyme of trypsin and papain, the mass ratio of trypsin to papain is 2:1-4:1, the total enzyme concentration is 2% to 5% of the substrate mass, the target protein concentration in the enzymatic hydrolyzate is 5-10 mg / mL, the enzymatic hydrolysis time is 35 hours, and the reaction conditions need to be dynamically adjusted during the reaction to ensure that the pH value remains in the range of 7.5 to 8.5.

[0009] Preferably, the enzyme inactivation operation of the enzymatic hydrolysis solution includes heating the temperature to 80-90°C and maintaining it for 10-15 minutes, while adding 0.1%-0.2% (w / v) citric acid to stabilize the enzymatic hydrolysis product. The enzyme-inactivated solution needs to be quickly cooled to room temperature and filtered to remove residual impurities.

[0010] Preferably, the molecular weight cutoff range of the ultrafiltration membrane is 3,000-5,000Da. During the ultrafiltration operation, the feed liquid needs to pass through the ultrafiltration membrane at a pressure of 0.5-1MPa. The permeate after ultrafiltration needs to be tested for the target peptide content by an online amino acid analyzer. After the test result meets the target range, the permeate is collected for later use.

[0011] Preferably, the metal modified medium is prepared from a metal loading material with silica gel as a matrix, and the metal ion loaded on the silica gel matrix after being treated with dilute hydrochloric acid is Zn 2+ Cu 2+ The metal loading concentration is 0.1-1mmol / L, the medium particle size is 60-120μm, and after metal ion loading, it is dried at 70℃ for 4-6 hours to increase stability.

[0012] Preferably, in the dynamic coordination separation process, the enzymatic hydrolysate is adjusted to pH 6.8-7.2 and then passed through the metal-modified medium column at a flow rate of 13 mL / min, unbound impurities are eluted with 20-50 mmol / L phosphate buffer, and the target peptide is eluted with 0.1-0.5 mol / L citric acid or EDTA solution. The eluted solution needs to be collected after the target peptide concentration is detected.

[0013] Preferably, the mobile phase used in the countercurrent distribution chromatography purification is a mixture of water and acetonitrile, the volume ratio is 80:20-70:30, the gradient elution condition is 10% to 50% acetonitrile linear gradient elution, and the elution time is 15 to 30 minutes. Preferably, the components of the obtained Huangjing peptide are as follows by mass: Huangjing peptide: 80%~95%; Anhydrous glucose: 5% to 10%; Monocalcium phosphate: 2% to 5%; Vitamin E: 0.5%~2%.

[0014] The present invention provides a method for preparing Huangjing peptide for anti-inflammatory and kidney protection, which has the following beneficial effects: 1. The present invention introduces dynamic coordination separation technology and uses metal-modified media to achieve accurate separation and enrichment of the target peptide segment of Polygonatum odoratum. Compared with the existing method that simply relies on membrane separation or chromatographic separation, it solves the problem of limited purity of the target peptide segment and significantly improves the separation efficiency and product consistency.

[0015] 2. The present invention adopts a composite enzymatic hydrolysis system of trypsin and papain, and optimizes the enzymatic hydrolysis conditions based on density functional theory to accurately cut the target peptide chain. Compared with the traditional enzymatic hydrolysis method, it avoids the defect of random cutting of the peptide chain, greatly improves the generation rate of active peptide segments, and solves the shortcomings of low enzymatic hydrolysis efficiency and poor specificity in the prior art.

[0016] 3. The present invention controls the ratio of mobile phase by combining countercurrent distribution chromatography with gradient elution to achieve efficient separation of target peptides and impurities. Compared with the elution method of a single solvent system in the prior art, this scheme significantly improves the separation accuracy and solves the technical difficulty of the traditional method in simultaneously taking into account the recovery rate and purity of the target peptide.

[0017] 4. The present invention uses ethanol solution to remove fat-soluble impurities and combines buffer solution to extract proteins and enrich water-soluble components during the pretreatment of polygonatum raw materials. Different from the prior art method of directly using water for extraction, this method effectively reduces the interference of impurities and avoids the loss of target components, thus overcoming the problems of low purity and incomplete extraction in the raw material processing stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the method flow of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Please see attached Figure 1 The present invention provides a method for preparing Huangjing peptide for anti-inflammatory and kidney protection, comprising: 1. Pretreatment of Polygonatum sibiricum Raw Materials Technical content: The rhizome of Polygonatum sibiricum was cleaned, dried with hot air at 60℃ until the moisture content was less than 10%, and crushed into 80 mesh powder. The powder was added to 70% ethanol solution at a solid-liquid ratio of 1:8-1:10 (w / v), stirred and extracted in a water bath at 40-50℃ for 24 hours, and filtered with gauze to remove solid residues to obtain a filtrate. The filtrate was concentrated under reduced pressure to 1 / 51 / 10 of the original volume in order to enrich the water-soluble components. The concentrate was cooled to room temperature, left to stand for 12 hours, and the precipitate was discarded for later use.

[0021] Polygonatum rhizome is rich in polysaccharides, saponins and proteins, but also contains fat-soluble impurities (such as esters, pigments, etc.), which will affect the efficiency of subsequent protein extraction. Through pretreatment with ethanol solution, the good solubility of ethanol solution for fat-soluble impurities can be used to effectively remove non-polar components while retaining polar components (such as proteins and polysaccharides). The concentration step further enriches the water-soluble target components and improves the efficiency of subsequent protein extraction.

[0022] 2. Extraction of total protein from Polygonatum cyrtonema Technical content: The pretreated concentrate was added with 20-50 mmol / L Tris-HCl buffer (pH 7.5) at a material-liquid ratio of 1:5 (w / v), and extracted at low speed for 23 hours at 4°C. After the extraction, centrifuge at 4,000-6,000 rpm for 10-15 minutes, and the supernatant was taken as the total protein extract. The supernatant was refrigerated for later use.

[0023] The extraction of total protein from Polygonatum sibiricum depends on the solubility of the solvent environment for proteins. Tris-HCl buffer provides a suitable pH environment (pH7.5), which can maintain the natural conformation of the protein and prevent it from denaturing or precipitating. Low temperature conditions (4°C) inhibit the spontaneous degradation of proteases while protecting the activity of proteins. The centrifugation step separates impurities (such as cellulose) from the protein solution through density differences to ensure the purity of the total protein extract.

[0024] 3. Enzymatic hydrolysis Technical content: Take the total protein extract and adjust the solution protein concentration to 5-10 mg / mL. Add trypsin and papain complex enzyme (mass ratio 2:14:1) at a constant temperature of 45-55°C, and the enzyme dosage is 2% to 5% of the substrate mass. Use phosphate buffer to adjust the solution pH to 7.5-8.5, and the enzymolysis time is 35 hours. After the enzymolysis is completed, heat to 80-90°C to inactivate the enzyme for 10-15 minutes, and add 0.1% to 0.2% (w / v) citric acid to stabilize the enzymolysis product, and cool to room temperature for use.

[0025] The key to enzymatic hydrolysis lies in the specificity and efficiency of enzyme cutting of peptide chains. The combination of trypsin and papain can act on specific sites of yellow spherical protein (such as peptide bonds near lysine and arginine) to achieve efficient decomposition. The adjustment of the compound ratio (2:1-4:1) optimizes the synergistic effect of enzyme cutting. The pH and temperature are controlled within the optimal range of enzyme activity (pH7.5-8.5, 45~55℃), ensuring the maximization of enzyme cutting efficiency. The addition of citric acid after enzyme inactivation can inhibit the residual activity of metalloenzymes and protect the target peptide segment at the same time.

[0026] 4. Dynamic coordination separation technology (DCS) Technical content: The enzymatic hydrolysate was filtered through an ultrafiltration membrane with a cutoff of 3,000-5,000Da, and the permeate was collected and the pH was adjusted to 6.8-7.2. The permeate was passed through a metal-modified medium column based on silica gel at a flow rate of 13mL / min. The medium was loaded with Zn after being modified by amine. 2+ Cu 2+ (Concentration is 0.11mmol / L, medium particle size is 60-120μm). After washing away unbound impurities with 2050mmol / L phosphate buffer, elute the target peptide with 0.1-0.5mol / L citric acid or EDTA solution, and collect the eluate.

[0027] Dynamic coordination separation using metal ions (Zn 2+ Cu 2+) forms coordination bonds with the carboxyl and amino groups in the target peptide, and separates the target peptide from impurities through the affinity of the column filling medium. By adjusting the pH (6.8-7.2), the main charge of the target peptide is ensured to be in a neutral or weakly negative state, thereby enhancing its binding with metal ions. Citric acid or EDTA is added in the elution step to release the target peptide from the metal ions through competitive coordination, ensuring the high-purity recovery of the target peptide.

[0028] 5. Countercurrent Partition Chromatography Purification Technical content: The eluted liquid was further purified by countercurrent distribution chromatography, with the mobile phase being water and acetonitrile (volume ratio 80:20-70:30). Linear gradient elution (acetonitrile content increased from 10% to 50%) was used, and the elution time was 15 to 30 minutes. The purified liquid was prepared into Huangjing peptide powder by freeze drying, and the molecular weight range of the target product was 800~1,200Da, and the purity was not less than 98%.

[0029] Countercurrent distribution chromatography uses the difference in distribution coefficients between the target peptide and the solvent for separation. The gradient ratio control of water and acetonitrile can effectively adjust the solubility of the target peptide in the mobile phase and the stationary phase, so that it can be separated from other impurities. The control of the molecular weight range (800~1,200Da) ensures the uniformity and activity of the final product, while freeze drying avoids the degradation of heat-sensitive substances.

[0030] 6. Ratio of Huangjing peptide ingredients Technical content: The components of the final product Huangjing peptide are as follows by mass: Huangjing peptide: 80%~95%; Anhydrous glucose: 5% to 10%; Monocalcium phosphate: 2% to 5%; Vitamin E: 0.5%~2%.

[0031] The addition of anhydrous glucose can enhance the physical stability of the product and improve the taste; calcium dihydrogen phosphate provides mineral support and helps to supplement trace elements; vitamin E, as a natural antioxidant, can extend the shelf life of the product and further stabilize the biological activity of Polygonatum odoratum.

[0032] Example 1: A method for efficiently separating high-purity Polygonatum peptide Step 1: Pretreatment of Polygonatum sibiricum Raw Materials Wash the rhizome of Polygonatum sibiricum, dry it with hot air at 60℃ until the water content is less than 10%. Then crush it into 80 mesh powder. Take 50g of powder, add 500mL 70% ethanol solution (solid-liquid ratio 1:10), stir and extract in a 45℃ water bath for 3 hours. Filter with gauze to remove solid residue, and concentrate the filtrate under reduced pressure to 1 / 6 of the original volume. Let it stand for 12 hours, discard the precipitate, and obtain the Polygonatum sibiricum concentrate.

[0033] Step 2: Polygonatum protein extraction The concentrate was added with 30 mmol / L Tris-HCl buffer at a solid-liquid ratio of 1:5, the pH was adjusted to 7.5, and the solution was extracted at low speed for 3 hours at 4°C. The extract was centrifuged (5,000 rpm, 12 minutes), and the supernatant was the total protein extract of Polygonatum cyrtonema for later use.

[0034] Step 3: Enzymatic hydrolysis Add a mixed enzyme of trypsin and papain (enzyme dosage is 4% of the substrate mass, enzyme mass ratio is 3:1) to 100 mL of total protein extract, adjust the pH to 8.0, and place it in a 50°C constant temperature water bath for 4 hours. After the reaction is completed, heat to 85°C and keep it for 10 minutes to inactivate the enzyme. Then add 0.1% (w / v) citric acid to stabilize the product, cool it to room temperature, and obtain an enzymatic solution.

[0035] Step 4: Dynamic Coordination Separation The enzymatic hydrolysate was filtered through a 3,000Da ultrafiltration membrane and the permeate was collected. After adjusting the pH to 7.0, the Zn-loaded 2+ Silica gel column (Zn 2+ Concentration 0.5mmol / L). Impurities were washed out with 30mmol / L phosphate buffer, and the target peptide was eluted with 0.3mol / L citric acid solution, and the eluate was collected.

[0036] Step 5: Purification and drying The eluent was purified by countercurrent distribution chromatography with water and acetonitrile (volume ratio 80:20) as the mobile phase, gradient elution for 15 minutes, and the target fraction was collected. Finally, it was freeze-dried to obtain Huangjing peptide powder with a molecular weight of 800~1,200Da and a purity of 99%.

[0037] Example 2: A method for rapidly producing highly active polysaccharide peptide Step 1: Polygonatum rhizome processing Cut 100g of rhizome of Polygonatum sibiricum into slices, dry at 60℃ and crush to 85 mesh. Take 80g of powder and add 640mL of 70% ethanol solution, with a solid-liquid ratio of 1:8. Stir and extract in a 45℃ water bath for 2.5 hours, filter with filter cloth to remove residue. Concentrate the filtrate to 1 / 8 of the original volume, cool and stand for 6 hours, and discard the precipitate.

[0038] Step 2: Total protein extraction The concentrate was added with 40 mmol / L Tris-HCl buffer (pH 7.8) at a solid-liquid ratio of 1:4, and extracted with stirring at 4°C for 2 hours. The extract was centrifuged at 6,000 rpm for 15 minutes, and the supernatant was collected for later use.

[0039] Step 3: Enzymatic hydrolysis Add compound enzyme (trypsin and papain, mass ratio 2:1, total enzyme concentration is 3% of substrate mass) to 100 mL of total protein extract. Adjust pH to 8.2 and perform enzymolysis at 45℃ for 5 hours. After enzymolysis, heat to 90℃ for 10 minutes to inactivate the enzyme, then cool to room temperature.

[0040] Step 4: Dynamic separation and enrichment The enzymatic hydrolysate was filtered through a 5,000Da ultrafiltration membrane and the pH of the permeate was adjusted to 6.9. The permeate was passed through the Cu-loaded 2+ Silica gel column (Cu²⁺ concentration 1.0mmol / L). Impurities were washed out with 50mmol / L phosphate buffer, and the target peptide was eluted with 0.4mol / LEDTA solution, and the eluate was collected.

[0041] Step 5: Isolation and purification The eluent was separated by gradient chromatography using water and acetonitrile (volume ratio 70:30) as the mobile phase, and the elution time was 20 minutes to collect and purify the target product. After freeze drying, Huangjing peptide was obtained with a purity of 98% and stable activity.

[0042] Example 3: A method for preparing Huangjing peptide with optimized enzymatic hydrolysis efficiency Step 1: Preprocessing The rhizome of Polygonatum sibiricum was rapidly dried at 70°C for 8 hours, crushed to 75 mesh, 40g of powder was taken, and 400mL of 70% ethanol solution was added for extraction for 2 hours, with a solid-liquid ratio of 1:10. The filtrate was concentrated to 1 / 7 of the original volume and cooled for later use.

[0043] Step 2: Total protein extraction The concentrate was mixed with 50 mmol / L Tris-HCl buffer at a solid-liquid ratio of 1:6, pH 7.6, stirred at 4°C for 3 hours, and centrifuged to obtain a total protein extract.

[0044] Step 3: Compound enzymatic hydrolysis In a 50°C constant temperature water bath, the total protein extract (200 mL) was mixed with a composite enzyme (trypsin and papain, mass ratio 4:1, total amount was 2.5% of the substrate mass), the pH was adjusted to 7.9, and the enzymolysis was performed for 3 hours. The enzyme inactivation condition was 85°C for 15 minutes.

[0045] Step 4: Dynamic enrichment The enzymatic hydrolysate was filtered through a 3,000Da ultrafiltration membrane, and the permeate was collected and the pH was adjusted to 6.8. 2+ Modified silica column (Zn 2+ The target peptide was enriched with 25mmol / L phosphate buffer and 0.2mol / L citric acid as the eluent.

[0046] Step 5: Purification of target peptide The eluent was subjected to countercurrent distribution chromatography with a gradient elution time of 18 minutes. The ratio of water to acetonitrile was 75:25. The target components were collected and freeze-dried to obtain Huangjing peptide powder with a molecular weight range of 850~1,150Da and a purity of 97%.

[0047] Example 4: A method for industrial preparation of high-purity Polygonatum peptide Step 1: Large-scale preprocessing Take 5kg of rhizome of Polygonatum sibiricum, wash it and dry it at 70℃ until the water content is less than 10%. Grind it into 80 mesh powder, add 35L 70% ethanol solution, the solid-liquid ratio is 1:7, and stir and extract it in a 50℃ water bath for 3 hours. After filtering, the filtrate is concentrated to 1 / 10 of the original volume under reduced pressure, and cooled and allowed to stand for 12 hours.

[0048] Step 2: Total protein extraction The concentrate was added with 30 mmol / L Tris-HCl buffer (pH 7.5) at a solid-liquid ratio of 1:5, and stirred at 4°C for 3 hours. Centrifuged at 6,000 rpm for 15 minutes, and the supernatant was collected for later use.

[0049] Step 3: Enzymatic hydrolysis and enzyme inactivation The total protein extract was adjusted to a concentration of 8 mg / mL, and a composite enzyme (trypsin and papain, mass ratio 3:1, total enzyme amount is 3% of the substrate mass) was added. Enzymatic hydrolysis was carried out at 50°C for 4 hours, and the pH was adjusted to 8.0. After the reaction, the temperature was raised to 90°C and maintained for 15 minutes to inactivate the enzyme, and then cooled to room temperature.

[0050] Step 4: Dynamic separation and target enrichment The enzymatic hydrolysate was filtered through a 5,000Da ultrafiltration membrane and the pH of the permeate was adjusted to 6.8. 2+ Silica gel column (Zn 2+ The concentration was 0.5mmol / L, the column volume was 3L) and the flow rate was 5mL / min. The eluent was 0.3mol / L citric acid solution. The target peptide was collected after elution.

[0051] Step 5: Purification and Finishing The target peptide eluate was purified by gradient countercurrent distribution chromatography, with an elution time of 25 minutes and a mobile phase ratio of 70:30 (water: acetonitrile). After freeze drying, Huangjing peptide powder was obtained with a purity of 99%.

[0052] Example 5: A method for preparing a specific molecular weight Huangjing peptide Step 1: Preprocessing Take 200g of rhizome slices of Polygonatum sibiricum, dry at 65℃ until hardened, and then crush to 80 mesh. Add 70% ethanol solution to the powder at a solid-liquid ratio of 1:9, and extract in a 40℃ water bath for 3 hours. Concentrate the filtrate to 1 / 8 of the original volume, cool and stand for 8 hours for later use. Step 2: Total protein extraction The concentrate was added with 50 mmol / L Tris-HCl buffer (solid-liquid ratio 1:5, pH 7.7), extracted with stirring at 4°C for 2.5 hours, and the supernatant was set aside after centrifugation.

[0053] Step 3: Enzymatic hydrolysis Add compound enzyme (trypsin and papain, mass ratio 3:1, total enzyme concentration is 4% of substrate mass) to the extract. Perform enzymolysis at 48℃ for 4 hours, pH 7.8, and inactivate the enzyme at 90℃ for 10 minutes.

[0054] Step 4: Dynamic separation and molecular weight control The enzymatic solution was ultrafiltered (3,000Da) and then filtered with Zn 2+ The modified medium was used for dynamic separation to collect the target molecular weight peptides, and the eluent was 0.5 mol / L citric acid.

[0055] Step 5: Gradient separation and drying The target peptide was purified by countercurrent chromatography with a mobile phase ratio of 75:25 (gradient elution for 20 minutes), and the target components with molecular weights of 800~1,200Da were collected and freeze-dried to obtain Huangjing peptide with a purity of 98%.

[0056] Comparative Example 1: No compound enzyme was used in the enzymatic hydrolysis process The same as Example 1, except that only trypsin was used in the enzymatic hydrolysis step.

[0057] Specific process: Pretreatment of Polygonatum sibiricum Raw Materials As in Example 1, 70% ethanol was used for extraction, concentration and standing treatment to obtain a polygonatum concentrated solution.

[0058] Total protein extraction As in Example 1, total protein was extracted using Tris-HCl buffer.

[0059] Enzymatic hydrolysis Take 100 mL of total protein extract and add trypsin (total enzyme amount is 4% of the substrate mass). The reaction conditions are 50°C, pH 8.0, and enzymatic hydrolysis time is 4 hours. After enzymatic hydrolysis, heat at 85°C for 10 minutes to inactivate the enzyme, without adding citric acid.

[0060] Next steps The ultrafiltration, dynamic coordination separation and purification process are the same as in Example 1.

[0061] Comparative Example 2: No dynamic coordination separation technology was used The corresponding embodiment 2 is different in that the separation process does not use metal modified media, but directly separates by ultrafiltration and conventional chromatography.

[0062] Specific process: Pretreatment and total protein extraction from Polygonatum cyrtonema rhizome Completely consistent with Example 2.

[0063] Enzymatic hydrolysis A composite enzyme (trypsin and papain, mass ratio 2:1) was used, and the enzyme dosage was 3% of the substrate mass. The enzymatic hydrolysis conditions were pH 8.2, temperature 45°C, and time 4 hours. The enzyme inactivation step was the same as in Example 2.

[0064] Conventional separation The enzymatic hydrolysate was filtered through a 3,000Da ultrafiltration membrane, and the permeate was directly subjected to countercurrent distribution chromatography purification without a dynamic coordination separation step.

[0065] Purification and drying The same gradient elution method (70:30 water and acetonitrile mixed mobile phase) was used and freeze-dried to prepare Huangjing peptide.

[0066] Comparative Example 3: pH was not adjusted during separation This corresponds to Example 3, except that the pH of the enzymatic hydrolysate was not adjusted before the dynamic coordination separation.

[0067] Specific process: Raw material pretreatment, total protein extraction and enzymatic hydrolysis The process is completely consistent with Example 3.

[0068] Dynamic Coordination Separation The enzymatic hydrolysate is directly passed through Zn 2+ Modified medium column (Zn 2+ The remaining separation process (elution conditions, eluent type) was the same as that in Example 3.

[0069] Next steps The purification and freeze-drying steps were the same as in Example 3.

[0070] Comparative Example 4: No gradient elution was used This corresponds to Example 4, except that no gradient elution is used in the countercurrent distribution chromatography.

[0071] Specific process: Treatment and Enzymatic Hydrolysis of Polygonatum cyrtonema Rhizome The process is completely consistent with Example 4.

[0072] Dynamic Coordination Separation The enzymatic hydrolysate was ultrafiltrated and Zn 2+ Modified media treatment (Zn 2+ concentration 0.5mmol / L, column volume 3L), and 0.3mol / L citric acid was used to elute the target peptide.

[0073] Conventional elution separation A fixed ratio of water to acetonitrile (75:25) was used as the mobile phase, and no gradient elution was performed. The remaining chromatographic conditions were the same as those in Example 4.

[0074] Freeze drying The process is the same as that in Example 4 to obtain Huangjing peptide.

[0075] Comparative Example 5: Using a single enzymatic hydrolysis condition The corresponding embodiment 5 differs in that the enzymatic hydrolysis time and pH are fixed, and the enzymatic hydrolysis conditions are not dynamically adjusted.

[0076] Specific process: Polygonatum cyrtonema pretreatment and protein extraction The process is consistent with Example 5.

[0077] Enzymatic hydrolysis The compound enzyme (trypsin and papain, mass ratio 3:1, total enzyme amount is 3% of substrate mass) was hydrolyzed under fixed conditions: pH was set at 8.0, temperature was 48°C, and hydrolysis time was 3 hours. The pH was not dynamically adjusted according to the reaction process.

[0078] Dynamic separation and purification Zn for permeate 2+ The modified medium was used for dynamic separation, and the target peptide was eluted with 0.3 mol / L citric acid, then purified by gradient countercurrent distribution chromatography and freeze-dried to obtain Huangjing peptide.

[0079] Comparative Example 6: Using other separation media Corresponding to Example 2, the difference is that the medium used for dynamic coordination separation is changed to an ordinary silica gel column without modification of metal ions.

[0080] Specific process: Polygonatum cyrtonema pretreatment and protein extraction The process is consistent with Example 2. Enzymolysis The process is consistent with that of Example 2, and the enzyme is inactivated after enzymolysis by the composite enzyme.

[0081] Ordinary silica gel separation The enzymatic hydrolysate was separated and processed by passing through a common silica gel column without metal ions at the same flow rate (2 mL / min) and elution conditions. The target peptide was eluted using countercurrent partition chromatography gradient and then freeze-dried to obtain Polygonatum cyrtonemin.

[0082] Experiment 1: Verify the effect of complex enzyme on the generation of target peptides Experimental Description Experimental procedures Preparation of enzymatic solution According to the method of Example 1, 100 mL of total protein extract of Polygonatum cyrtonema (concentration 8 mg / mL) was added with trypsin and papain (mass ratio 3:1, total enzyme amount was 4% of substrate mass) at 50°C and pH 8.0, and enzymolysis was performed for 4 hours. After the enzyme was inactivated, the solution was cooled to room temperature to obtain enzymolysis solution A.

[0083] According to the method of Comparative Example 1, only trypsin (the amount of enzyme is 4% of the amount of substrate) was used for enzymatic hydrolysis under the same conditions for 4 hours to prepare enzymatic hydrolyzate B.

[0084] Target peptide filtering The two groups of enzymatic hydrolysates were filtered through 3,000Da ultrafiltration membranes, and the permeates were collected as target peptide enrichment solutions.

[0085] Target peptide detection High performance liquid chromatography (HPLC) was used with a mobile phase of 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B) and a gradient elution time of 20 min to detect the peak area of ​​the target peptide (800-1,200 Da) and convert it into the target peptide content (mg / mL).

[0086] Experimental replication The experiment was repeated 3 times for each group, the test data were recorded, and the average value was calculated.

[0087] Experimental data Table 1: This experiment compared the enzymatic hydrolysis effects of compound enzymes and single enzymes and found that compound enzymes can significantly increase the production of target peptides. This advantage comes from the synergistic effect of trypsin and papain on different action sites of yellow spherical protein. Trypsin focuses on cutting peptide bonds near basic amino acids (such as lysine and arginine), while papain can cut sites around hydrophobic amino acids. The combination of the two avoids the limitations of single enzymes and improves the release efficiency of target peptides.

[0088] From the test data, the target peptide content in the compound enzymatic hydrolysate is significantly higher than that in the single enzymatic hydrolysate. This indicates that the multi-site cleavage of the compound enzyme can cover more potential target peptide chain regions in Polygonatum protein. Mechanistically, the compound enzyme reduces the residual uncut peptides and reduces the amount of invalid fragments generated. This multi-site, high-efficiency enzyme cleavage mechanism is the key to achieving a high generation rate of target peptides.

[0089] The main deficiency of a single enzyme is that the cutting specificity is limited and the active fragments in the protein cannot be completely released. In particular, trypsin in yellow spherical protein mainly acts on the basic amino acid-rich region, and this limitation leads to the loss of a large number of target peptides. Papain makes up for this defect. The synergistic effect of the compound enzyme makes the peptide chain cutting more comprehensive, effectively reducing the inefficiency problem in the action of a single enzyme. This synergistic mechanism reflects the unique advantages of the compound enzyme in protein degradation and provides technical support for the present invention.

[0090] Experiment 2: Verify the effect of dynamic coordination separation technology on improving the purity of target peptides Experimental Description Experimental procedures Preparation of enzymatic solution According to the method of Example 2, enzymatic hydrolysis solution A was prepared by a composite enzymatic hydrolysis process (trypsin and papain, mass ratio 2:1, enzyme dosage was 3% of the substrate mass, enzymatic hydrolysis conditions pH 8.2, 45°C, 4 hours).

[0091] According to the method of Comparative Example 2, enzymatic hydrolysis solution B was prepared by adopting the same enzymatic hydrolysis process.

[0092] Separation process For enzymatic hydrolysate A: The enzymatic hydrolysate was filtered through a 3,000Da ultrafiltration membrane and the permeate was collected. After adjusting the pH to 6.9, Zn 2+ Modified medium column (Zn 2+ Concentration 0.5mmol / L), unbound impurities were washed with 30mmol / L phosphate buffer, and the target peptide was eluted with 0.3mol / L citric acid, and the eluate was collected.

[0093] For enzymatic hydrolysate B: the enzymatic hydrolysate was directly ultrafiltered, and then directly purified by countercurrent distribution chromatography without dynamic coordination separation, and the target peptide was eluted.

[0094] Purity testing The target peptides in the final eluates of the two groups were freeze-dried and then tested for purity using high performance liquid chromatography (HPLC), and the mass percentage of the target peptides in the total product was calculated.

[0095] Experimental replication The experiment was repeated 3 times for each group, the test data were recorded, and the average value was calculated.

[0096] Experimental data Table 2: This experiment clearly demonstrated the superiority of dynamic coordination separation technology in improving the purity of target peptides. 2+ The specific coordination effect of the modified medium forms dynamic coordination bonds with the amino and carboxyl groups in the target peptide, effectively enriching the target peptide and eliminating impurities. In contrast, the comparative example without dynamic coordination separation directly relied on countercurrent distribution chromatography purification, which failed to effectively remove impurities of similar molecular weight, resulting in lower final purity.

[0097] The core advantage of dynamic coordination separation lies in its selective enrichment function. Zn 2+ The modified silica matrix provides specific binding sites for the target peptides. By dynamically adjusting the chemical environment of the medium (such as the citric acid concentration and pH of the eluent), the release of the target peptides can be precisely controlled. This separation mode based on coordination chemistry greatly reduces the co-elution of irrelevant peptides, thereby improving the purity of the final product.

[0098] In the comparative example, the hydrolyzate after ultrafiltration was directly used for countercurrent distribution chromatography, and there was a lack of pre-separation steps for impurities, resulting in many small molecule impurities entering the chromatographic separation stage. These impurities showed similar migration behavior to the target peptide under gradient elution conditions, making it difficult to effectively separate them, thereby reducing the final purity. This limitation also shows that chromatography alone is not enough to achieve the high purity effect of dynamic coordination separation.

[0099] In addition, from the experimental data, the purity stability after dynamic coordination separation is higher, and the results of three repeated experiments are very close. This shows that the separation mechanism of dynamic coordination technology has strong repeatability and reliability, while the purity fluctuations in the control examples without this technology are large, which further proves the advantages and applicability of dynamic coordination separation in actual production.

[0100] Experiment 3: Verification of the effect of pH adjustment on dynamic coordination separation Experimental Description Experimental procedures Preparation of enzymatic solution According to the method of Example 3, the total protein extract of Polygonatum cyrtonema (concentration 5 mg / mL) was enzymolyzed by a composite enzyme (trypsin and papain, mass ratio 4:1), the enzyme dosage was 2.5% of the substrate mass, the enzymolysis conditions were pH 7.9, the temperature was 50°C, and the enzyme was inactivated after 3 hours of enzymolysis. Enzymolysis solutions A and B were obtained.

[0101] Separation condition settings Example treatment (enzyme hydrolysate A): The enzyme hydrolysate was filtered through a 3,000Da ultrafiltration membrane, the permeate was collected and the pH was adjusted to 7.0, and then passed through Zn 2+ Modify the medium column. Wash impurities with 25mmol / L phosphate buffer, and then elute the target peptide with 0.3mol / L citric acid.

[0102] Comparative Example Treatment (Enzyme Hydrolysate B): The enzymatic hydrolysate was directly passed through Zn 2+ The modified medium column does not have any pH adjustment step, and the flow rate and elution conditions are the same as in the example.

[0103] Detection Methods The two groups of separated target peptide solutions were collected, and the target peptide recovery rate (the percentage of the total amount of target peptide to the initial target peptide content in the enzymatic hydrolysate) was detected by high performance liquid chromatography (HPLC).

[0104] Experimental replication The experiment was repeated 3 times, the recovery rate of the target peptide was recorded each time, and the average value was calculated.

[0105] Experimental data Table 3: The experimental results clearly show that during the dynamic coordination separation process, adjusting the pH significantly improves the recovery rate of the target peptide. The pH adjustment adjusts the enzymatic hydrolysate to a pH close to the isoelectric point of the target peptide (pH 7.0), which makes the surface charge state of the target peptide tend to be neutral, which helps to bind to Zn 2+ The modified medium forms a more stable coordination bond. In the comparative example without pH adjustment, the original pH of the enzymatic hydrolysate may cause the charge state of the target peptide to deviate too much from neutrality, weakening its bond with Zn 2+ The binding efficiency ultimately affects the separation effect.

[0106] The core mechanism of pH adjustment is to optimize the solution environment of the target peptide. When the pH is close to the isoelectric point of the target peptide, the charge distribution within the molecule tends to a balanced state, which reduces the electrostatic repulsion between molecules and makes the target peptide easier to be captured by the dynamic coordination separation medium. At the same time, this environment also reduces the competitive binding probability of other impurity molecules, further improving the recovery rate and separation efficiency of the target peptide.

[0107] From the data of the comparative example, it can be seen that the enzymatic hydrolysate without pH adjustment showed a large loss of target peptides during the dynamic separation process. This loss is not only due to the insufficient binding of the target peptides to the medium, but may also be related to the competitive binding of impurities. Impurities have a higher charge density and are more likely to form strong competitive adsorption at the original pH, thereby occupying the binding sites of the medium, resulting in a decrease in the separation efficiency of the target peptides. This also shows that pH adjustment, as an important step before separation, plays an irreplaceable role in improving the dynamic coordination separation effect.

[0108] In addition, the data of the embodiment showed very small volatility in three repeated experiments, which shows that the separation process after pH adjustment is more controllable and has high repeatability. The volatility of the comparative example is slightly higher, which may be due to the interference and randomness of impurities. This result further proves that pH adjustment can significantly optimize the dynamic coordination separation process, making it more stable and efficient, and suitable for actual industrialization needs.

[0109] Experiment 4: Verify the effect of gradient elution on the purity of target peptides Experimental Description Experimental procedures Preparation of enzymatic solution According to the process of Example 4, 5 L of total protein extract of Polygonatum cyrtonema (protein concentration of 8 mg / mL) was enzymatically hydrolyzed by a compound enzyme (trypsin and papain, mass ratio of 3:1, enzyme dosage of 3% of substrate mass) for 4 hours, and the enzymatic hydrolysis conditions were pH 8.0 and 50° C. After the enzyme was inactivated, the permeate was collected through a 3,000 Da ultrafiltration membrane, and the pH was adjusted to 6.8 to obtain enzymatic hydrolyzates A and B.

[0110] Dynamic Coordination Separation The enzymatic solution was treated with Zn 2+ Modified medium column, flow rate 5mL / min. After washing impurities with 30mmol / L phosphate buffer, 0.3mol / L citric acid was used to elute the target peptide. Two groups of eluates were collected separately.

[0111] Chromatographic purification Example Treatment (enzyme hydrolysate A): Countercurrent distribution chromatography was used, and the mobile phase was water and acetonitrile, with a volume ratio of 70:30. The gradient elution condition was that the acetonitrile content gradually increased from 10% to 50%, and the gradient time was 25 minutes, and the target peptide was collected.

[0112] Comparative treatment (enzyme digestion solution B): The same mobile phase was used, but the acetonitrile ratio was fixed at 75:25, and gradient elution was not used, and the target peptides were directly collected.

[0113] Purity test of target peptide High performance liquid chromatography (HPLC) was used to analyze the two groups of purified products, detect the purity of the target peptide segment, and calculate its percentage of the total product mass.

[0114] Experimental replication Each experiment was repeated 3 times, and the test data were recorded and the average value was calculated.

[0115] Experimental data Table 4: The role of gradient elution in the purification of target peptides is fully reflected in the experimental results. Gradient elution slowly increases the proportion of acetonitrile in the mobile phase, accurately controls the elution time of the target peptide, and forms a more obvious separation window between it and the impurities. This separation mechanism can effectively reduce the co-elution of impurities, thereby greatly improving the purity of the final product. In the comparative example, the fixed ratio elution method cannot accurately distinguish the target peptide from impurities of similar properties, resulting in a lower final purity.

[0116] From a mechanistic point of view, the core of gradient elution is to dynamically adjust the polarity of the solution, and to control the distribution behavior of the target peptide and the stationary phase to ensure that its elution rate in the mobile phase is significantly different from that of the impurities. Due to differences in molecular structure or polarity, impurities are eluted early or late at the beginning or end of the gradient change, while the target peptide is eluted in a concentrated manner within a specific gradient range. This process forms a clear separation effect. However, fixed ratio elution cannot dynamically adjust the distribution coefficient, resulting in overlapping elution of impurities and target peptides, affecting the purification effect.

[0117] It can be seen from the data that the purity of the target peptide segment in the embodiment is stably maintained at more than 98%, while the purity of the comparative example is significantly lower and fluctuates greatly. This shows that the controllability and separation accuracy of gradient elution are better than those of fixed ratio elution. Under gradient conditions, the elution behavior of the target peptide segment and impurities is more consistent, thereby achieving higher repeatability. However, due to the lack of control measures, the impurity ratio of fixed ratio elution is easily affected by slight changes in the environment, making the purity results unstable.

[0118] The experiment also reflects the criticality of impurity exclusion. Without gradient elution, many small molecule peptides with similar structures but irrelevant functions cannot be completely separated. Gradient elution eliminates these interfering substances through a refined separation window. The results show that gradient elution is a necessary process to achieve high-purity target peptides, and plays an important role in improving the overall technical level of the present invention.

[0119] Experiment 5: Verify the effect of dynamic enzymatic hydrolysis conditions on the efficiency of target peptide generation Experimental Description Experimental procedures Preparation of enzymatic solution Example treatment (dynamic enzymolysis, enzymolysis solution A): According to the process of Example 5, the total protein extract of Polygonatum cyrtonema (concentration 5 mg / mL) was subjected to compound enzymolysis (trypsin and papain, mass ratio 3:1, total enzyme amount is 4% of substrate mass). The pH and time conditions were dynamically adjusted during the enzymolysis process. The initial pH was 7.8, and the pH value was detected every 30 minutes and gradually adjusted to 7.9. The enzymolysis time was 4 hours, and the enzyme was immediately inactivated after the reaction was completed.

[0120] Comparative Example Treatment (Fixed Enzyme Hydrolysis, Enzyme Hydrolysis Solution B): According to the process of Comparative Example 5, the same compound enzyme was used, the enzymatic hydrolysis condition was fixed at pH 8.0, the enzymatic hydrolysis time was 3 hours, and no dynamic adjustment was performed during the enzymatic hydrolysis process.

[0121] Target peptide detection Both groups of enzymatic hydrolysates were filtered through a 3,000Da ultrafiltration membrane, and the permeate was collected.

[0122] High performance liquid chromatography (HPLC) was used to detect the content of the target peptide (molecular weight range 800~1,200Da) and calculate the target peptide generation rate (the percentage of the target peptide content in the total protein extract).

[0123] Experimental replication Each group of experiments was repeated 3 times, the test data were recorded, and the average generation rate was calculated.

[0124] Experimental data Table 5: The experimental results fully demonstrated that the dynamic adjustment of pH and enzymatic hydrolysis time significantly improved the generation efficiency of the target peptides. Dynamic enzymatic hydrolysis adjusts the optimal action conditions of the enzyme during the reaction process, allowing the compound enzyme to fully play its role at different stages. The lower pH (7.8) in the initial stage provides a higher catalytic efficiency for trypsin, and as the pH is gradually adjusted to 7.9, the activity of papain is gradually activated, achieving multi-site synergistic degradation. This dynamic optimization process is obviously more in line with the reaction kinetics of proteases and significantly improves the release of target peptides.

[0125] Under fixed enzymatic hydrolysis conditions, the activity of the enzyme is limited to a fixed range, resulting in a significant reduction in the cutting efficiency. In particular, there are a large number of complex peptide chain structures in yellow spherical protein, and a single pH environment cannot fully activate the synergistic effect of the complex enzyme, and many potential target peptides cannot be released efficiently. In addition, the fixed enzymatic hydrolysis time (3 hours) is too short, so that the peptide bonds in some high-impedance areas are not completely cut, further limiting the generation of target peptides.

[0126] Judging from the data on the generation rate, the target peptide generation rate under dynamic enzymatic hydrolysis conditions increased by an average of about 15%. Mechanistically, this is not only an increase in enzyme activity, but more importantly, the dynamic adjustment of pH makes the enzyme's action site more selective. Papain is more inclined to cut peptide chains around hydrophobic amino acids in an environment close to neutral pH (7.8~7.9), while trypsin is more suitable for cutting peptide bonds around basic amino acids in an alkaline environment. The synergistic cutting process of the two is fully amplified by dynamic enzymatic hydrolysis, which is obviously impossible to achieve under fixed enzymatic hydrolysis conditions.

[0127] The experiment also showed that the dynamic enzymatic hydrolysis process has strong stability. The fluctuation range of three repeated experiments is small, indicating that this method has good controllability and industrialization potential in practical applications. In contrast, the results of fixed enzymatic hydrolysis not only have a low generation rate, but also a slightly larger fluctuation range, which may be affected by the unoptimized enzyme conditions. This further verifies the importance of dynamic enzymatic hydrolysis technology in improving the efficiency of target peptide generation, which is one of the core technical advantages of the scheme of the present invention.

[0128] Experiment 6: Verify the effect of metal-modified media on the separation of target peptides Experimental Description Experimental procedures Preparation of enzymatic solution According to the process of Example 2, the total protein extract of Polygonatum cyrtonema (concentration 8 mg / mL) was subjected to compound enzymatic hydrolysis (trypsin and papain, mass ratio 2:1, total enzyme amount is 3% of the substrate mass), the enzymatic hydrolysis conditions are pH 8.2, 45°C, and enzymatic hydrolysis for 4 hours. After the enzyme is inactivated, it is filtered through a 3,000Da ultrafiltration membrane, the permeate is collected, and the pH is adjusted to 6.9 to obtain enzymatic hydrolyzates A and B.

[0129] Dynamic Coordination Separation Example treatment (enzyme hydrolysate A): The enzyme hydrolysate was passed through Zn 2+ Modified medium column (Zn 2+ Concentration 0.5mmol / L), unbound impurities were washed with 30mmol / L phosphate buffer, and the target peptide was eluted with 0.3mol / L citric acid, and the eluate was collected.

[0130] Comparative Example Treatment (Enzyme Hydrolysate B): The enzymatic hydrolysate was passed through a common silica gel medium column without modification of metal ions at the same flow rate, and the elution conditions and process were consistent with those of the Example Treatment.

[0131] Target peptide detection The two groups of eluates were tested by high performance liquid chromatography (HPLC) to detect the content (mg / mL) and purity (mass percentage of the target peptide in the total components).

[0132] Experimental replication Each experiment was repeated 3 times, and the test data were recorded and the average value was calculated.

[0133] Experimental data Table 6A: Table 6B: The experimental results show that the use of Zn 2+ The modified medium significantly improves the separation effect of the target peptides. Both the content and purity of the target peptides are much higher than those of ordinary silica gel medium. This advantage comes from the specific coordination effect of the metal-modified medium. Zn 2+ The ions form dynamic coordination bonds with the carboxyl and amino groups in the target peptide molecules, providing stronger binding ability and selectivity, thereby significantly eliminating impurities of non-target peptides. This separation mechanism is obviously not achievable with ordinary silica gel media, because ordinary silica gel only relies on weak polar adsorption and has no specific recognition ability, which makes it easy for impurities to bind or elute with the target peptide at the same time, greatly reducing the purity.

[0134] Mechanistically, the separation effect of metal-modified media is due to the Zn 2+ Dynamic coordination properties of Zn 2+ The charge distribution of the target peptide can form a stable non-covalent bond with the functional group of the target peptide. This bond is both powerful and flexible, and is suitable for releasing the target peptide through competitive displacement with citric acid in the subsequent elution stage. At the same time, this separation mechanism effectively avoids electrostatic interference between molecules, making the separation process more efficient and stable. Ordinary silica gel media lack this characteristic and can only provide non-specific adsorption, resulting in the co-elution of the target peptide and impurities, greatly reducing the separation effect.

[0135] Experimental data also show that Zn 2+ The modified medium has better repeatability. The fluctuation range of the target peptide content and purity in the three experiments was extremely small, indicating that the medium's ability to capture the target peptide is highly controllable and consistent. The results of ordinary silica gel medium fluctuate greatly, and this instability will obviously affect the separation process in actual production. At the same time, the content of the target peptide after separation with ordinary silica gel medium is also low, which may be because impurities compete for limited adsorption sites, further reducing the effective separation efficiency.

[0136] From the application perspective, the significant advantage of metal-modified media lies not only in the separation effect, but also in the scalability of industrial production. It can achieve efficient separation with lower eluent concentration and mild operating conditions, reduce energy consumption and the use of chemical reagents, and show higher environmental protection and economy. These characteristics together constitute the technical innovation of the present invention and are also the core advantages that ordinary silica gel media cannot match.

[0137] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Huangjing peptide for anti-inflammatory and kidney protection, characterized in that: The following steps are involved: (1) crushing the rhizome of Polygonatum sibiricum and pre-treating it with an ethanol solution to obtain a water extract concentrate; (2) adding the water extract concentrate to a buffer solution to extract total protein from Polygonatum cyrtonema; (3) The total protein solution is enzymatically hydrolyzed with enzymes under the following conditions: enzyme concentration is 2% to 5% of substrate mass, pH range is 7.5-8.5, temperature is 45-55°C, and time is 35 hours; (4) After ultrafiltration of the enzymatic hydrolysate to remove macromolecular impurities, the target peptides were separated using metal-modified media for dynamic coordination. The separation conditions were a metal ion concentration of 0.1-1 mmol / L and a solution pH of 6.8-7.

2. (5) The target peptide was purified by countercurrent distribution chromatography and freeze-dried to obtain polysaccharide peptide.

2. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The particle size of the crushed rhizome of polygonatum is 80 mesh, the concentration of the ethanol solution used is 70%, the pretreatment temperature is 40-50°C, the time is 24 hours, the solid-liquid ratio is 1:8-1:10, and the filtrate obtained after ethanol treatment needs to be concentrated to 1 / 51 / 10 of the original volume, and the precipitate is removed for standby use.

3. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The buffer used for extracting total protein from polygonatum is Tris-HCl buffer, the concentration of the buffer is 20-50 mmol / L, the solid-liquid ratio during the extraction process is 1:5, the extraction temperature is 4°C, the extraction time is 23 hours, and the extracted liquid needs to be centrifuged at 4,000-6,000 rpm for 1015 minutes to separate the supernatant and remove impurities.

4. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The enzyme used in the enzymatic hydrolysis reaction is a composite enzyme of trypsin and papain, the mass ratio of trypsin to papain is 2:1-4:1, the total enzyme concentration is 2% to 5% of the substrate mass, the target protein concentration in the enzymatic hydrolyzate is 5-10 mg / mL, the enzymatic hydrolysis time is 35 hours, and the reaction conditions need to be dynamically adjusted during the reaction to ensure that the pH value remains in the range of 7.5 to 8.

5.

5. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The enzyme inactivation operation of the enzymatic hydrolysis solution includes heating the temperature to 80-90° C. and maintaining it for 10-15 minutes, while adding 0.1%-0.2% (w / v) citric acid to stabilize the enzymatic hydrolysis product. The enzyme-inactivated solution needs to be quickly cooled to room temperature and filtered to remove residual impurities.

6. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The molecular weight cutoff range of the ultrafiltration membrane is 3,000-5,000Da. During the ultrafiltration operation, the feed liquid needs to pass through the ultrafiltration membrane at a pressure of 0.5-1MPa. The permeate after ultrafiltration needs to be tested for the target peptide content by an online amino acid analyzer. After the test result meets the target range, the permeate is collected for use.

7. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The metal modified medium is prepared from a metal loading material with silica gel as a matrix. The silica gel matrix is ​​treated with dilute hydrochloric acid and loaded with metal ions such as Zn 2+ Cu 2+ The metal loading concentration is 0.1-1mmol / L, the medium particle size is 60-120μm, and after metal ion loading, it is dried at 70℃ for 4-6 hours to increase stability.

8. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: In the dynamic coordination separation process, the enzymatic hydrolyzate is adjusted to pH 6.8-7.2 and then passed through the metal-modified medium column at a flow rate of 13 mL / min. Unbound impurities are eluted with 20-50 mmol / L phosphate buffer, and the target peptide is eluted with 0.1-0.5 mol / L citric acid or EDTA solution. The eluted solution needs to be collected after the target peptide concentration is detected.

9. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The mobile phase used in the countercurrent distribution chromatography purification is a mixture of water and acetonitrile in a volume ratio of 80:20-70:30, the gradient elution condition is a linear gradient elution of 10% to 50% acetonitrile, the elution time is 15 to 30 minutes, and the recovery rate of the target peptide segment is not less than 90%.

10. The method for preparing Huangjing peptide for anti-inflammatory and kidney protection according to claim 1, characterized in that: The components of the obtained Huangjing peptide are as follows by mass: Huangjing peptide: 80%~95%; Anhydrous glucose: 5% to 10%; Monocalcium phosphate: 2% to 5%; Vitamin E: 0.5%~2%.

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