A preparation method of chondroitin sulfate from sturgeon and its application in a dressing for promoting the healing of diabetic chronic wounds

Abstract

The present invention relates to the technical field of chondroitin sulfate extraction. To solve the problems in the prior art that the extraction and purification process of chondroitin sulfate is complex, a large amount of chemical reagents are required and the loss of chondroitin sulfate is relatively large, a preparation method of sturgeon chondroitin sulfate is disclosed, which includes subjecting sturgeon cartilage particles to thermal liquefaction treatment and then performing enzymatic hydrolysis to obtain an enzymatic hydrolysate; filtering and purifying the enzymatic hydrolysate to obtain a sturgeon chondroitin sulfate solution. This method uses less chemical reagents, has simple steps, and has a high recovery rate for the purification of chondroitin sulfate, can realize the green industrial production of chondroitin sulfate, and at the same time can obtain a by-product of cartilage collagen bioactive peptide. The present invention also provides an application of a diabetic chronic wound healing dressing containing sturgeon chondroitin sulfate. This dressing has good hemostatic and antibacterial properties, biocompatibility, biodegradability, tissue adhesion properties and mechanical properties, can promote the healing of diabetic chronic wounds, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of chondroitin sulfate extraction, and particularly to a preparation method of sturgeon chondroitin sulfate and its application in a dressing for promoting the healing of diabetic chronic wounds. Background Art

[0002] The skin is the first line of defense to protect the human body from external bacteria and viruses, and its integrity is extremely important for human safety. Diabetic ulcers are a type of disordered and intractable chronic wound, which greatly increases the pain of patients' lives. According to the latest Global Diabetes Map (IDF Diabetes Atlas) (ninth edition) released by the International Diabetes Federation (IDF), it is estimated that by 2045, the number of diabetic patients will reach 693 million. The probability of diabetic patients developing ulcers is 15% - 25%, 66% of the patients will relapse ulcers 5 years after cure, 12% of the patients will have their limbs amputated after ulcer relapse, and 70% of the amputated patients are due to the ulcer not being treated in a timely and effective manner. Therefore, it is urgent to further develop the preparation of dressings for intervening in the healing of diabetic chronic wounds.

[0003] Natural chondroitin sulfate has received extensive attention in the field of medical dressings due to its unique advantages of biocompatibility, biodegradability, and its potential antibacterial and macrophage-regulating activities. Chondroitin sulfate (CS) is a type of glycosaminoglycan rich in sulfate groups and carboxyl groups, and carrying strong negative charges. It is mainly present in the matrix of tissues such as cartilage, ligaments, tendons, and arterial walls of humans and animals. The industrial production process for isolating chondroitin sulfate from cartilage has been implemented for many years and generally includes the following four steps: (1) chemical hydrolysis of cartilage; (2) decomposition of glycosaminoglycan and core protein; (3) removal of proteins and collection of chondroitin sulfate; (4) purification of chondroitin sulfate. The first two stages are mainly carried out in an alkaline solution containing high concentrations of sodium hydroxide, urea, cysteine, or guanidine hydrochloride, and then glycosaminoglycan is selectively precipitated by cationic quaternary ammonium salts, non-ionic detergents, potassium thiocyanate, or alcohol solutions. Proteins can be removed by trichloroacetic acid, and finally purified by chromatography to obtain chondroitin sulfate meeting the purity requirements. However, the traditional chondroitin sulfate extraction process usually involves chemicals such as high concentrations of alkali, urea, and guanidine hydrochloride. Although it promotes the hydrolysis of cartilage and the separation of glycosaminoglycan and core protein, it also exacerbates environmental pollution. To further pursue sustainability, some studies, such as the invention patents with publication numbers CN102190740A and CN104017108A, have proposed various methods such as dilute alkali-enzymatic hydrolysis method and mechanochemical-assisted extraction method to replace the traditional process. The main steps are: (1) adding alkali for adjustment and then adding biological enzymes to hydrolyze cartilage and proteins; (2) selectively precipitating chondroitin sulfate using an alcohol solution; (3) neutralizing the reaction solution; (4) using ultrafiltration-dialysis technology for molecular weight separation and then purifying chondroitin sulfate. Among them, commonly used hydrolytic proteases include papain, alkaline protease, trypsin, pepsin, subtilisin, and the use of a combination of multiple enzymes. The existing extraction methods have reduced environmental pollution to a certain extent, but there are still problems such as the use of chemical reagents and complex purification processes, resulting in a large loss of chondroitin sulfate and difficulties in industrial production. Summary of the Invention

[0004] In order to overcome the problems in the prior art that the extraction and purification process of chondroitin sulfate is complex, requires the use of a large amount of chemical reagents, and causes a large loss of chondroitin sulfate, the present invention provides a method for preparing sturgeon chondroitin sulfate. This preparation method is efficient and lossless in the extraction process of chondroitin sulfate, with simple overall operation, suitable for industrial production, less green pollution, and in line with the current concept of sustainable development. The present invention also provides a diabetic chronic wound healing dressing containing sturgeon chondroitin sulfate. This dressing has good hemostatic and antibacterial properties, biocompatibility, biodegradability, tissue adhesion properties, and mechanical properties, can promote the healing of diabetic chronic wounds, and has extremely high industrial application prospects.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A preparation method of chondroitin sulfate from sturgeon cartilage, comprising the following steps:

[0007] (1) Thermally liquefy the sturgeon cartilage particles;

[0008] (2) Add water and protease to the treated sturgeon cartilage particles for enzymatic hydrolysis. After complete enzymatic hydrolysis, heat to inactivate the enzyme to obtain an enzymatic hydrolysate;

[0009] (3) Filter and purify the enzymatic hydrolysate to obtain a chondroitin sulfate solution from sturgeon cartilage.

[0010] The extraction raw materials of chondroitin sulfate are basically traditional terrestrial animals, and terrestrial animals have certain safety hazards, such as infectious diseases like mad cow disease, swine flu, foot-and-mouth disease, avian influenza, etc. Therefore, chondroitin sulfate from aquatic raw material sources has great advantages. Sturgeon is one of the oldest, most primitive, largest in size, and longest-lived cartilaginous fish species currently living on Earth. China is the country with the largest sturgeon aquaculture production in the world, which has increased from 11,000 tons in the early 2000s to 108,000 tons in 2021. Sturgeon caviar is the main product of sturgeon and has extremely high economic value. As a processing by-product, sturgeon cartilage has a low utilization rate, a single product structure, and low added value. The present invention selects sturgeon cartilage as the raw material for preparing chondroitin sulfate, which has extremely high industrial application prospects. Crushing sturgeon cartilage and then subjecting it to thermal liquefaction treatment or steam explosion treatment can effectively destroy the structure of fish bones and cause liquefaction, which can promote the cleavage of glycosidic bonds and improve the subsequent enzymatic hydrolysis efficiency. There is no need to adjust the pH before enzymatic hydrolysis, the amount of chemical reagents used is small, and the subsequent neutralization step can be omitted. The chondroitin sulfate prepared by the present invention is basically the same in structure as the shark chondroitin sulfate (Shark-CS) on the market, except for the differences caused by species diversity, and is also similar to the chondroitin sulfate isolated from sturgeon cartilage by the dilute alkali-enzymatic hydrolysis-chemical precipitation method. The recovery rate of chondroitin sulfate obtained in this preparation process is high, and sturgeon cartilage collagen peptides can also be obtained simultaneously.

[0011] Preferably, the sturgeon cartilage particles in step (1) are obtained by boiling fresh sturgeon cartilage until the bone and meat are separated and then crushing, or by mixing dried sturgeon cartilage with water to a solid-liquid ratio of 1:(3 - 4) and then crushing.

[0012] Preferably, the thermal liquefaction treatment in step (1) is to carry out thermal liquefaction on the sturgeon cartilage particles, and the thermal liquefaction conditions are a pressure of 0.04 - 0.10 MPa, a temperature of 105 - 115 °C, and a time of 1.5 - 2.5 h.

[0013] The thermal liquefaction process is extremely susceptible to the Maillard reaction, so the thermal liquefaction temperature cannot be too high.

[0014] Preferably, in the step (3), the protease is a composite neutral protease, which is composed of flavor protease, neutral protease and papain with a mass ratio of (1-2):(1-2):(2-3), and the addition amount of the protease is 0.2-0.8%.

[0015] Preferably, in the step (3), the material-liquid ratio of the processed sturgeon cartilage particles to water is 1:(3-6), the temperature during enzymatic hydrolysis is 45-60 °C, and the enzymatic hydrolysis time is 6-9 h; after heating to 90-100 °C, keep warm for 10-20 min to inactivate the enzyme.

[0016] Preferably, in the step (4), the filtration and purification process is to sequentially pass the enzymatic hydrolysate through ceramic membrane filtration, RO reverse osmosis membrane concentration, and 10 kDa spiral wound membrane ultrafiltration separation.

[0017] A preparation method of a dressing for promoting the healing of chronic diabetic wounds comprises the following steps:

[0018] 1) After freeze-drying and re-dissolving the sturgeon chondroitin sulfate solution, add soluble calcium salt and carboxymethyl chitosan for cross-linking to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan composite filler;

[0019] 2) Freeze-thaw the carboxymethyl chitosan for 1-6 times and then freeze-dry it to obtain a carboxymethyl chitosan sponge;

[0020] 3) Mix the carboxymethyl chitosan sponge with the filler to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan / carboxymethyl chitosan composite sponge hydrogel dressing.

[0021] Sturgeon chondroitin sulfate can promote the proliferation and migration of fibroblasts, activate macrophages, up-regulate the expression of CD31, VEGF and PCNA, etc., and can promote epidermal and dermal hyperplasia, angiogenesis, and accelerate the healing of skin wounds. In the present invention, sturgeon chondroitin sulfate is cross-linked under the action of Ca 2+ and then reacts with the amino group in carboxymethyl chitosan based on Schiff base reaction to generate double bonds to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan complex. The complex is used as a filler and filled into the carboxymethyl chitosan sponge to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan / carboxymethyl chitosan composite sponge hydrogel dressing. Among them, Ca 2+ in the filler can be used as a coagulation factor to release and activate the active mode participating in cascade coagulation; the addition of carboxymethyl chitosan further enhances the antibacterial activity and mechanical stability of the hydrogel.

[0022] Preferably, in the step 1), the molar ratio of calcium ions in sturgeon chondroitin sulfate to the soluble calcium salt is (1-2):(3-5), and the mass ratio of sturgeon chondroitin sulfate to carboxymethyl chitosan is (0.5-1):1.

[0023] Preferably, the freeze-thaw process in step (2) is to dissolve carboxymethyl chitosan and then freeze it at a low temperature of -10 to -20°C, then freeze it at an ultra-low temperature of -30 to -60°C, and finally lyophilize it.

[0024] Preferably, the mass ratio of carboxymethyl chitosan sponge to filler in step (3) is 1:(1 - 3).

[0025] Therefore, the present invention has the following beneficial effects: (1) In the preparation process of sturgeon chondroitin sulfate, the amount of chemical reagents used is small, it is environmentally friendly, the steps are simple, the loss of chondroitin sulfate is small, and the purification recovery rate is high, which can realize the green industrial production of chondroitin sulfate, and at the same time, the by-product of cartilage collagen bioactive peptide can be obtained; (2) Using sturgeon cartilage as the raw material for the preparation of chondroitin sulfate, the source is wide, the cost is low, and the biosafety hazard is low; (3) Combining sturgeon chondroitin sulfate with carboxymethyl chitosan to obtain a dressing for promoting the healing of diabetic chronic wounds, this dressing has good antibacterial activity and mechanical stability. When used for the treatment of diabetic patients' chronic wounds, it can avoid wound infection and accelerate wound healing. Description of the Drawings

[0026] Figure 1 It is the structural diagram of chondroitin sulfate.

[0027] Figure 2 It is the microscopic structural diagram of the cartilage particles obtained in Example 1 and Comparative Examples 1 - 2 of the present invention.

[0028] Figure 3 It is the change diagram of the migration activity of L929 cells.

[0029] Figure 4 It is the morphological change diagram of RAW264.7 cells.

[0030] Figure 5 It is the influence and heat map of the healing of mouse wounds.

[0031] Figure 6 It is the immunohistochemical staining diagram of the wound tissue section. Detailed Embodiments

[0032] The present invention will be further described below in conjunction with the drawings and specific implementation methods.

[0033] Example 1

[0034] A preparation method of sturgeon chondroitin sulfate, the steps are as follows:

[0035] (1) Sturgeon cartilage segmentation: Add fresh sturgeon cartilage to water and heat it until the bone and meat are separated, take it out, and break it into granular form to obtain sturgeon cartilage particles for standby;

[0036] (2) Pretreatment by heat liquefaction technology (HP): Place the sturgeon cartilage particles obtained in step (1) in a high-pressure reactor, heat to a temperature of 110 °C, and keep warm for 2 h to obtain heat-liquefied cartilage liquid. The brightness value of the heat-liquefied fish bone liquid obtained under this condition is 65;

[0037] (3) Enzymatic hydrolysis: Dilute the heat-liquefied cartilage liquid obtained in step (2) with water according to a solid-liquid ratio of 1:4, add compound neutral protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: temperature 55 °C, time 9 h, enzyme addition amount 0.2%; After the enzymatic hydrolysis is complete, inactivate the enzyme at 100 °C for 10 min to obtain an enzymatic hydrolysate. The degree of hydrolysis of the protein under this condition is 29%;

[0038] (4) Ultrafiltration separation: Pass the enzymatic hydrolysate obtained in step (3) through ceramic membrane filtration, RO reverse osmosis membrane concentration, and 10 kDa spiral wound membrane ultrafiltration separation in sequence to obtain sturgeon chondroitin sulfate solution.

[0039] The sturgeon chondroitin obtained in Example 1 was detected by high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RI), Folin-phenol method, sulfuric acid-carbazole method, gelatin turbidimetry method and high performance liquid chromatography respectively.

[0040] The average molecular weight of the chondroitin sulfate obtained in Example 1 was 56.67±0.4 kDa, the protein content was 9.66±1.3%, the uronic acid content was 29.60±0.44%, the sulfate group content was 13.11±3.2%, the monosaccharides were composed of glucuronic acid (GlcA, 39.88%), galactosamine (GalNAc, 32.96%), galactose (Gal, 18.64%) and glucosamine (GlcNAc, 8.53%), and the disaccharides were composed of ΔDi6S (58.38%), ΔDi4S (27.34) and ΔDi0S (14.29%), which proved that the extracted polysaccharide was chondroitin sulfate. And the chondroitin sulfate obtained in Example 1 was mainly composed of C-type (CS-C) disaccharide units, followed by A-type disaccharide units (CS-A) and O-type disaccharide units (CS-O). The sulfate group was located at the 6th or 4th position of N-acetylgalactosamine, compared with Figure 1 The chemical structure of chondroitin sulfate can prove that the product obtained in Example 1 is a highly sulfated chondroitin sulfate with high medicinal value. Compared with the commercially available shark chondroitin sulfate (Shark-CS), except for the differences caused by species diversity, the rest of the structures are basically the same, similar to the chondroitin sulfate isolated from sturgeon cartilage by the dilute alkali-enzymatic hydrolysis-chemical precipitation method, and the recovery rate reaches 93.68%. It can be seen that the loss of sturgeon chondroitin sulfate in the preparation method of the present invention is small.

[0041] Comparative Example 1

[0042] A preparation method of chondroitin sulfate from sturgeon, the steps are as follows:

[0043] (1) Sturgeon cartilage segmentation: Add fresh sturgeon cartilage to water and heat it until the bone and meat are separated. Take it out and break it into granular form to obtain sturgeon cartilage particles for standby;

[0044] (2) Pretreatment by heat liquefaction technology (HP): Place the sturgeon cartilage particles obtained in step (1) in a high-pressure reactor, heat to 130 °C, and keep warm for 2 h to obtain heat-liquefied cartilage liquid. The brightness value of the heat-liquefied fish bone liquid obtained under this condition is 37.5;

[0045] (3) Enzymatic hydrolysis: Dilute the heat-liquefied cartilage liquid obtained in step (2) with water according to the solid-liquid ratio of 1:2, add compound neutral protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: temperature 40 °C, time 6 h, enzyme addition amount 0.1%; After the enzymatic hydrolysis is complete, inactivate the enzyme at 100 °C for 10 min to obtain an enzymatic hydrolysate. The degree of hydrolysis of protein under this condition is 17;

[0046] (4) Ultrafiltration separation: Pass the enzymatic hydrolysate obtained in step (3) through ceramic membrane filtration, RO reverse osmosis membrane concentration, and 10 kDa spiral wound membrane ultrafiltration separation in sequence to obtain chondroitin sulfate solution from sturgeon.

[0047] After detection, the recovery rate of chondroitin sulfate obtained in Comparative Example 1 is 73.89%.

[0048] Comparative Example 2

[0049] (1) Sturgeon cartilage segmentation: Add fresh sturgeon cartilage to water and heat it until the bone and meat are separated. Take it out and break it into granular form to obtain sturgeon cartilage particles for standby;

[0050] (2) Pretreatment by steam explosion technology (SE): Place the sturgeon cartilage particles obtained in step (1) in a steam exploder and process it at 1 MPa for 240 s. The brightness value of the steam-exploded cartilage liquid obtained under this condition is 78;

[0051] (3) Enzymatic hydrolysis: After pre-freezing the steam-exploded cartilage liquid obtained in step (2), place it in a vacuum freeze-drying oven and freeze-dry it at -80 °C, vacuum degree 20 Pa, and 48 h. Then dilute it with water according to the solid-liquid ratio of 1:40, add compound neutral protease for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: temperature 55 °C, time 9 h, enzyme addition amount 1.0%; After the enzymatic hydrolysis is complete, inactivate the enzyme at 100 °C for 10 min to obtain an enzymatic hydrolysate. The degree of hydrolysis of protein under this condition is 21%;

[0052] (4) Ultrafiltration separation: Pass the enzymatic hydrolysate obtained in step (3) through ceramic membrane filtration, RO reverse osmosis membrane concentration, and 10 kDa spiral wound membrane ultrafiltration separation in sequence to obtain chondroitin sulfate solution from sturgeon.

[0053] After detection, the average molecular weight of chondroitin obtained in Comparative Example 2 was 62.0 ± 0.18 kDa, the protein content was 23.02 ± 0.3%, the glucuronic acid content was 28.24 ± 0.99%, the sulfate radical content was 12.1 ± 0.3%, and the recovery rate was 80.60%.

[0054] The microscopic structures of the chondroitin granules of sturgeon obtained in step (2) of Example 1 and the chondroitin granules of sturgeon without pretreatment are as Figure 2 shown. The chondroitin granules of sturgeon after thermal liquefaction treatment in Example 1 had the best effect, showing uniform fragmented shapes, which could effectively destroy the structure of fish bones and was beneficial to subsequent enzymatic hydrolysis treatment. The thermal liquefaction conditions in Comparative Example 1 were different from those in Example 1, and its destructive effect on fish bones was worse than that in Example 1. The recovery rate of chondroitin sulfate of sturgeon finally obtained was also lower than that in Example 1. And Comparative Example 2 showed that the effect of steam explosion was worse than that of thermal liquefaction.

[0055] Example 2

[0056] A dressing for promoting the healing of diabetic chronic wounds is prepared by the following steps:

[0057] 1) The chondroitin sulfate solution of sturgeon obtained in Example 1 was freeze-dried, redissolved with water at a mass ratio of 3 wt%, then calcium chloride and carboxymethyl chitosan were added and mixed. The mass ratio of chondroitin sulfate of sturgeon, calcium chloride and carboxymethyl chitosan was 2:3:2, and stirred evenly to obtain a chondroitin sulfate of sturgeon-Ca-carboxymethyl chitosan composite filler;

[0058] 2) Carboxymethyl chitosan was dissolved, cryogenically frozen at -20 °C, then ultra-low temperature frozen at -60 °C, and finally freeze-dried, repeated 6 times to obtain a carboxymethyl chitosan sponge;

[0059] 3) The carboxymethyl chitosan sponge and the chondroitin sulfate of sturgeon-Ca-carboxymethyl chitosan composite filler were mixed in equal proportion in an aqueous solution, and the mass content in the aqueous solution was 5 wt%. After dissolution, it was cryogenically frozen at -20 °C, then ultra-low temperature frozen at -60 °C, and finally freeze-dried, repeated 5 times to obtain a chondroitin sulfate of sturgeon-Ca-carboxymethyl chitosan / carboxymethyl chitosan composite sponge hydrogel dressing.

[0060] The effects of the dressing obtained in Example 2 on the cell proliferation and migration activities of mouse fibroblasts (L929) were detected by cell experiments; and the healing performance of the dressing on the chronic wounds of diabetic mice was tested by animal experiments, and the changes of inflammatory factors in the serum of mice were monitored. The results showed that the thermally liquefied chondroitin sulfate dressing of sturgeon had no toxic effect on L929 cells in the concentration range of 6.25 to 100 μg / mL; the proliferation rate of the chondroitin sulfate dressing of sturgeon incubated with cells was positively correlated with the incubation time; using the cell scratch test, the results of the effect on the migration of L929 cells are asFigure 3 shown; the results of the effects on macrophage immunomodulatory activity are as Figure 4 shown. After treatment with the dressing of Example 2, the cell morphology changed from round to spindle-shaped and flattened, indicating that the dressing of Example 2 has the effect of activating macrophages; MTT assay showed that the dressing of Example 2 is non-toxic to RAW264.7 cells; the results of the mouse wound healing experiment are as Figure 5 shown. Both the dressing of Example 2 and the positive control have the effect of promoting wound healing. Immunohistochemical analysis was performed on the wound skin tissue of mice to evaluate the expression of CD31, VEGF, and PCNA. The results are as Figure 6 shown. The expressions of CD31, VEGF, and PCNA in the treatment group with the dressing of Example 2 and the positive group were significantly up-regulated, and the effect was better than that of the positive control group. This indicates that the dressing of Example 2 has a significant angiogenesis-promoting effect on mouse wounds. The up-regulation of the cell proliferation biomarker PCNA indicates that the dressing of Example 2 can promote cell proliferation, which is consistent with the results of L929 cell proliferation.

[0061] The above detection results prove that the diabetic chronic wound healing dressing of the present invention has a good wound healing effect, can stop bleeding as soon as possible, promote epidermal and dermal hyperplasia and angiogenesis by promoting fibroblast proliferation, migration, activating macrophages, and up-regulating the expressions of CD31, VEGF, and PCNA, effectively reduce the inflammatory reaction of the wound, and accelerate the healing of mouse skin wounds. The diabetic chronic wound healing dressing of the present invention has good industrial application prospects.

Claims

1. A preparation method of chondroitin sulfate from sturgeon, characterized in that, It includes the following steps: (1) Thermally liquefy the sturgeon cartilage particles; (2) Add water and protease to the treated sturgeon cartilage particles for enzymatic hydrolysis. After complete enzymatic hydrolysis, heat to inactivate the enzyme to obtain an enzymatic hydrolysate. The protease is composed of flavor protease, neutral protease, and papain with a mass ratio of (1~2):(1~2):(2~3), and the addition amount of the protease is 0.2~0.8%; (3) Filter and purify the enzymatic hydrolysate to obtain a sturgeon chondroitin sulfate solution.

2. The preparation method of chondroitin sulfate from sturgeon according to claim 1, characterized in that, In the step (1), the sturgeon cartilage particles are obtained by boiling fresh sturgeon cartilage until the bone and meat are separated and then crushing, or by mixing dried sturgeon cartilage with water to a solid-liquid ratio of 1:(3~4) and then crushing.

3. The preparation method of chondroitin sulfate from sturgeon according to claim 1, characterized in that, The thermal liquefaction treatment in the step (1) is to perform thermal liquefaction on the sturgeon cartilage particles. The thermal liquefaction conditions are a pressure of 0.04~0.10 MPa, a temperature of 105~115 °C, and a time of 1.5~2.5 h.

4. The preparation method of chondroitin sulfate from sturgeon according to claim 1, characterized in that, In the step (2), the material-liquid ratio of the treated sturgeon cartilage particles to water is 1:(3~6). The temperature during enzymatic hydrolysis is 45~60 °C, and the enzymatic hydrolysis time is 6~9 h; heat to 90~100 °C and keep warm for 10~20 min to inactivate the enzyme.

5. The preparation method of chondroitin sulfate from sturgeon according to claim 1, characterized in that, In the step (3), the filtration and purification process is to pass the enzymatic hydrolysate through ceramic membrane filtration, RO reverse osmosis membrane concentration, and 10 kDa spiral wound membrane ultrafiltration separation in sequence.

6. Use of chondroitin sulfate from sturgeon prepared by the preparation method according to any one of claims 1 to 5 in the preparation of a dressing for promoting the healing of chronic diabetic wounds, characterized in that, It includes the following steps: 1) After freeze-drying and re-dissolving the sturgeon chondroitin sulfate solution, add soluble calcium salt and carboxymethyl chitosan for cross-linking to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan composite filler; 2) Freeze-thaw carboxymethyl chitosan 1~6 times and then freeze-dry to obtain a carboxymethyl chitosan sponge; 3) Mix the carboxymethyl chitosan sponge with the filler to obtain a sturgeon chondroitin sulfate-Ca-carboxymethyl chitosan / carboxymethyl chitosan composite sponge hydrogel dressing.

7. The application according to claim 6, characterized in that, In the step 1), the molar ratio of sturgeon chondroitin sulfate to calcium ions in the soluble calcium salt is (1~2):(3~5), and the mass ratio of sturgeon chondroitin sulfate to carboxymethyl chitosan is (0.5~1):

1.

8. The application according to claim 6, characterized in that, The freeze-thaw process in the step 2) is to dissolve carboxymethyl chitosan and freeze it at a low temperature of -10~-20 °C, then freeze it at an ultra-low temperature of -30~-60 °C, and finally freeze-dry.

9. The application according to claim 6, characterized in that, In the step 3), the mass ratio of the carboxymethyl chitosan sponge to the filler is 1:(1~3).

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