Preparation method of a yellow-stem peptide for anti-inflammatory kidney protection

Abstract

The application relates to the technical field of bioactive peptide preparation, and discloses a preparation method of a rhizoma polygonati peptide for anti-inflammatory and kidney protection, which comprises the following steps: (1) crushing rhizoma polygonati and then pretreating the rhizoma polygonati with an ethanol solution to obtain a water extraction concentrate; (2) adding the water extraction concentrate into a buffer solution to extract total rhizoma polygonati protein; (3) subjecting the total protein solution to enzymolysis with an enzyme, wherein the enzymolysis conditions are as follows: 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; (4) after the enzymolysis solution is subjected to ultrafiltration to remove macromolecular impurities, a target peptide segment is separated through metal-modified medium dynamic coordination, the target peptide segment is purified through countercurrent distribution chromatography, and the rhizoma polygonati peptide is prepared through freeze-drying. The application is better used as anti-inflammatory and kidney protection raw materials through the complex enzyme synergistic enzymolysis, multi-site dynamic regulation and dynamic coordination separation technology, and is used for preventing and assisting in treating kidney-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioactive peptide preparation, in particular to a preparation method of a polygonatum sibiricum peptide for anti-inflammatory and kidney protection. BACKGROUND

[0002] The polygonatum sibiricum peptide has attracted much attention in the functional food and pharmaceutical fields in recent years due to its significant anti-inflammatory and kidney protection functions. As an active peptide segment with a clear molecular weight range (800-1,200 Da), the polygonatum sibiricum peptide protects kidney cells through mechanisms such as regulating inflammatory factors and resisting oxidative stress. At present, the preparation of the polygonatum sibiricum peptide mainly includes the steps of raw material pretreatment, protein extraction, enzymolysis, separation, and purification. However, these processes still have many limitations in the actual production efficiency, target peptide segment generation amount, and purity of the final product, which limits the large-scale application of the polygonatum sibiricum peptide.

[0003] In the prior art, a single enzyme is used for protein degradation under fixed conditions in the enzymolysis stage, which fails to fully utilize the synergy of compound enzymes, resulting in a low generation rate of the target peptide segment. At the same time, ultrafiltration combined with conventional chromatography separation technology is mainly used in the separation and purification stage, which mainly relies on the molecular weight or physical properties for coarse separation and lacks specific selection of the target peptide segment, resulting in a low purity of the final product. In addition, the lack of dynamic regulation means seriously limits the enzymolysis efficiency, separation effect, and stability and efficiency of the overall process. The prior art has deficiencies in the enzymolysis synergy mechanism, separation precision, and process stability, and it is difficult to achieve efficient preparation and high-purity separation of the target peptide segment. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a preparation method of a polygonatum sibiricum peptide for anti-inflammatory and kidney protection, which solves the problems of low generation efficiency of the target peptide segment of the polygonatum sibiricum peptide, insufficient separation purity, and lack of dynamic regulation of the process in the prior art.

[0005] To achieve the above purpose, the present application is implemented by the following technical scheme: a preparation method of a polygonatum sibiricum peptide for anti-inflammatory and kidney protection, comprising the following steps:

[0006] (1) The polygonatum sibiricum rhizome is crushed and pretreated with an ethanol solution to obtain a water extraction concentrate;

[0007] (2) The water extraction concentrate is added to a buffer solution to extract total polygonatum sibiricum protein;

[0008] (3) The total protein solution is subjected to enzymolysis with an enzyme, and the enzymolysis conditions are as follows: enzyme concentration is 2%-5% of the mass of the substrate, pH range is 7.5-8.5, temperature is 45-55℃, and time is 35 hours;

[0009] (4) After removing macromolecular impurities from the enzymatic hydrolysate by ultrafiltration, the target peptide is separated by dynamic coordination using metal-modified medium, and the separation conditions are as follows: the concentration of metal ions is 0.1-1 mmol / L, and the pH of the solution is 6.8-7.2.

[0010] (5) The target peptide is purified by reverse flow partition chromatography, and the prepared Huangjing peptide is obtained after freeze-drying.

[0011] Preferably, the particle size of the crushed rhizome of Polygonatum sibiricum 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 / 5 of the original volume and the precipitate is removed for use.

[0012] Preferably, the buffer used for extracting total protein from Polygonatum sibiricum is Tris-HCl buffer, and the concentration of the buffer is 20-50 mmol / L. The solid-liquid ratio during extraction is 1:5, the extraction temperature is 4°C, and the extraction time is 23 hours. The liquid after extraction needs to be centrifuged at 4,000-6,000 rpm for 15 minutes to separate the supernatant and remove impurities.

[0013] Preferably, the enzyme used in the enzymatic reaction is a complex enzyme of trypsin and papain, and the mass ratio of trypsin to papain is 2:1-4:1. The total enzyme concentration is 2%-5% of the mass of the substrate, the concentration of the target protein in the enzymatic hydrolysate is 5-10 mg / mL, and the enzymatic hydrolysis time is 35 hours. During the reaction process, the reaction conditions need to be dynamically adjusted to ensure that the pH value remains in the range of 7.5-8.5.

[0014] Preferably, the enzyme inactivation operation of the enzymatic hydrolysate includes heating to 80-90°C and maintaining for 10-15 minutes, while adding 0.1%-0.2% (w / v) of citric acid to stabilize the enzymatic hydrolysate. The solution after enzyme inactivation needs to be quickly cooled to room temperature and filtered to remove residual impurities.

[0015] Preferably, the molecular weight cut-off range of the ultrafiltration membrane is 3,000-5,000 Da, and the pressure of the solid-liquid mixture passing through the ultrafiltration membrane during the ultrafiltration operation is 0.5-1 MPa. The permeate after ultrafiltration needs to be detected for the content of the target peptide by an online amino acid analyzer, and the permeate is collected for use after the detection result meets the target range.

[0016] Preferably, the metal-modified medium is prepared by loading metal ions on a silica gel matrix, and the metal ions loaded on the silica gel matrix after treatment with dilute hydrochloric acid are Zn 2+ or Cu 2+ . The concentration of the metal-loaded material is 0.1-1 mmol / L, the particle size of the medium is 60-120 μm, and the metal ions are dried at 70°C for 4-6 hours to increase stability after being loaded.

[0017] Preferably, in the dynamic coordination separation process, the enzymatic solution is adjusted to pH 6.8-7.2 and passed through the metal-modified medium column at a flow rate of 13 mL / min, the unbound impurities are eluted by 20-50 mmol / L phosphate buffer, and the target peptide segment is eluted by 0.1-0.5 mol / L citric acid or EDTA solution. The eluted solution needs to be detected for target peptide concentration before collection.

[0018] Preferably, the mobile phase used in the reverse flow distribution chromatography purification is a mixture of water and acetonitrile with a volume ratio of 80:20-70:30, and the gradient elution condition is a linear gradient elution of 10% to 50% acetonitrile with an elution time of 15-30 minutes. Preferably, the obtained Huangjing peptide components account for 80%-95% by mass fraction:

[0019] Huangjing peptide: 80%-95%;

[0020] Anhydrous glucose: 5%-10%;

[0021] Calcium dihydrogen phosphate: 2%-5%;

[0022] Vitamin E: 0.5%-2%.

[0023] The present application provides a preparation method of Huangjing peptide for anti-inflammatory kidney protection. The present application has the following beneficial effects:

[0024] 1. The present application introduces dynamic coordination separation technology, uses metal-modified medium to realize precise separation and enrichment of Huangjing peptide target peptide segment, compared with the method of simply relying on membrane separation or chromatographic separation in the prior art, solves the problem of limited purity of target peptide segment, and significantly improves the separation efficiency and product consistency.

[0025] 2. The present application adopts a complex enzyme hydrolysis system of trypsin and papain, and optimizes the enzyme hydrolysis conditions based on density functional theory, precisely cuts the target peptide chain, compared with the traditional enzyme hydrolysis method, avoids the defect of random cutting of peptide chain, greatly improves the generation rate of active peptide segment, and solves the problems of low enzyme hydrolysis efficiency and poor specificity in the prior art.

[0026] 3. The present application realizes efficient separation of target peptide segment and impurities by reverse flow distribution chromatography combined with gradient elution and control of mobile phase proportion, compared with the elution method of single solvent system in the prior art, the present application significantly improves the separation precision, solves the technical difficulty that the recovery rate and purity of target peptide segment are difficult to be considered at the same time in the traditional method.

[0027] 4、The present application is different from the prior art by using ethanol solution to remove fat-soluble impurities during the pretreatment of rhizoma polygonati raw materials, combining with buffer to extract proteins and enrich water-soluble components, the method effectively reduces impurity interference, avoids the loss of target components, and overcomes the problems of low purity and incomplete extraction in the raw material treatment stage. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The present application is different from the prior art by using ethanol solution to remove fat-soluble impurities during the pretreatment of rhizoma polygonati raw materials, combining with buffer to extract proteins and enrich water-soluble components, the method effectively reduces impurity interference, avoids the loss of target components, and overcomes the problems of low purity and incomplete extraction in the raw material treatment stage. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the specification of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] Please refer to the drawings in the specification of the present application Figure 1 The present application provides a preparation method of rhizoma polygonati peptide for anti-inflammatory and kidney protection, comprising:

[0031] 1. Pretreatment of rhizoma polygonati raw materials

[0032] Technical content:

[0033] The rhizoma polygonati is washed and dried at 60℃ to a moisture content of less than 10%, and then crushed to a powder of 80 mesh. The powder is added to 70% ethanol solution at a solid-liquid ratio of 1:8-1:10 (w / v), and stirred in a water bath at 40-50℃ for 24 hours. The solid residue is removed by filtration to obtain a filtrate. The filtrate is concentrated under reduced pressure to 1 / 5-1 / 10 of the original volume to enrich water-soluble components. The concentrated solution is cooled to room temperature and left to stand for 12 hours, and the precipitate is discarded for later use.

[0034] The rhizoma polygonati contains rich 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 to fat-soluble impurities can effectively remove non-polar components while retaining polar components (such as proteins and polysaccharides). The concentration step further enriches the water-soluble target components, improving the efficiency of subsequent protein extraction.

[0035] 2. Extraction of total rhizoma polygonati protein

[0036] Technical content:

[0037] The pretreated concentrated solution was added to 20-50 mmol / L Tris-HCl buffer (pH 7.5) at a solid-liquid ratio of 1:5 (w / v) and extracted at low speed under 4°C for 23 hours. After extraction, the solution was centrifuged at 4,000-6,000 rpm for 10-15 minutes, and the supernatant was taken as the total protein extract. The supernatant was stored in the refrigerator.

[0038] The extraction of total protein from Huangjing depends on the solubility of the solvent environment for the protein. The Tris-HCl buffer provides a suitable pH environment (pH 7.5) that can maintain the natural conformation of the protein and prevent its denaturation or precipitation. Low temperature conditions (4°C) inhibit the spontaneous degradation of proteases, while protecting the activity of the protein. The centrifugation step separates impurities (such as cellulose) from the protein solution by density difference, ensuring the purity of the total protein extract.

[0039] 3. Enzymatic reaction

[0040] Technical content:

[0041] The total protein extract was adjusted to a protein concentration of 5-10 mg / mL. Under constant temperature conditions of 45-55°C, a complex enzyme of trypsin and papain (mass ratio 2:14:1) was added, with an enzyme dosage of 2%-5% of the substrate mass. The solution pH was adjusted to 7.5-8.5 with phosphate buffer, and the enzymatic hydrolysis time was 35 hours. After enzymatic hydrolysis, the enzyme was inactivated by heating to 80-90°C for 10-15 minutes, and 0.1%-0.2% (w / v) citric acid was added to stabilize the enzymatic hydrolysate, which was then cooled to room temperature for use.

[0042] The key to enzymatic hydrolysis is the specificity and efficiency of enzyme cleavage of the peptide chain. The combination of trypsin and papain complex enzyme can act on specific sites of Huangjing protein (such as the peptide bond near lysine and arginine), achieving efficient decomposition. The adjustment of the complex ratio (2:1-4:1) optimizes the synergistic effect of enzyme cleavage. The pH and temperature are controlled within the optimal range of enzyme activity (pH 7.5-8.5, 45-55°C), ensuring the maximum efficiency of enzyme cleavage. The addition of citric acid after enzyme inactivation can inhibit the residual activity of metalloenzymes, while also protecting the target peptide segments.

[0043] 4. Dynamic coordination separation technology (DCS)

[0044] Technical content:

[0045] The enzymatic hydrolysate was filtered using an ultrafiltration membrane with a 3,000-5,000 Da cutoff, and the permeate was collected and adjusted to a pH of 6.8-7.2. The permeate was passed through a metal-modified silica gel medium column at a flow rate of 13 mL / min. The medium was modified by amine groups and loaded with Zn 2+ or Cu 2+(Concentration: 0.11 mmol / L, particle size: 60-120 μm). After washing away unbound impurities with 2050 mmol / L phosphate buffer, elute the target peptide with 0.1–0.5 mol / L citric acid or EDTA solution and collect the eluent.

[0046] Dynamic coordination separation utilizes metal ions (Zn) 2+ or Cu 2+ The target peptide forms coordination bonds with the carboxyl and amino groups in the target peptide, separating the target peptide from impurities through the affinity of the column packing medium. By adjusting the pH (6.8–7.2), the major charge of the target peptide is ensured to be in a neutral or weakly negative state, thereby enhancing its binding to metal ions. Citric acid or EDTA is added during the elution step to release the target peptide from the metal ions through competitive coordination, ensuring high-purity recovery of the target peptide.

[0047] 5. Countercurrent partition chromatography purification

[0048] Technical content:

[0049] The eluted liquid was further purified by countercurrent partition chromatography using water and acetonitrile (volume ratio 80:20-70:30). A linear gradient elution was employed (acetonitrile content increased from 10% to 50%), with elution times of 15–30 minutes. The purified liquid was freeze-dried to prepare Polygonatum peptide powder. The target product had a molecular weight range of 800–1,200 Da and a purity of not less than 98%.

[0050] Countercurrent partition chromatography separates target peptides based on the difference in partition coefficients between the target peptide and the solvent. Gradient ratio control of water and acetonitrile effectively regulates the solubility of the target peptide in the mobile and stationary phases, enabling its separation from other impurities. Control of the molecular weight range (800–1,200 Da) ensures the homogeneity and activity of the final product, while freeze-drying prevents degradation of heat-sensitive substances.

[0051] 6. Composition ratio of Polygonatum peptides

[0052] Technical content:

[0053] The components of the final product, Polygonatum peptide, are as follows by mass percentage:

[0054] Polygonatum peptide: 80%–95%;

[0055] Anhydrous glucose: 5%–10%;

[0056] Calcium dihydrogen phosphate: 2%–5%;

[0057] Vitamin E: 0.5%–2%.

[0058] The addition of anhydrous glucose enhances the physical stability of the product and improves its taste; calcium dihydrogen phosphate provides mineral support and helps replenish trace elements; vitamin E, as a natural antioxidant, extends the product's shelf life and further stabilizes the bioactivity of polygonatum peptides.

[0059] Example 1: A method for efficiently separating high-purity Polygonatum odoratum peptides

[0060] Step 1: Pretreatment of Polygonatum sibiricum raw materials

[0061] Clean the rhizomes of Polygonatum sibiricum and dry them with hot air at 60℃ until the moisture content is less than 10%. Then pulverize them into 80-mesh powder. Take 50g of powder and add 500mL of 70% ethanol solution (solid-liquid ratio 1:10), and extract by stirring in a water bath at 45℃ for 3 hours. Filter with gauze to remove solid residue, concentrate the filtrate under reduced pressure to 1 / 6 of the original volume, let it stand for 12 hours, discard the precipitate, and obtain concentrated Polygonatum sibiricum solution.

[0062] Step 2: Extraction of Polygonatum protein

[0063] Add the concentrate to 30 mmol / L Tris-HCl buffer at a solid-liquid ratio of 1:5, adjust the pH to 7.5, and extract at 4°C with low-speed stirring for 3 hours. Centrifuge the extract (5,000 rpm, 12 minutes), and the supernatant is the total protein extract of Polygonatum sibiricum, which is ready for use.

[0064] Step 3: Enzymatic hydrolysis

[0065] Add a mixture of trypsin and papain (4% of the substrate mass, enzyme ratio 3:1) to 100 mL of total protein extract, adjust the pH to 8.0, and incubate in a 50°C water bath for 4 hours. After the reaction, heat to 85°C and hold for 10 minutes to inactivate the enzyme. Then add 0.1% (w / v) citric acid to stabilize the product, cool to room temperature, and obtain the enzymatic hydrolysate.

[0066] Step 4: Dynamic coordination separation

[0067] The enzymatic hydrolysate was filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected. After adjusting the pH to 7.0, it was passed through a Zn-loaded membrane at a flow rate of 2 mL / min. 2+ silica gel matrix column (Zn 2+ (Concentration 0.5 mmol / L). Wash away impurities with 30 mmol / L phosphate buffer, then elute the target peptide with 0.3 mol / L citric acid solution, and collect the eluent.

[0068] Step 5: Purification and Drying

[0069] The eluent was purified by countercurrent partition chromatography using water and acetonitrile (80:20 v / v) as the mobile phase, with gradient elution for 15 minutes, and the target component was collected. Finally, it was freeze-dried to obtain Polygonatum peptide powder with a molecular weight of 800–1,200 Da and a purity of 99%.

[0070] Example 2: A method for rapid production of highly active Polygonatum odoratum peptide

[0071] Step 1: Processing of Polygonatum rhizome

[0072] Slice 100g of Polygonatum rhizome, dry at 60℃, and then pulverize to 85 mesh. Take 80g of powder and add 640mL of 70% ethanol solution (solid-to-liquid ratio 1:8). Extract by stirring in a water bath at 45℃ for 2.5 hours, and filter to remove residue using a filter cloth. Concentrate the filtrate to 1 / 8 of its original volume, cool and let stand for 6 hours, and discard the precipitate.

[0073] Step 2: Total protein extraction

[0074] The concentrate was added to 40 mmol / L Tris-HCl buffer (pH 7.8) at a feed-to-solid ratio of 1:4, and extracted at 4°C with stirring for 2 hours. The extract was centrifuged at 6,000 rpm for 15 minutes, and the supernatant was collected for later use.

[0075] Step 3: Complex enzymatic hydrolysis

[0076] Add a compound enzyme (trypsin and papain, mass ratio 2:1, total enzyme concentration 3% of substrate mass) to 100 mL of total protein extract. Adjust pH to 8.2 and incubate at 45°C for 5 hours. After incubation, heat to 90°C and hold for 10 minutes to inactivate the enzyme, then cool to room temperature.

[0077] Step 4: Dynamic Separation and Enrichment

[0078] The enzymatic hydrolysate was filtered through a 5,000 Da ultrafiltration membrane, and the pH of the permeate was adjusted to 6.9. The permeate was then passed through a Cu-loaded ultrafiltration membrane at a flow rate of 3 mL / min. 2+ A silica gel column (Cu²⁺ concentration 1.0 mmol / L) was used. Impurities were washed away with 50 mmol / L phosphate buffer, and the target peptide was eluted with 0.4 mol / L EDTA solution. The eluent was collected.

[0079] Step 5: Separation and purification

[0080] The eluent was obtained by gradient chromatography using water and acetonitrile (70:30 v / v) as the mobile phase for 20 minutes. The purified target product was collected and freeze-dried to obtain Polygonatum peptide with a purity of 98% and stable activity.

[0081] Example 3: A method for preparing Polygonatum peptide with optimized enzymatic hydrolysis efficiency

[0082] Step 1: Preprocessing

[0083] The rhizome of Polygonatum was rapidly dried at 70℃ for 8 hours, pulverized to 75 mesh, and 40g of powder was extracted with 400mL of 70% ethanol solution for 2 hours at a material-to-liquid ratio of 1:10. The filtrate was concentrated to 1 / 7 of its original volume and cooled for later use.

[0084] Step 2: Total protein extraction

[0085] The concentrate was mixed with 50 mmol / L Tris-HCl buffer at a feed-to-liquid ratio of 1:6, at pH 7.6, stirred at 4°C for 3 hours, and centrifuged to obtain the total protein extract.

[0086] Step 3: Compound enzymatic hydrolysis

[0087] In a constant temperature water bath at 50℃, the total protein extract (200 mL) was mixed with a compound enzyme (trypsin and papain, mass ratio 4:1, total amount 2.5% of substrate mass), the pH was adjusted to 7.9, and enzymatic hydrolysis was carried out for 3 hours. Enzyme inactivation was performed at 85℃ for 15 minutes.

[0088] Step 4: Dynamic enrichment

[0089] The enzymatic hydrolysate was filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected and adjusted to pH 6.8. Zn was then used. 2+ Modified silicone pillars (Zn 2+ The target peptide was enriched at a concentration of 0.3 mmol / L. The column wash buffer was 25 mmol / L phosphate buffer, and the elution buffer was 0.2 mol / L citric acid.

[0090] Step 5: Purification of the target peptide

[0091] The eluent was subjected to countercurrent partition chromatography with a gradient elution time of 18 minutes. The water to acetonitrile ratio was 75:25. The target component was collected and freeze-dried to obtain Polygonatum odoratum peptide powder with a molecular weight range of 850–1,150 Da and a purity of 97%.

[0092] Example 4: A method for industrial-scale preparation of high-purity Polygonatum peptides

[0093] Step 1: Large-scale preprocessing

[0094] Take 5 kg of Polygonatum rhizome, wash it, and dry it at 70℃ until the moisture content is less than 10%. Crush it into 80-mesh powder, add 35 L of 70% ethanol solution (solid-to-liquid ratio 1:7), and extract by stirring in a 50℃ water bath for 3 hours. After filtration, concentrate the filtrate under reduced pressure to 1 / 10 of the original volume, and let it cool and stand for 12 hours.

[0095] Step 2: Total protein extraction

[0096] Add the concentrate to 30 mmol / L Tris-HCl buffer (pH 7.5) at a feed-to-solid ratio of 1:5 and stir at 4°C for 3 hours. Centrifuge at 6,000 rpm for 15 minutes and collect the supernatant for later use.

[0097] Step 3: Enzymatic hydrolysis and enzyme inactivation

[0098] The total protein extract concentration was adjusted to 8 mg / mL, and a compound enzyme (trypsin and papain, mass ratio 3:1, total enzyme amount 3% of substrate mass) was added. Enzymatic hydrolysis was performed at 50℃ for 4 hours, and the pH was adjusted to 8.0. After the reaction, the temperature was raised to 90℃ and held for 15 minutes to inactivate the enzyme, then cooled to room temperature.

[0099] Step 4: Dynamic Separation and Target Enrichment

[0100] The enzymatic hydrolysate was filtered through a 5,000 Da ultrafiltration membrane, and the pH of the permeate was adjusted to 6.8. Zn was then loaded. 2+ Silicone column (Zn) 2+ The concentration was 0.5 mmol / L, column volume was 3 L, and the flow rate was 5 mL / min. The elution buffer was 0.3 mol / L citric acid solution. The target peptide was collected after elution.

[0101] Step 5: Purification and Finished Product

[0102] The target peptide eluent was purified by gradient countercurrent partition chromatography for 25 minutes using a mobile phase ratio of 70:30 (water:acetonitrile). The eluent was then freeze-dried to obtain Polygonatum peptide powder with a purity of 99%.

[0103] Example 5: A method for preparing a specific molecular weight polygonatum peptide

[0104] Step 1: Preprocessing

[0105] Take 200g of sliced ​​Polygonatum rhizome, dry at 65℃ until hardened, and then pulverize to 80 mesh. Add the powder to a 70% ethanol solution at a ratio of 1:9, and extract in a 40℃ water bath for 3 hours. Concentrate the filtrate to 1 / 8 of its original volume, cool and let stand for 8 hours for later use. Step 2: Total protein extraction

[0106] Add 50 mmol / L Tris-HCl buffer (solid-to-solid ratio 1:5, pH 7.7) to the concentrate, stir and extract at 4°C for 2.5 hours, centrifuge and use the supernatant for later use.

[0107] Step 3: Enzymatic hydrolysis

[0108] Add a compound enzyme (trypsin and papain, mass ratio 3:1, total enzyme concentration 4% of substrate mass) to the extract. Incubate at 48℃ for 4 hours at pH 7.8, then inactivate the enzyme at 90℃ for 10 minutes.

[0109] Step 4: Dynamic Separation and Molecular Weight Control

[0110] The enzymatic hydrolysate was ultrafiltered (3,000 Da), and then filtered with Zn. 2+ Dynamic separation was performed using the modified medium to collect the target molecular weight peptides, with 0.5 mol / L citric acid as the eluent.

[0111] Step 5: Gradient separation and drying

[0112] The target peptide was purified by countercurrent chromatography with a mobile phase ratio of 75:25 (gradient elution for 20 minutes). The target fraction with a molecular weight of 800~1,200 Da was collected and freeze-dried to obtain Polygonatum peptide with a purity of 98%.

[0113] Comparative Example 1: No compound enzyme was used during the enzymatic hydrolysis process.

[0114] The difference from Example 1 is that only trypsin is used in the enzymatic hydrolysis step.

[0115] Specific procedures:

[0116] Pretreatment of Polygonatum sibiricum raw materials

[0117] Similar to Example 1, 70% ethanol was used for extraction, concentration, and settling to obtain a concentrated extract of Polygonatum odoratum.

[0118] Total protein extraction

[0119] As in Example 1, total protein was extracted using Tris-HCl buffer.

[0120] Enzymatic hydrolysis

[0121] Take 100 mL of total protein extract and add trypsin (total enzyme amount is 4% of the substrate mass). The reaction conditions are 50℃, pH 8.0, and enzymatic hydrolysis time is 4 hours. After enzymatic hydrolysis, inactivate the enzyme by heating at 85℃ for 10 minutes, without adding citric acid.

[0122] Next steps

[0123] The ultrafiltration, dynamic coordination separation and purification process is the same as in Example 1.

[0124] Comparative Example 2: No dynamic coordination separation technology used

[0125] The difference with respect to Example 2 is that the separation process does not use a metal-modified medium, but is directly separated by ultrafiltration and conventional chromatography.

[0126] Specific procedures:

[0127] Pretreatment of Polygonatum rhizome and extraction of total protein

[0128] Completely consistent with Example 2.

[0129] Enzymatic hydrolysis

[0130] A compound enzyme (trypsin and papain, mass ratio 2:1) was used, with the enzyme amount being 3% of the substrate mass. The enzymatic hydrolysis conditions were pH 8.2, temperature 45°C, and time 4 hours. The enzyme inactivation procedure was the same as in Example 2.

[0131] conventional separation

[0132] The enzymatic hydrolysate was filtered through a 3,000 Da ultrafiltration membrane, and the permeate was directly purified by countercurrent partition chromatography without undergoing a dynamic coordination separation step.

[0133] Purification and drying

[0134] Polygonatum peptides were prepared by freeze-drying using the same gradient elution method (70:30 water and acetonitrile mixed mobile phase).

[0135] Comparative Example 3: pH was not adjusted during the separation process.

[0136] The difference from Example 3 is that the pH of the enzymatic hydrolysate was not adjusted before dynamic coordination separation.

[0137] Specific procedures:

[0138] Raw material pretreatment, total protein extraction and enzymatic hydrolysis

[0139] The process is exactly the same as in Example 3.

[0140] Dynamic coordination separation

[0141] The enzymatic hydrolysate is directly passed through Zn 2+ Modified medium column (Zn) 2+ The concentration was 0.5 mmol / L, and the pH was not adjusted. The remaining separation procedures (elution conditions, eluent type) were consistent with those in Example 3.

[0142] Next steps

[0143] The purification and freeze-drying steps were the same as in Example 3.

[0144] Comparative Example 4: No gradient elution used

[0145] The difference from Example 4 is that gradient elution was not used in the countercurrent partition chromatography.

[0146] Specific procedures:

[0147] Processing and enzymatic hydrolysis of Polygonatum rhizome

[0148] The process is exactly the same as in Example 4.

[0149] Dynamic coordination separation

[0150] The enzymatic hydrolysate was subjected to ultrafiltration and Zn 2+Modification of the medium (Zn) 2+ The concentration was 0.5 mmol / L, column volume was 3 L), and the target peptide was eluted with 0.3 mol / L citric acid.

[0151] Conventional elution separation

[0152] A fixed ratio of water to acetonitrile (75:25) was used as the mobile phase, and gradient elution was not performed. All other chromatographic conditions remained the same as in Example 4.

[0153] freeze-drying

[0154] The process is the same as in Example 4, and polygonatum peptide is obtained.

[0155] Comparative Example 5: Using a single enzymatic hydrolysis condition

[0156] The difference from Example 5 is that the enzymatic hydrolysis time and pH are fixed, and the enzymatic hydrolysis conditions are not dynamically adjusted.

[0157] Specific procedures:

[0158] Polygonatum pretreatment and protein extraction

[0159] The process is the same as in Example 5.

[0160] Enzymatic hydrolysis

[0161] A compound enzyme (trypsin and papain, mass ratio 3:1, total enzyme amount 3% of substrate mass) was used for enzymatic hydrolysis under fixed conditions: pH set at 8.0, temperature at 48℃, and hydrolysis time at 3 hours. The pH was not dynamically adjusted based on the reaction process.

[0162] Dynamic separation and purification

[0163] Zn permeate 2+ Dynamic separation was performed using the modified medium. The target peptide was eluted with 0.3 mol / L citric acid, then purified by gradient countercurrent partition chromatography, and freeze-dried to obtain Polygonatum peptide.

[0164] Comparative Example 6: Using other separation media

[0165] The difference in Example 2 is that the medium used for dynamic coordination separation is changed to an ordinary silica gel column without modified metal ions.

[0166] Specific procedures:

[0167] Polygonatum pretreatment and protein extraction

[0168] The procedure is the same as in Example 2. Enzymatic hydrolysis

[0169] The process is the same as in Example 2, with enzyme inactivation following enzymatic hydrolysis of the compound enzyme.

[0170] Ordinary silicone separation

[0171] The enzymatic hydrolysate was separated by passing it through a standard silica gel column without metal ion loading at the same flow rate (2 mL / min) and elution conditions. Purification and drying followed.

[0172] The target peptide was eluted using a countercurrent partition chromatography gradient, followed by freeze-drying to obtain Polygonatum peptide.

[0173] Experiment 1: Verifying the effect of complex enzymes on the formation of target peptides

[0174] Experimental instructions

[0175] Experimental steps

[0176] Preparation of enzymatic hydrolysate

[0177] Following the method in Example 1, 100 mL of total protein extract from Polygonatum sibiricum (concentration 8 mg / mL) was subjected to enzymatic hydrolysis with trypsin and papain (mass ratio 3:1, total enzyme amount 4% of substrate mass) at 50°C and pH 8.0 for 4 hours. After enzyme inactivation, the solution was cooled to room temperature to obtain hydrolysate A.

[0178] Following the method of Comparative Example 1, enzyme hydrolysate B was prepared by using only trypsin (4% of the substrate mass) under the same conditions for 4 hours.

[0179] Target peptide filtration

[0180] Both sets of enzymatic hydrolysates were filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected as the target peptide enrichment solution.

[0181] Target peptide detection

[0182] 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 minutes. The peak area of ​​the target peptide (800~1,200 Da) was detected and converted into the target peptide content (mg / mL).

[0183] Experimental repetition

[0184] Repeat the experiment three times for each group, record the test data, and calculate the average value.

[0185] Experimental data

[0186] Table 1:

[0187]

[0188] This experiment compared the enzymatic hydrolysis effects of a compound enzyme with those of a single enzyme, revealing that the compound enzyme significantly increased the production of the target peptide. This advantage stems from the synergistic effect of trypsin and papain at their different sites of action on protamine. Trypsin focuses on cleaving peptide bonds near basic amino acids (such as lysine and arginine), while papain cleaves sites surrounding hydrophobic amino acids. The combination of the two avoids the limitations of single-enzyme action and improves the release efficiency of the target peptide.

[0189] The test data showed that the content of target peptides in the compound enzyme hydrolysate was significantly higher than that in the single enzyme hydrolysate. This indicates that the multi-site cleavage of the compound enzyme can cover more potential target peptide chain regions in protamine. Mechanistically, the compound enzyme reduces the residue of uncleaved peptides and simultaneously reduces the generation of invalid fragments. This multi-site, highly efficient enzymatic cleavage mechanism is key to achieving a high generation rate of target peptides.

[0190] The main limitation of single enzymes lies in their limited cleavage specificity, failing to completely release the active fragments in proteins. In particular, trypsin in protamine primarily acts on regions rich in basic amino acids, leading to the loss of a significant amount of the target peptide. Papain, however, overcomes this deficiency. The synergistic effect of the combined enzymes results in more comprehensive peptide chain cleavage, effectively reducing the inefficiency of single enzyme action. This synergistic mechanism demonstrates the unique advantages of combined enzymes in protein degradation, providing technical support for this invention.

[0191] Experiment 2: Verifying the effect of dynamic coordination separation technology on improving the purity of target peptides

[0192] Experimental instructions

[0193] Experimental steps

[0194] Preparation of enzymatic hydrolysate

[0195] Following the method in Example 2, enzymatic hydrolysate A (trypsin and papain, mass ratio 2:1, enzyme amount 3% of substrate mass, hydrolysis conditions pH 8.2, 45℃, 4 hours) was prepared using a compound enzymatic hydrolysis process.

[0196] Following the method of Comparative Example 2, enzyme hydrolysate B was prepared using the same enzymatic hydrolysis process.

[0197] Separation process

[0198] For enzymatic hydrolysate A: The hydrolysate was filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected. After adjusting the pH to 6.9, it was filtered through a Zn filter at a flow rate of 2 mL / min. 2+ Modified medium column (Zn) 2+The target peptide was washed with 30 mmol / L phosphate buffer (concentration 0.5 mmol / L) to remove unbound impurities, and then eluted with 0.3 mol / L citric acid. The eluent was collected.

[0199] For enzyme hydrolysate B: After passing the enzyme hydrolysate directly through ultrafiltration, without dynamic coordination separation, it was directly purified by countercurrent partition chromatography and the target peptide was obtained by elution.

[0200] Purity testing

[0201] The purity of the target peptides in the two final eluents was determined by high performance liquid chromatography (HPLC) after freeze-drying, and the mass percentage of the target peptides in the total product was calculated.

[0202] Experimental repetition

[0203] Repeat the experiment three times for each group, record the test data, and calculate the average value.

[0204] Experimental data

[0205] Table 2:

[0206]

[0207] This experiment clearly demonstrates the superiority of dynamic coordination separation technology in improving the purity of target peptides. After ultrafiltration of the enzymatic hydrolysate, dynamic coordination separation utilizes Zn... 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 sample, which did not use dynamic coordination separation and relied directly on countercurrent partition chromatography for purification, failed to effectively remove impurities of similar molecular weight, resulting in lower final purity.

[0208] 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, and the release of these peptides can be precisely controlled by dynamically adjusting the chemical environment of the medium (such as the citric acid concentration and pH of the eluent). This coordination chemistry-based separation method significantly reduces the co-elution of irrelevant peptides, thereby improving the purity of the final product.

[0209] In the comparative example, the ultrafiltration enzymatic hydrolysate was directly used for countercurrent partition chromatography, lacking a pre-separation step for impurities. This resulted in many small molecule impurities entering the chromatographic separation stage. These impurities exhibited similar migration behavior to the target peptide under gradient elution conditions, making them difficult to separate effectively and thus reducing the final purity. This limitation also indicates that chromatographic techniques alone are insufficient to achieve the high purity results of dynamic coordination separation.

[0210] Furthermore, experimental data show that the purity stability after dynamic coordination separation is higher, with very similar results in three repeated experiments. This indicates that the separation mechanism of dynamic coordination technology has strong repeatability and reliability, while the purity fluctuates significantly in the control group that did not use this technology, further demonstrating the advantages and applicability of dynamic coordination separation in actual production.

[0211] Experiment 3: Verifying the effect of pH adjustment on dynamic coordination separation.

[0212] Experimental instructions

[0213] Experimental steps

[0214] Preparation of enzymatic hydrolysate

[0215] Following the method in Example 3, the total protein extract of Polygonatum sibiricum (concentration 5 mg / mL) was enzymatically hydrolyzed using a compound enzyme (trypsin and papain, mass ratio 4:1). The enzyme dosage was 2.5% of the substrate mass, and the hydrolysis conditions were pH 7.9, temperature 50°C, and enzyme inactivation after 3 hours of hydrolysis. Hydrolysates A and B were obtained.

[0216] Separation condition settings

[0217] Example processing (enzymatic hydrolysate A): The enzymatic hydrolysate was filtered through a 3,000 Da ultrafiltration membrane. The permeate was collected and the pH was adjusted to 7.0. Then, it was filtered through a Zn filter at a flow rate of 3 mL / min. 2+ Modify the medium column. Wash impurities with 25 mmol / L phosphate buffer, then elute the target peptide with 0.3 mol / L citric acid.

[0218] Comparative treatment (enzyme hydrolysate B): The enzyme hydrolysate was directly passed through Zn 2+ The modified medium column was used without any pH adjustment steps, and the flow rate and elution conditions were the same as in the example.

[0219] Detection methods

[0220] Two sets of separated target peptide solutions were collected, and the recovery rate of the target peptides (the percentage of the total target peptides relative to the initial target peptide content in the enzymatic hydrolysate) was detected by high performance liquid chromatography (HPLC).

[0221] Experimental repetition

[0222] The experiment was repeated three times, and the recovery rate of the target peptide was recorded each time and the average value was calculated.

[0223] Experimental data

[0224] Table 3:

[0225]

[0226] Experimental results clearly show that pH adjustment significantly improves the recovery rate of the target peptide during dynamic coordination separation. Adjusting the pH of the enzymatic hydrolysate to near the isoelectric point of the target peptide (pH 7.0) makes the surface charge state of the target peptide more neutral, which facilitates its interaction with Zn. 2+ The modified medium forms more stable coordination bonds. In contrast, in the control group without pH adjustment, the original pH of the enzymatic hydrolysate may cause the target peptide's charge state to deviate too far from neutral, weakening its affinity for Zn. 2+ The binding efficiency ultimately affects the separation effect.

[0227] The core mechanism of pH adjustment lies in optimizing 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 be in equilibrium. This reduces the electrostatic repulsion between molecules, making it easier for the target peptide 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.

[0228] The comparative data show that the unadjusted enzymatic hydrolysate exhibits significant loss of target peptides during dynamic separation. This loss is not only due to insufficient binding of the target peptides to the medium but may also be related to competitive binding with impurities. Impurities carry a high charge density and are more likely to form strongly competitive adsorption at the original pH, thus occupying the binding sites on the medium and leading to a decrease in the separation efficiency of the target peptides. This also demonstrates that pH adjustment, as a crucial step before separation, plays an irreplaceable role in improving the dynamic coordination separation effect.

[0229] Furthermore, the data from the example experiment showed very little fluctuation across three repeated experiments, indicating that the separation process after pH adjustment is more controllable and highly reproducible. The comparative example, however, showed slightly higher fluctuations, possibly due to interference from impurities and greater randomness. This result further demonstrates that pH adjustment can significantly optimize the dynamic coordination separation process, making it more stable and efficient, and suitable for practical industrial applications.

[0230] Experiment 4: Verifying the effect of gradient elution on the purity of the target peptide

[0231] Experimental instructions

[0232] Experimental steps

[0233] Preparation of enzymatic hydrolysate

[0234] Following the procedure in Example 4, 5 L of total protein extract from Polygonatum sibiricum (protein concentration 8 mg / mL) was taken and enzymatically hydrolyzed for 4 hours with a compound enzyme (trypsin and papain, mass ratio 3:1, enzyme dosage 3% of substrate mass) at pH 8.0 and 50°C. After enzyme inactivation, the permeate was collected through a 3,000 Da ultrafiltration membrane, and the pH was adjusted to 6.8 to obtain hydrolysates A and B.

[0235] Dynamic coordination separation

[0236] Enzymatic hydrolysate through Zn 2+ The modified medium column was used at a flow rate of 5 mL / min. After washing away impurities with 30 mmol / L phosphate buffer, the target peptide was eluted with 0.3 mol / L citric acid. Two sets of eluents were collected separately.

[0237] Chromatographic purification

[0238] Example processing (enzymatic hydrolysate A): Countercurrent partition chromatography was used with a mobile phase of water and acetonitrile at a volume ratio of 70:30. Gradient elution conditions were applied, with the acetonitrile content gradually increasing from 10% to 50% over a gradient time of 25 minutes, and the target peptide was collected.

[0239] Comparative treatment (enzymatic hydrolysate B): The same mobile phase was used, but the acetonitrile ratio was fixed at 75:25. Gradient elution was not used, and the target peptide was collected directly.

[0240] Target peptide purity detection

[0241] High-performance liquid chromatography (HPLC) was used to analyze the two groups of purified products, detect the purity of the target peptide, and calculate its percentage of the total product mass.

[0242] Experimental repetition

[0243] Each experiment was repeated 3 times, the test data were recorded, and the average value was calculated.

[0244] Experimental data

[0245] Table 4:

[0246]

[0247] The role of gradient elution in the purification of target peptides was fully demonstrated in the experimental results. Gradient elution, by slowly increasing the proportion of acetonitrile in the mobile phase, precisely controls the elution time of the target peptide, creating a more distinct separation window between it and impurities. This separation mechanism effectively reduces co-elution of impurities, thereby significantly improving the purity of the final product. In contrast, the fixed-ratio elution method used in the comparative example could not accurately distinguish between the target peptide and impurities with similar properties, resulting in lower final purity.

[0248] Mechanistically, the core of gradient elution lies in dynamically adjusting the polarity of the solution. By controlling the partitioning behavior of the target peptide with the stationary phase, it ensures that the elution rate of the target peptide in the mobile phase is significantly different from that of impurities. Due to differences in molecular structure or polarity, impurities are eluted earlier or later at the beginning or end of the gradient change, while the target peptide is concentrated in a specific gradient range, resulting in a clear separation effect. In contrast, fixed-ratio elution cannot dynamically adjust the partition coefficient, leading to overlapping elution of impurities and target peptides, which affects the purification effect.

[0249] The data shows that the purity of the target peptide in the example remained consistently above 98%, while the purity in the comparative example was significantly lower and fluctuated considerably. This indicates that gradient elution offers better controllability and separation precision than fixed-ratio elution. Under gradient conditions, the elution behavior of the target peptide and impurities is more consistent, resulting in higher reproducibility. Fixed-ratio elution, lacking controllable mechanisms, makes the impurity ratio susceptible to minor environmental changes, leading to unstable purity results.

[0250] The experiments also highlighted the criticality of impurity removal. Without gradient elution, many structurally similar but functionally unrelated small peptides could not be completely separated. Gradient elution, through a refined separation window, eliminated these interfering substances. The results demonstrate that gradient elution is an essential process for achieving high-purity target peptides and plays a vital role in enhancing the overall technical level of this invention.

[0251] Experiment 5: Verifying the effect of dynamic enzymatic hydrolysis conditions on the efficiency of target peptide generation.

[0252] Experimental instructions

[0253] Experimental steps

[0254] Preparation of enzymatic hydrolysate

[0255] Example Processing (Dynamic Enzymatic Hydrolysis, Hydrolysate A): Following the procedure in Example 5, the total protein extract of Polygonatum sibiricum (concentration 5 mg / mL) was subjected to compound enzymatic hydrolysis (trypsin and papain, mass ratio 3:1, total enzyme amount 4% of substrate mass). pH and time conditions were dynamically adjusted during the hydrolysis process. The initial pH was 7.8, and the pH was checked every 30 minutes and gradually adjusted to 7.9. The hydrolysis time was 4 hours, and the enzyme was immediately inactivated after the reaction was completed.

[0256] Comparative treatment (fixed enzyme hydrolysis, enzyme hydrolysate B): Following the procedure of Comparative Example 5, the same compound enzyme was used, and the enzyme hydrolysis conditions were fixed at pH 8.0 and the hydrolysis time was 3 hours. No dynamic adjustments were made during the enzyme hydrolysis process.

[0257] Target peptide detection

[0258] Both sets of enzymatic hydrolysates were filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected.

[0259] The content of target peptides (molecular weight range 800~1,200 Da) was detected by high performance liquid chromatography (HPLC), and the target peptide yield (the percentage of target peptide content to the total protein content of the total protein extract) was calculated.

[0260] Experimental repetition

[0261] Each experiment was repeated 3 times, the detection data were recorded, and the average generation rate was calculated.

[0262] Experimental data

[0263] Table 5:

[0264]

[0265] Experimental results clearly demonstrate that dynamically adjusting pH and hydrolysis time significantly improves the generation efficiency of the target peptide. Dynamic hydrolysis, by adjusting the optimal enzyme activity conditions during the reaction process, allows the complex enzymes to fully exert their effects at different stages. The lower initial pH (7.8) provides higher catalytic efficiency for trypsin, while as the pH is gradually adjusted to 7.9, the activity of papain is progressively activated, achieving synergistic degradation at multiple sites. This dynamic optimization process is clearly more in line with the reaction kinetics of proteases, significantly increasing the release of the target peptide.

[0266] Under fixed enzymatic hydrolysis conditions, enzyme activity is limited to a fixed range, resulting in a significant reduction in cleavage efficiency. In particular, the complex peptide chain structure in protamine means that a single pH environment cannot fully activate the synergistic effect of the complex enzymes, preventing the efficient release of many potential target peptides. Furthermore, the short fixed enzymatic hydrolysis time (3 hours) results in incomplete cleavage of peptide bonds in some high-resistance regions, further limiting the amount of target peptides generated.

[0267] Based on the generation rate data, the average generation rate of target peptides increased by about 15% under dynamic enzymatic hydrolysis conditions. Mechanistically, this is not merely due to increased enzyme activity, but more importantly, the dynamic adjustment of pH makes the enzyme's action site more selective. Papain tends to cleave peptide chains around hydrophobic amino acids in a near-neutral pH environment (7.8–7.9), while trypsin is more suitable for cleaving peptide bonds around basic amino acids in an alkaline environment. The synergistic cleavage process of both is fully amplified by dynamic enzymatic hydrolysis, which is clearly impossible to achieve under fixed enzymatic hydrolysis conditions.

[0268] Experiments also show that the dynamic enzymatic hydrolysis process has strong stability. The fluctuation range of the 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 lower yield but also a slightly larger fluctuation range, which may be affected by unoptimized enzyme conditions. This further verifies the importance of dynamic enzymatic hydrolysis technology in improving the efficiency of target peptide generation, and is one of the core technical advantages of the present invention.

[0269] Experiment 6: Verifying the effect of metal-modified media on the separation of target peptides

[0270] Experimental instructions

[0271] Experimental steps

[0272] Preparation of enzymatic hydrolysate

[0273] Following the procedure in Example 2, the total protein extract of Polygonatum sibiricum (concentration 8 mg / mL) was subjected to enzymatic hydrolysis with a mixture of trypsin and papain (mass ratio 2:1, total enzyme amount 3% of substrate mass) under the following conditions: pH 8.2, 45°C, for 4 hours. After enzyme inactivation, the solution was filtered through a 3,000 Da ultrafiltration membrane, and the permeate was collected. The pH was adjusted to 6.9 to obtain hydrolysates A and B.

[0274] Dynamic coordination separation

[0275] Example treatment (enzymatic hydrolysate A): The enzymatic hydrolysate was passed through Zn at a flow rate of 2 mL / min. 2+ Modified medium column (Zn) 2+ The target peptide was washed with 30 mmol / L phosphate buffer (concentration 0.5 mmol / L) to remove unbound impurities, and then eluted with 0.3 mol / L citric acid. The eluent was collected.

[0276] Comparative treatment (enzyme hydrolysate B): The enzyme hydrolysate was passed through a standard silica gel column without modified metal ions at the same flow rate, and the elution conditions and process were consistent with those in the example.

[0277] Target peptide detection

[0278] The content (mg / mL) and purity (mass percentage of target peptide in total components) of the two eluents were determined by high performance liquid chromatography (HPLC).

[0279] Experimental repetition

[0280] Each experiment was repeated 3 times, the test data were recorded, and the average value was calculated.

[0281] Experimental data

[0282] Table 6A:

[0283]

[0284] Table 6B:

[0285]

[0286] Experimental results show that using Zn 2+ The modified medium significantly improved the separation of target peptides, with both the content and purity of target peptides being far superior to those obtained with ordinary silica gel media. This advantage stems from the specific coordination effect of the metal-modified medium. Zn 2+ Ions form dynamic coordination bonds with the carboxyl and amino groups in the target peptide molecule, providing stronger binding capacity and selectivity, thereby significantly eliminating impurities that are not target peptides. This separation mechanism obviously cannot be achieved with ordinary silica gel media, because ordinary silica gel relies only on weak polar adsorption and lacks specific recognition ability, which makes it easy for impurities to bind to or elute the target peptide at the same time, resulting in a significant reduction in purity.

[0287] Mechanistically, the separation effect of the metal-modified medium benefits from Zn 2+ The dynamic coordination characteristics of Zn. At pH 6.9, Zn... 2+ The charge distribution of the silica gel allows it to form stable non-covalent bonds with the functional groups of the target peptide. This binding is both strong and flexible, making it suitable for releasing the target peptide through competitive displacement with citric acid in the subsequent elution stage. Simultaneously, this separation mechanism effectively avoids electrostatic interference between molecules, making the separation process more efficient and stable. Ordinary silica gel media lacks this property, only providing non-specific adsorption, leading to co-elution of the target peptide with impurities, significantly reducing the separation efficiency.

[0288] Experimental data also show that Zn 2+ The modified medium exhibited better reproducibility. The fluctuation range of target peptide content and purity was minimal across the three experiments, indicating that the medium's capture ability for the target peptide was highly controllable and consistent. In contrast, the results from ordinary silica gel media showed greater fluctuations, and this instability would obviously affect the separation process in actual production. Furthermore, the target peptide content after separation using ordinary silica gel media was also lower, possibly because impurities competed for limited adsorption sites, further reducing the effective separation efficiency.

[0289] From an application perspective, the significant advantages of metal-modified media lie not only in their separation efficiency but also in their scalability for industrial production. They enable highly efficient separation with lower eluent concentrations and milder operating conditions, reducing energy consumption and the use of chemical reagents, thus exhibiting greater environmental friendliness and economic efficiency. These characteristics collectively constitute the technological innovation of this invention and represent a core advantage that ordinary silica gel media cannot match.

[0290] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Polygonatum peptide for anti-inflammatory and kidney-protective purposes, characterized in that, Includes the following steps: (1) After crushing the rhizome of Polygonatum odoratum, it was pretreated with ethanol solution to obtain a concentrated water extract; (2) Add the concentrated aqueous extract to the buffer solution to extract total protein from Polygonatum sibiricum; (3) The total protein solution is subjected to enzymatic hydrolysis with enzymes. The enzymatic hydrolysis conditions are: enzyme concentration of 2%-5% of substrate mass, pH range of 7.5-8.5, temperature of 45-55℃, and time of 3-5 hours. (4) After removing macromolecular impurities by ultrafiltration of the enzymatic hydrolysate, the target peptide is separated by dynamic coordination using a metal-modified medium. The separation conditions are: metal ion concentration of 0.1-1 mmol / L and solution pH of 6.8-7.

2. (5) The target peptide was purified by countercurrent partition chromatography and freeze-dried to obtain Polygonatum peptide; The enzyme used in the enzymatic hydrolysis reaction is a combination of trypsin and papain, with a mass ratio of trypsin to papain of 2:1-4:

1. The total enzyme concentration is 2% to 5% of the substrate mass, the target protein concentration in the hydrolysate is 5-10 mg / mL, and the hydrolysis time is 3-5 hours. During the reaction, the reaction conditions need to be dynamically adjusted to ensure that the pH value is maintained within the range of 7.5 to 8.

5. The metal-modified medium is prepared from a metal-supported material based on silica gel. The silica gel matrix is ​​treated with dilute hydrochloric acid and then loaded with Zn metal ions. 2+ or Cu 2+ The metal loading concentration was 0.1-1 mmol / L, the medium particle size was 60-120 μm, and the metal ion loading was dried at 70℃ for 4-6 hours to increase stability. In the dynamic coordination separation process, after adjusting the pH to 6.8-7.2, the enzymatic hydrolysate is passed through a 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 is collected after detecting the concentration of the target peptide. The mobile phase used in the countercurrent partition chromatography purification is a mixture of water and acetonitrile in a volume ratio of 80:20-70:

30. The gradient elution conditions are linear gradient elution of 10% to 50% acetonitrile, with an elution time of 15 to 30 minutes. The recovery rate of the target peptide is not less than 90%.

2. The method for preparing Polygonatum peptide for anti-inflammatory and kidney-protective purposes according to claim 1, characterized in that, The particle size of the Polygonatum rhizome pulverized is 80 mesh, the concentration of the ethanol solution used is 70%, the pretreatment temperature is 40-50℃, the time is 2-3 hours, the material-liquid ratio is 1:8-1:10, the filtrate obtained after ethanol treatment needs to be concentrated to 1 / 6-1 / 10 of the original volume and the precipitate removed for later use.

3. The method for preparing Polygonatum peptide for anti-inflammatory and kidney-protective purposes according to claim 1, characterized in that, The buffer used for extracting total protein from Polygonatum sibiricum is Tris-HCl buffer with a concentration of 20-50 mmol / L. The material-to-liquid ratio during extraction is 1:5, the extraction temperature is 4℃, and the extraction time is 2-3 hours. The extracted liquid needs to be centrifuged at 4,000-6,000 rpm for 10-15 minutes to separate the supernatant and remove impurities.

4. The method for preparing Polygonatum peptide for anti-inflammatory and kidney-protective purposes according to claim 1, characterized in that, The enzyme inactivation process of the enzymatic hydrolysate includes heating 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 hydrolysate. The inactivated solution needs to be rapidly cooled to room temperature and filtered to remove residual impurities.

5. The method for preparing Polygonatum peptide for anti-inflammatory and kidney-protective purposes according to claim 1, characterized in that, The ultrafiltration membrane has a molecular weight cutoff range of 3,000-5,000 Da. During the ultrafiltration operation, the feed solution needs to pass through the ultrafiltration membrane at a pressure of 0.5-1 MPa. The permeate after ultrafiltration needs to be tested for the content of the target peptide by an online amino acid analyzer. After the test results meet the target range, the permeate is collected for later use.

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