Preparation process of high-purity sodium chondroitin sulfate with low molecular weight

Abstract

The present invention provides a preparation process for sodium chondroitin sulfate with high purity and low molecular weight, comprising the following steps: (1) raw material crushing; (2) soaking and boiling; after cooling, adding sodium chloride and sodium hydroxide solution, and performing multiple microwave radiation treatments, while performing ultrasonic treatment during the microwave radiation treatment; (3) enzymatic hydrolysis with alkaline protease and papain to obtain a filtrate; (4) purification; (5) alcohol precipitation to obtain a first precipitate; (6) enzymatic hydrolysis with chondroitin sulfate lyase, and performing multiple microwave radiation treatments on the obtained filtrate, while performing ultrasonic treatment during the microwave radiation treatment to obtain a treated solution; (7) ultrafiltration; (8) alcohol precipitation to obtain a second precipitate; (9) dehydration and drying; the present invention can prepare sodium chondroitin sulfate with high purity and low molecular weight, and has a high overall yield.

Description

Technical Field

[0001] The present invention relates to the technical field of sodium chondroitin sulfate, and particularly relates to a preparation process of high-purity and low-molecular-weight sodium chondroitin sulfate. Background Art

[0002] Sodium chondroitin sulfate is mainly the sulfate sodium salt of a copolymer of N-acetylgalactosamine (2-acetamido-2-deoxy-β-D-galactopyranose) and D-glucuronic acid. The hexoses in the copolymer are alternately linked by β-1,3 and β-1,4 glycosidic bonds. Sodium chondroitin sulfate is mostly derived from porcine cartilage, porcine laryngeal bone, chicken breast cartilage, bovine or ovine nasal bone, laryngeal bone, costal cartilage, trachea, lunar bone and miscellaneous cartilage of various parts, as well as fish cartilage, etc., and is prepared through processes such as cartilage degradation, core protein hydrolysis, deproteinization, recovery, and purification.

[0003] Among them, low-molecular-weight sodium chondroitin sulfate is a form of sodium chondroitin sulfate with a relatively low molecular weight. Low-molecular-weight sodium chondroitin sulfate is prepared from sodium chondroitin sulfate through specific degradation methods (such as oxidative degradation method, acid degradation method, microwave-assisted degradation method, and enzymatic degradation method, etc.). Its molecular weight is smaller. Compared with ordinary sodium chondroitin sulfate, low-molecular-weight sodium chondroitin sulfate has a lower molecular weight, which makes it have better permeability and bioavailability in some applications. Due to the reduction of the molecular weight, low-molecular-weight sodium chondroitin sulfate may exhibit higher biological activity, such as stronger anti-inflammatory, anticoagulant, and cartilage repair-promoting effects.

[0004] Therefore, the development of the preparation of low-molecular-weight sodium chondroitin sulfate is a research and development trend. Although there are already some methods for preparing low-molecular-weight sodium chondroitin sulfate, in some methods, multiple chemical agents other than sodium chloride will be used. For example, the domestic patent with the application number 202010815590.9 discloses a preparation method of low-molecular-weight sodium chondroitin sulfate, which uses porcine-derived sodium chondroitin sulfate as the raw material and prepares low-molecular-weight sodium chondroitin sulfate through processes such as quaternary ammonium salt salinization, benzyl esterification, degradation, oxidative decolorization, fractional control, and freeze-drying. However, in this method, chemical reagents such as benzethonium chloride and benzyl chloride are used, which will introduce new impurities into sodium chondroitin sulfate, thus affecting the use performance of sodium chondroitin sulfate. In addition, it is difficult to simultaneously take into account the purity and yield of the product in the current preparation of low-molecular-weight sodium chondroitin sulfate. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation process of high-purity and low-molecular-weight sodium chondroitin sulfate, which will not introduce unconventional impurities during the preparation process of sodium chondroitin sulfate, can prepare high-purity low-molecular-weight sodium chondroitin sulfate, and has a high overall yield.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] A preparation process of high-purity chondroitin sulfate sodium with low molecular weight, comprising the following steps:

[0008] (1) Dry the pretreated cartilage raw material, then carry out coarse crushing, sieve it to obtain coarse crushed material; then carry out liquid nitrogen impregnation treatment on the obtained coarse crushed material; carry out air flow crushing on the coarse crushed material after liquid nitrogen treatment to obtain fine crushed cartilage material;

[0009] (2) Immerse the fine crushed cartilage material in water, then boil it; then cool it to room temperature, add sodium chloride, and add sodium hydroxide solution to adjust the pH value to 10-11, and then carry out multiple microwave radiation treatments on the obtained mixed material liquid, and carry out ultrasonic treatment while each microwave radiation treatment; then cool it to room temperature to obtain a mixed treatment material liquid;

[0010] (3) Carry out alkaline protease enzymolysis and papain enzymolysis on the mixed treatment material liquid in sequence; then carry out enzyme inactivation treatment, and filter to obtain filtrate one;

[0011] (4) First carry out microfiltration on the filtrate one; then adsorb it with a macroporous resin chromatography column, and elute it with sodium chloride solution, and collect the eluate;

[0012] (5) Carry out vacuum concentration on the eluate, add ethanol to the obtained concentrated solution, let it stand, and filter to obtain precipitate one;

[0013] (6) Add sodium chloride solution to the precipitate one, mix it, carry out chondroitin sulfate lyase enzymolysis on the obtained mixed solution, then carry out enzyme inactivation treatment, and filter to obtain filtrate two; carry out multiple microwave radiation treatments on the filtrate two, and carry out ultrasonic treatment while each microwave radiation treatment to obtain a treatment solution;

[0014] (7) Carry out ultrafiltration treatment on the treatment solution with an ultrafiltration membrane to obtain an ultrafiltrate;

[0015] (8) Adjust the pH of the ultrafiltrate with hydrochloric acid, add ethanol, stir and let it stand, then filter to obtain precipitate two;

[0016] (9) Dehydrate the precipitate two with ethanol, filter and dry it to obtain the chondroitin sulfate sodium.

[0017] Preferably, in step (1), the pretreated cartilage raw material is obtained by the following method: after washing the cartilage raw material, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material;

[0018] The mesh number of the sieving is 60-100 meshes;

[0019] When carrying out the liquid nitrogen impregnation treatment, the impregnation time is 20-40 min.

[0020] Preferably, in step (2), the addition amount of water is 5 - 6 times the mass of the finely crushed cartilage material;

[0021] The soaking time is 3 - 5 h, and the boiling time is 30 - 60 min; the addition amount of sodium chloride is 2.5 - 4% of the mass of the finely crushed cartilage material.

[0022] Preferably, in step (2), the power of the microwave radiation treatment is 1000 - 1500 W, the time for each microwave radiation treatment is 3 - 5 min, and the intermittent time between two adjacent microwave radiation treatments is 1 - 2 min; the number of times of the microwave radiation treatment is 10 - 15 times; ultrasonic treatment is carried out simultaneously during each microwave radiation treatment, and the power of the ultrasonic treatment is 500 - 1000 W.

[0023] Preferably, step (3) specifically includes the following steps: maintaining the temperature of the mixed treatment liquid between 35 - 38 °C, adjusting the pH value to 8.5 - 9.5, then adding alkaline protease at 0.3 - 0.4% of the mass of the finely crushed cartilage material, and carrying out constant-temperature enzymatic hydrolysis for 2 - 3 h; maintaining the temperature between 35 - 38 °C, adding papain at 0.3 - 0.4% of the mass of the finely crushed cartilage material, and carrying out constant-temperature enzymatic hydrolysis for 2 - 3 h; after the enzymatic hydrolysis is completed, heating to 95 - 100 °C for enzyme inactivation, maintaining for 5 - 10 min, then cooling to room temperature, and filtering to obtain a filtrate.

[0024] Preferably, in step (4), microfiltration is carried out using a 0.2 - μm microfiltration module;

[0025] The macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of the macroporous resin is 2.5 - 3.5 times the mass of the finely crushed cartilage material; the mass fraction of the sodium chloride solution is 10 - 15%, the mass ratio of the sodium chloride solution to the macroporous resin is 3 - 4:1, and the elution time is 2.5 - 3.5 h.

[0026] Preferably, step (5) specifically includes the following steps: carrying out vacuum concentration on the eluate at 60 - 70 °C to make the obtained concentrated solution be 30 - 35% of the volume of the eluate, then adding ethanol with a volume fraction of more than 95% at 8 - 10 times the volume of the concentrated solution, standing for 3 - 5 h, and then filtering to obtain precipitate I.

[0027] Preferably, in step (6), a sodium chloride solution with a mass 10 - 12 times that of precipitate I is added; the mass fraction of the sodium chloride solution is 2.5 - 3%;

[0028] The enzymatic hydrolysis of the obtained mixture with chondroitinase includes the following steps: adjusting the pH value of the obtained mixture to 6.5 - 7.0 with hydrochloric acid, maintaining the temperature between 35 - 38 °C, adding chondroitinase at 0.05 - 0.1% of the mass of the finely ground cartilage material, and carrying out enzymatic hydrolysis at a constant temperature for 2 - 3 h;

[0029] In step (6), the power of the microwave radiation treatment is 1000 - 1500 W; the time for each microwave radiation treatment is 3 - 5 min, and the intermittent time between adjacent microwave radiation treatments is 1 - 2 min; the number of microwave radiation treatments is 8 - 12 times; ultrasonic treatment is carried out simultaneously during each microwave radiation treatment, and the power of the ultrasonic treatment is 500 - 1000 W;

[0030] Preferably, in step (7), the cut-off molecular weight of the ultrafiltration membrane is 1000 - 5000 Da.

[0031] Preferably, in step (8), adjusting the pH value of the ultrafiltrate to 5.8 - 6.0 with hydrochloric acid; adding ethanol to make the volume fraction of ethanol in the obtained solution 85 - 90%, the stirring time is 1 - 1.5 h, and the standing time is 7 - 10 h;

[0032] Step (9) specifically includes the following steps: soaking the precipitate II twice with ethanol having a volume fraction of 92 - 95% for 2 - 3 h each time, filtering and then washing with absolute ethanol, and then placing it in a vacuum dryer at 50 - 55 °C to obtain sodium chondroitin.

[0033] The beneficial effects of the present invention are:

[0034] 1. When preparing sodium chondroitin in the present invention, before enzymatic hydrolysis with alkaline protease and papain, by simultaneously carrying out intermittent microwave radiation and ultrasonic treatment on the mixture solution of the finely ground cartilage material after adding alkali, the mechanical scission of macromolecular substances is enhanced, the degradation effect is strengthened, and thus more low-molecular-weight sodium chondroitin is obtained. And after subsequent enzymatic hydrolysis with chondroitinase, intermittent microwave radiation and ultrasonic treatment are carried out again, and the microwave radiation and ultrasonic treatment act synergistically again, which can further promote the degradation of sodium chondroitin and increase the yield of low-molecular-weight sodium chondroitin. And through research, the intermittent microwave radiation and ultrasonic treatment process adopted in the present invention can significantly improve the purity of the obtained low-molecular-weight sodium chondroitin compared with the continuous microwave radiation and ultrasonic treatment process.

[0035] 2. The present invention impregnates the coarsely ground cartilage raw material with liquid nitrogen, which can enhance the porosity of the ground material and increase its brittleness. Then, airflow pulverization is carried out to obtain a cartilage finely ground material with multiple pores and small particle size, which can improve the degradation efficiency and further increase the yield of sodium chondroitin.

[0036] 3. In the process of preparing sodium chondroitin sulfate, after enzymatic hydrolysis, microfiltration, macroporous resin adsorption, and the first alcohol precipitation treatment are carried out in sequence, and then secondary enzymatic hydrolysis, ultrafiltration, secondary alcohol precipitation treatment and other processes are carried out. Through the cooperation of the above processes, sodium chondroitin sulfate with relatively high purity and low molecular weight can be obtained. Detailed implementation manners

[0037] To make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] Embodiment 1:

[0039] A preparation process for high-purity low-molecular-weight sodium chondroitin sulfate includes the following steps:

[0040] (1) After washing the cartilage raw material, pig laryngeal bone, heating it to boiling, removing the floating oil, taking it out and washing it, the pre-treated cartilage raw material is obtained; the pre-treated cartilage raw material is dried, then coarsely pulverized, passed through a 100-mesh sieve to obtain a coarsely pulverized material; then the obtained coarsely pulverized material is subjected to liquid nitrogen impregnation treatment for 30 minutes; the coarsely pulverized material after liquid nitrogen treatment is subjected to air flow pulverization to obtain a finely pulverized cartilage material.

[0041] (2) The finely pulverized cartilage material is soaked in water 6 times its mass for 4 hours, then boiled and continued for 50 minutes; then cooled to room temperature, sodium chloride accounting for 4% of the mass of the finely pulverized cartilage material is added, and the pH value is adjusted to 10.5 by adding a sodium hydroxide solution. Then the obtained mixed material liquid is subjected to 15 times of microwave radiation treatment, and ultrasonic treatment is carried out simultaneously during each microwave radiation treatment; then cooled to room temperature to obtain a mixed treatment material liquid;

[0042] Among them, the power of the microwave radiation treatment is 1000 W, the time of each microwave radiation treatment is 5 minutes, and the intermittent time between adjacent two microwave radiation treatments is 1.5 minutes; the power of the ultrasonic treatment is 800 W.

[0043] (3) Keep the temperature of the mixed treatment liquid between 35 - 38 °C, adjust the pH value to 8.5 with hydrochloric acid, then add alkaline protease at 0.3% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 3 h; keep the temperature between 35 - 38 °C, add papain at 0.35% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 2 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, maintain for 5 min, then cool to room temperature, and filter to obtain filtrate one.

[0044] (4) Microfilter filtrate one with a 0.2 μm microfiltration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a 12% sodium chloride solution by mass fraction, and collect the eluate; among them, the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 3 times the mass of the finely crushed cartilage material; the mass ratio of this sodium chloride solution to macroporous resin D315 is 3:1, and the elution time is 3.5 h.

[0045] (5) Carry out vacuum concentration of the eluate at 67 - 70 °C to make the obtained concentrated solution be 35% of the volume of the eluate, then add ethanol with a volume fraction of 96% which is 10 times the volume of the concentrated solution, let it stand for 3 h, and then filter to obtain precipitate one.

[0046] (6) Add a 3% sodium chloride solution by mass fraction which is 10 times the mass of precipitate one, mix to obtain a mixed solution; adjust the pH value of the obtained mixed solution to 6.5 with hydrochloric acid, and keep the temperature between 35 - 38 °C, add chondroitin sulfate lyase at 0.05% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 3 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, maintain for 5 min, then cool to room temperature, and filter to obtain filtrate two;

[0047] Carry out 10 times of microwave radiation treatment on the obtained filtrate two, carry out ultrasonic treatment simultaneously during each microwave radiation treatment, and obtain a treatment solution;

[0048] Among them, the power of the microwave radiation treatment is 1000 W, the time of each microwave radiation treatment is 5 min, and the intermittent time between adjacent two microwave radiation treatments is 1.5 min; the power of the ultrasonic treatment is 800 W.

[0049] (7) Ultrafilter the treatment solution with an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0050] (8) Adjust the pH value of the ultrafiltrate to 5.8 with hydrochloric acid, add ethanol, and make the volume fraction of ethanol in the obtained solution be 85%, stir for 1.5 h and then let it stand for 10 h, and then filter to obtain precipitate two.

[0051] (9) Soak the precipitate II twice with ethanol with a volume fraction of 92% for 3 hours each time. After filtration, wash it with absolute ethanol, and then place it in a vacuum dryer at 50 - 53 °C to obtain sodium chondroitin sulfate.

[0052] Example 2:

[0053] A preparation process of high-purity low-molecular-weight sodium chondroitin sulfate, comprising the following steps:

[0054] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then carry out coarse crushing, and pass through an 80-mesh sieve to obtain coarse crushed material; then carry out liquid nitrogen impregnation treatment on the obtained coarse crushed material for 40 minutes; carry out air flow crushing on the coarse crushed material after liquid nitrogen treatment to obtain fine crushed cartilage material.

[0055] (2) Soak the fine crushed cartilage material in water 5 times its mass for 4 hours, then boil it and continue for 40 minutes; then cool it to room temperature, add sodium chloride 2.5% of the mass of the fine crushed cartilage material, and add sodium hydroxide solution to adjust the pH value to 11, and then carry out 10 times of microwave radiation treatment on the obtained mixed material liquid, and carry out ultrasonic treatment while performing each microwave radiation treatment; then cool it to room temperature to obtain the mixed treated material liquid;

[0056] Among them, the power of the microwave radiation treatment is 1500 W, the time of each microwave radiation treatment is 4 minutes, and the intermittent time between adjacent two microwave radiation treatments is 2 minutes; the power of the ultrasonic treatment is 800 W.

[0057] (3) Keep the temperature of the mixed treated material liquid between 35 - 38 °C, adjust the pH value to 8.5 with hydrochloric acid, add alkaline protease 0.4% of the mass of the fine crushed cartilage material, and carry out constant temperature enzymatic hydrolysis for 3 hours; keep the temperature between 35 - 38 °C, add papain 0.4% of the mass of the fine crushed cartilage material, and carry out constant temperature enzymatic hydrolysis for 3 hours; after the enzymatic hydrolysis is completed, raise the temperature to 95 - 97 °C for enzyme inactivation, keep it for 10 minutes, and then cool it to room temperature, filter to obtain filtrate I.

[0058] (4) Carry out microfiltration on filtrate I with a 0.2 μm microfiltration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a sodium chloride solution with a mass fraction of 10%, and collect the eluate; among them, the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 3.5 times the mass of the fine crushed cartilage material; the mass ratio of this sodium chloride solution to macroporous resin D315 is 4:1, and the elution time is 3 hours.

[0059] (5) Concentrate the eluent under reduced pressure at 60 - 63 °C to make the resulting concentrated solution 32% of the volume of the eluent. Then add ethanol with a volume fraction of 96% that is 8 times the volume of the concentrated solution, let it stand for 5 h, and then filter to obtain precipitate one.

[0060] (6) Add a sodium chloride solution with a mass fraction of 3% that is 10 times the mass of precipitate one and mix to obtain a mixed solution. Adjust the pH value of the resulting mixed solution to 6.5 with hydrochloric acid and keep the temperature between 35 - 38 °C. Add chondroitin sulfate lyase at 0.08% of the mass of the chondrocyte fine powder and carry out constant-temperature enzymatic hydrolysis for 2 h. After the enzymatic hydrolysis is completed, raise the temperature to 95 - 97 °C for enzyme inactivation, keep it for 10 min, then cool to room temperature and filter to obtain filtrate two.

[0061] Perform 10 times of microwave radiation treatment on the resulting filtrate two. During each microwave radiation treatment, carry out ultrasonic treatment simultaneously to obtain a treated solution.

[0062] Among them, the power of the microwave radiation treatment is 1500 W, the time of each microwave radiation treatment is 4 min, and the intermittent time between adjacent two microwave radiation treatments is 2 min; the power of the ultrasonic treatment is 800 W.

[0063] (7) Ultrafilter the treated solution with an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0064] (8) Adjust the pH value of the ultrafiltrate to 6.0 with hydrochloric acid, add ethanol to make the volume fraction of ethanol in the resulting solution 85%, stir for 1 h, then let it stand for 9 h, and then filter to obtain precipitate two.

[0065] (9) Soak the precipitate two twice with ethanol with a volume fraction of 92% for 3 h each time. After filtration, wash it with absolute ethanol, and then place it in a vacuum dryer at 50 - 53 °C to obtain sodium chondroitin sulfate.

[0066] Example 3:

[0067] A preparation process of high-purity low-molecular-weight sodium chondroitin sulfate, comprising the following steps:

[0068] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material. Dry the pretreated cartilage raw material, then carry out coarse crushing, pass through a 60-mesh sieve to obtain coarse crushed material. Then carry out liquid nitrogen impregnation treatment on the obtained coarse crushed material for 40 min. Carry out airflow crushing on the coarse crushed material after liquid nitrogen treatment to obtain chondrocyte fine powder.

[0069] (2) Soak the finely ground cartilage material in water six times its mass for 5 h, then boil it for 50 min and continue boiling; then cool it to room temperature, add sodium chloride at 3% of the mass of the finely ground cartilage material, and add sodium hydroxide solution to adjust the pH value to 11. Then, subject the obtained mixed material liquid to 15 times of microwave radiation treatment, and perform ultrasonic treatment simultaneously during each microwave radiation treatment; then cool it to room temperature to obtain a mixed treatment material liquid.

[0070] Among them, the power of the microwave radiation treatment is 1200 W, the time of each microwave radiation treatment is 4 min, and the intermittent time between two adjacent microwave radiation treatments is 2 min; the power of the ultrasonic treatment is 1000 W.

[0071] (3) Keep the temperature of the mixed treatment material liquid between 35 - 38 °C, adjust the pH value to 9.5 with hydrochloric acid, then add alkaline protease at 0.3% of the mass of the finely ground cartilage material, and perform constant-temperature enzymatic hydrolysis for 2 h; keep the temperature between 35 - 38 °C, add papain at 0.3% of the mass of the finely ground cartilage material, and perform constant-temperature enzymatic hydrolysis for 3 h; after the enzymatic hydrolysis is completed, raise the temperature to 95 - 97 °C for enzyme inactivation, keep it for 9 min, then cool it to room temperature and filter to obtain filtrate one.

[0072] (4) Subject filtrate one to microfiltration using a 0.2 - μm microfiltration module; then adsorb it using a macroporous resin chromatography column, and then elute it using a sodium chloride solution with a mass fraction of 12%, and collect the eluate; among them, the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 2.5 times the mass of the finely ground cartilage material; the mass ratio of this sodium chloride solution to macroporous resin D315 is 4:1, and the elution time is 3 h.

[0073] (5) Concentrate the eluate under reduced pressure at 65 - 68 °C so that the obtained concentrated solution is 33% of the volume of the eluate, then add ethanol with a volume fraction of 96 ten times the volume of the concentrated solution, let it stand for 5 h, and then filter to obtain precipitate one.

[0074] (6) Add a sodium chloride solution with a mass fraction of 3% twelve times the mass of precipitate one, mix to obtain a mixed solution; adjust the pH value of the obtained mixed solution to 7.0 with hydrochloric acid, and keep the temperature between 35 - 38 °C, add chondroitin sulfate lyase at 0.1% of the mass of the finely ground cartilage material, and perform constant-temperature enzymatic hydrolysis for 3 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, keep it for 6 min, then cool it to room temperature and filter to obtain filtrate two;

[0075] Subject the obtained filtrate two to 8 times of microwave radiation treatment, and perform ultrasonic treatment simultaneously during each microwave radiation treatment to obtain a treatment solution.

[0076] Among them, the power of the microwave radiation treatment is 1200 W, the time for each microwave radiation treatment is 4 min, and the intermittent time between two adjacent microwave radiation treatments is 2 min; the power of the ultrasonic treatment is 1000 W.

[0077] (7) Ultrafilter the treatment liquid with an ultrafiltration membrane to obtain an ultrafiltrate; the molecular weight cut-off of this ultrafiltration membrane is 5000 Da.

[0078] (8) Adjust the pH value of the ultrafiltrate to 6.0 with hydrochloric acid, add ethanol, and make the volume fraction of ethanol in the resulting solution 90%. Stir for 1.5 h and then let it stand for 8 h, and then filter to obtain precipitate II.

[0079] (9) Soak the precipitate II twice with ethanol with a volume fraction of 95% for 2 h each time. After filtration, wash it with absolute ethanol, and then place it in a vacuum dryer at 53 - 55 °C to obtain sodium chondroitin sulfate.

[0080] Example 4:

[0081] A preparation process of high-purity low-molecular-weight sodium chondroitin sulfate, comprising the following steps:

[0082] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it, and obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then carry out coarse crushing, pass through a 100-mesh sieve to obtain a coarsely crushed material; then carry out liquid nitrogen impregnation treatment on the obtained coarsely crushed material for 20 min; carry out air flow crushing on the coarsely crushed material after liquid nitrogen treatment to obtain a finely crushed cartilage material.

[0083] (2) Soak the finely crushed cartilage material in water 5 times its mass for 3 h, then boil it and continue for 60 min; then cool it to room temperature, add sodium chloride 2.5% of the mass of the finely crushed cartilage material, and add sodium hydroxide solution to adjust the pH value to 11, and then carry out 12 times of microwave radiation treatment on the obtained mixed material liquid, and carry out ultrasonic treatment at the same time during each microwave radiation treatment; then cool it to room temperature to obtain a mixed treatment material liquid;

[0084] Among them, the power of the microwave radiation treatment is 1500 W, the time for each microwave radiation treatment is 3 min, and the intermittent time between two adjacent microwave radiation treatments is 1 min; the power of the ultrasonic treatment is 800 W.

[0085] (3) Keep the temperature of the mixed treatment liquid between 35 - 38 °C, adjust the pH value to 9 with hydrochloric acid, then add alkaline protease accounting for 0.4% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 3 h; keep the temperature between 35 - 38 °C, add papain accounting for 0.3% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 2.5 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, maintain for 8 min, then cool to room temperature, and filter to obtain filtrate one.

[0086] (4) Microfilter filtrate one with a 0.2 μm microfiltration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a 15% sodium chloride solution by mass fraction, and collect the eluate; the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 3.5 times the mass of the finely crushed cartilage material; the mass ratio of this sodium chloride solution to macroporous resin D315 is 3:1, and the elution time is 2.5 h.

[0087] (5) Carry out vacuum concentration of the eluate at 60 - 63 °C to make the obtained concentrated solution be 33% of the volume of the eluate, then add ethanol with a volume fraction of 96% which is 8 times the volume of the concentrated solution, let it stand for 5 h, and then filter to obtain precipitate one.

[0088] (6) Add a 2.5% sodium chloride solution by mass fraction which is 10 times the mass of precipitate one, mix to obtain a mixed solution; adjust the pH value of the obtained mixed solution to 6.5 with hydrochloric acid, and keep the temperature between 35 - 38 °C, add chondroitin sulfate lyase accounting for 0.05% of the mass of the finely crushed cartilage material, and carry out enzymatic hydrolysis at a constant temperature for 3 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, maintain for 5 min, then cool to room temperature, and filter to obtain filtrate two;

[0089] Carry out 12 times of microwave radiation treatment on the obtained filtrate two, carry out ultrasonic treatment simultaneously during each microwave radiation treatment, and obtain a treatment solution;

[0090] Among them, the power of the microwave radiation treatment is 1500 W, the time of each microwave radiation treatment is 3 min, the intermittent time between adjacent two microwave radiation treatments is 1 min; the power of the ultrasonic treatment is 800 W.

[0091] (7) Ultrafilter the treatment solution with an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0092] (8) Adjust the pH value of the ultrafiltrate to 5.8 with hydrochloric acid, add ethanol, and make the ethanol volume fraction in the obtained solution be 88%, stir for 1 h and then let it stand for 7 h, and then filter to obtain precipitate two.

[0093] (9) Soak the precipitate II twice with ethanol with a volume fraction of 95% for 3 hours each time. After filtration, wash it with absolute ethanol, and then place it in a vacuum dryer at 53 - 55 °C to obtain sodium chondroitin sulfate.

[0094] Example 5:

[0095] A preparation process of high - purity and low - molecular - weight sodium chondroitin sulfate, comprising the following steps:

[0096] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it again to obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then carry out coarse crushing, pass through a 100 - mesh sieve to obtain the coarsely crushed material; then carry out liquid nitrogen impregnation treatment on the obtained coarsely crushed material for 30 minutes; carry out air - flow crushing on the coarsely crushed material after liquid nitrogen treatment to obtain the finely crushed cartilage material.

[0097] (2) Soak the finely crushed cartilage material in water 5 times its mass for 5 hours, then boil it and continue for 30 minutes; then cool it to room temperature, add sodium chloride accounting for 4% of the mass of the finely crushed cartilage material, and add sodium hydroxide solution to adjust the pH value to 10, then carry out 10 times of microwave radiation treatment on the obtained mixed material liquid, and carry out ultrasonic treatment simultaneously during each microwave radiation treatment; then cool it to room temperature to obtain the mixed - treated material liquid;

[0098] Among them, the power of the microwave radiation treatment is 1000 W, the time of each microwave radiation treatment is 5 minutes, and the intermittent time between adjacent two microwave radiation treatments is 2 minutes; the power of the ultrasonic treatment is 500 W.

[0099] (3) Keep the temperature of the mixed - treated material liquid between 35 - 38 °C, adjust the pH value to 8.5 with hydrochloric acid, then add alkaline protease accounting for 0.35% of the mass of the finely crushed cartilage material, and carry out constant - temperature enzymatic hydrolysis for 3 hours; keep the temperature between 35 - 38 °C, add papain accounting for 0.3% of the mass of the finely crushed cartilage material, and carry out constant - temperature enzymatic hydrolysis for 3 hours; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, keep it for 6 minutes, then cool it to room temperature, filter to obtain filtrate I.

[0100] (4) Micro - filter filtrate I with a 0.2 - μm micro - filtration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a sodium chloride solution with a mass fraction of 10%, and collect the eluate; among them, the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 3 times the mass of the finely crushed cartilage material; the mass ratio of the sodium chloride solution to macroporous resin D315 is 3:1, and the elution time is 3.5 hours.

[0101] (5) Concentrate the eluent under reduced pressure at 67 - 70 °C to make the resulting concentrated solution 30% of the volume of the eluent. Then add ethanol with a volume fraction of 96% that is 9 times the volume of the concentrated solution, let it stand for 3 h, and then filter to obtain Precipitate I.

[0102] (6) Add a sodium chloride solution with a mass fraction of 2.8% that is 10 times the mass of Precipitate I and mix to obtain a mixed solution. Adjust the pH value of the resulting mixed solution to 7.0 with hydrochloric acid, keep the temperature between 35 - 38 °C, add chondroitin sulfate lyase at 0.1% of the mass of the finely ground cartilage material, and carry out constant-temperature enzymatic hydrolysis for 2.5 h. After the enzymatic hydrolysis ends, raise the temperature to 95 - 97 °C for enzyme inactivation, keep it for 8 min, then cool to room temperature and filter to obtain Filtrate II.

[0103] Subject the obtained Filtrate II to 11 times of microwave radiation treatment, and perform ultrasonic treatment simultaneously during each microwave radiation treatment to obtain a treated solution.

[0104] Among them, the power of the microwave radiation treatment is 1000 W, the time of each microwave radiation treatment is 5 min, and the intermittent time between adjacent two microwave radiation treatments is 2 min; the power of the ultrasonic treatment is 500 W.

[0105] (7) Subject the treated solution to ultrafiltration treatment with an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0106] (8) Adjust the pH value of the ultrafiltrate to 6.0 with hydrochloric acid, add ethanol to make the volume fraction of ethanol in the resulting solution 88%, stir for 1.5 h and then let it stand for 10 h, and then filter to obtain Precipitate II.

[0107] (9) First soak Precipitate II twice with ethanol with a volume fraction of 92% for 3 h each time, filter and then wash with absolute ethanol, and then place it in a vacuum dryer at 53 - 55 °C to obtain sodium chondroitin sulfate.

[0108] Comparative Example 1:

[0109] A preparation process of sodium chondroitin sulfate, comprising the following steps:

[0110] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then perform coarse crushing, pass through a 100-mesh sieve to obtain a coarsely crushed material; then subject the obtained coarsely crushed material to liquid nitrogen impregnation treatment for 30 min; subject the coarsely crushed material after liquid nitrogen treatment to air flow crushing to obtain a finely ground cartilage material.

[0111] (2) Soak the finely crushed cartilage material in water six times its mass for 4 h, then boil it for 50 min; then cool it to room temperature, add sodium chloride accounting for 4% of the mass of the finely crushed cartilage material, and add sodium hydroxide solution to adjust the pH value to 10.5, then stir for 100 min to obtain a mixed treatment liquid.

[0112] (3) Keep the temperature of the mixed treatment liquid between 35 - 38 °C, adjust the pH value to 8.5 with hydrochloric acid, then add alkaline protease accounting for 0.3% of the mass of the finely crushed cartilage material, and carry out constant-temperature enzymatic hydrolysis for 3 h; keep the temperature between 35 - 38 °C, add papain accounting for 0.35% of the mass of the finely crushed cartilage material, and carry out constant-temperature enzymatic hydrolysis for 2 h; after the enzymatic hydrolysis ends, raise the temperature to 100 °C for enzyme inactivation, keep it for 5 min, then cool it to room temperature and filter to obtain filtrate one.

[0113] (4) Microfilter filtrate one with a 0.2 - μm microfiltration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a 12% sodium chloride solution by mass fraction, and collect the eluate; the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is three times the mass of the finely crushed cartilage material; the mass ratio of this sodium chloride solution to macroporous resin D315 is 3:1, and the elution time is 3.5 h.

[0114] (5) Carry out vacuum concentration on the eluate at 67 - 70 °C to make the obtained concentrated solution be 35% of the volume of the eluate, then add ethanol with a volume fraction of 96% ten times the volume of the concentrated solution, let it stand for 3 h, and then filter to obtain precipitate one.

[0115] (6) Add a 3% sodium chloride solution by mass fraction ten times the mass of precipitate one and mix to obtain a mixed solution; adjust the pH value of the obtained mixed solution to 6.5 with hydrochloric acid, and keep the temperature between 35 - 38 °C, add chondroitin sulfate lyase accounting for 0.05% of the mass of the finely crushed cartilage material, and carry out constant-temperature enzymatic hydrolysis for 3 h; after the enzymatic hydrolysis ends, raise the temperature to 100 °C for enzyme inactivation, keep it for 5 min, then cool it to room temperature and filter to obtain filtrate two;

[0116] (7) Ultrafilter filtrate two with an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0117] (8) Adjust the pH value of the ultrafiltrate to 5.8 with hydrochloric acid, add ethanol, and make the volume fraction of ethanol in the obtained solution be 85%, stir for 1.5 h and then let it stand for 10 h, and then filter to obtain precipitate two.

[0118] (9) Soak the second precipitate twice with ethanol at a volume fraction of 85% for 3 hours each time. After filtration, wash it with absolute ethanol, and then place it in a vacuum dryer at 50 - 53 °C to obtain sodium chondroitin sulfate.

[0119] Comparative Example 2:

[0120] A preparation process of sodium chondroitin sulfate, comprising the following steps:

[0121] (1) After washing the cartilage raw material of pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then perform coarse crushing, pass through a 100 - mesh sieve to obtain the coarsely crushed material; then perform liquid nitrogen impregnation treatment on the obtained coarsely crushed material for 30 minutes; perform air flow crushing on the coarsely crushed material after liquid nitrogen treatment to obtain the finely crushed cartilage material.

[0122] (2) Soak the finely crushed cartilage material in water at 6 times its mass for 4 hours, then boil it and continue for 50 minutes; then cool it to room temperature, add sodium chloride at 4% of the mass of the finely crushed cartilage material, and add sodium hydroxide solution to adjust the pH value to 10.5, then perform continuous microwave radiation treatment on the obtained mixed material liquid for 75 minutes, and perform ultrasonic treatment while performing microwave radiation treatment, where the power of the microwave radiation treatment is 1000 W and the power of the ultrasonic treatment is 800 W; then cool it to room temperature to obtain the mixed treatment material liquid.

[0123] (3) Keep the temperature of the mixed treatment material liquid between 35 - 38 °C, adjust the pH value to 8.5, then add alkaline protease at 0.3% of the mass of the finely crushed cartilage material, and perform constant - temperature enzymatic hydrolysis for 3 hours; keep the temperature between 35 - 38 °C, add papain at 0.35% of the mass of the finely crushed cartilage material, and perform constant - temperature enzymatic hydrolysis for 2 hours; after the enzymatic hydrolysis, raise the temperature to 100 °C for enzyme inactivation, keep it for 5 minutes, then cool it to room temperature, filter to obtain the first filtrate.

[0124] (4) Micro - filter the first filtrate with a 0.2 - μm micro - filtration module; then adsorb it with a macroporous resin chromatography column, and then elute it with a sodium chloride solution with a mass fraction of 12%, and collect the eluate; where the macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of macroporous resin D315 is 3 times the mass of the finely crushed cartilage material; the mass ratio of the sodium chloride solution to macroporous resin D315 is 3:1, and the elution time is 3.5 hours.

[0125] (5) Perform vacuum concentration on the eluate at 67 - 70 °C to make the obtained concentrated solution 35% of the volume of the eluate, then add ethanol with a volume fraction of 96% at 10 times the volume of the concentrated solution, let it stand for 3 hours, and then perform filtration to obtain the first precipitate.

[0126] (6) Add a sodium chloride solution with a mass fraction of 3% that is 10 times the mass of the first precipitate, mix to obtain a mixed solution; adjust the pH value of the obtained mixed solution to 6.5 with hydrochloric acid, maintain the temperature between 35 - 38 °C, add chondroitin sulfate lyase at 0.05% of the mass of the finely ground cartilage material, and perform enzymatic hydrolysis at a constant temperature for 3 h; after the enzymatic hydrolysis is completed, raise the temperature to 100 °C for enzyme inactivation, maintain for 5 min, then cool to room temperature, filter to obtain the second filtrate;

[0127] Perform continuous microwave radiation treatment on the obtained second filtrate for 50 min. During the microwave radiation treatment, ultrasonic treatment is carried out simultaneously. The power of the microwave radiation treatment is 1000 W, and the power of the ultrasonic treatment is 800 W to obtain a treated solution.

[0128] (7) Ultrafilter the treated solution using an ultrafiltration membrane to obtain an ultrafiltrate; the cut-off molecular weight of this ultrafiltration membrane is 5000 Da.

[0129] (8) Adjust the pH value of the ultrafiltrate to 5.8 with hydrochloric acid, add ethanol, and make the volume fraction of ethanol in the obtained solution 85%. Stir for 1.5 h and then let it stand for 10 h, then filter to obtain the second precipitate.

[0130] (9) Soak the second precipitate twice with ethanol with a volume fraction of 85%, each time for 3 h. After filtration, wash with absolute ethanol, and then place it in a vacuum dryer at 50 - 53 °C to obtain sodium chondroitin sulfate.

[0131] Comparative Example 3:

[0132] A preparation process of sodium chondroitin sulfate, comprising the following steps:

[0133] (1) After washing the cartilage raw material, pig laryngeal bone, heat it to boiling, remove the floating oil, take it out and wash it to obtain the pretreated cartilage raw material; dry the pretreated cartilage raw material, then perform coarse grinding, pass through a 100-mesh sieve to obtain a coarsely ground material; perform air flow grinding on the coarsely ground material to obtain a finely ground cartilage material.

[0134] Steps (2), (3), (4), (5), (6), (7), (8), and (9) are the same as those in Example 1.

[0135] Test the yield, purity, chloride content, and molecular weight (MW) of the sodium chondroitin sulfate obtained in Examples 1 - 5 and Comparative Examples 1 - 3. The specific test results are shown in Table 1.

[0136] Table 1 Yield, purity, and chloride content of sodium chondroitin sulfate

[0137]

[0138] As can be seen from Table 1, the chondroitin sulfate sodium obtained by the preparation process in Examples 1-5 of the present invention has a low molecular weight while also having a high yield and high purity. By comparing Example 1 with Comparative Example 1, it can be seen that the present invention uses intermittent microwave radiation treatment combined with ultrasonic treatment in step (2) and step (6). Compared with not performing microwave radiation treatment combined with ultrasonic treatment, the yield of low molecular weight chondroitin sulfate sodium can be significantly improved. By comparing Example 1 with Comparative Example 1, it can be seen that the process of using intermittent microwave radiation treatment combined with ultrasonic treatment can make the purity of the obtained low molecular weight chondroitin sulfate sodium significantly higher than that of the continuous microwave radiation treatment combined with ultrasonic treatment process. By comparing Example 1 with Comparative Example 3, it can be seen that the liquid nitrogen impregnation treatment of the coarsely crushed material has a certain effect on improving the yield of low molecular weight chondroitin sulfate sodium.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for preparing high-purity low molecular weight sodium chondroitin sulfate, characterized in that: The following steps are involved: (1) drying the pretreated cartilage raw material, and then coarsely crushing and sieving to obtain a coarse crushed material; then subjecting the obtained coarse crushed material to a liquid nitrogen immersion treatment; and subjecting the coarse crushed material after the liquid nitrogen treatment to a jet milling to obtain a cartilage fine crushed material; (2) soaking the finely ground cartilage in water and then boiling it; then cooling it to room temperature, adding sodium chloride, and adding sodium hydroxide solution to adjust the pH value to 10-11, and then subjecting the resulting mixed liquid to multiple microwave irradiation treatments, and performing ultrasonic treatment at the same time during each microwave irradiation treatment; then cooling it to room temperature to obtain a mixed treated liquid; The power of the microwave radiation treatment is 1000-1500W, the time of each microwave radiation treatment is 3-5min, and the interval time between two adjacent microwave radiation treatments is 1-2min; the number of microwave radiation treatments is 10-15 times; each microwave radiation treatment is simultaneously performed with ultrasonic treatment, and the power of the ultrasonic treatment is 500-1000W; (3) maintaining the temperature of the mixed treated liquid at 35-38° C., adjusting the pH value to 8.5-9.5, adding 0.3-0.4% alkaline protease by weight of the finely ground cartilage material, and performing constant temperature enzymolysis for 2-3 hours; maintaining the temperature at 35-38° C., adding 0.3-0.4% papain by weight of the finely ground cartilage material, and performing constant temperature enzymolysis for 2-3 hours; after the enzymolysis is completed, heating to 95-100° C. to inactivate the enzyme, maintaining the temperature for 5-10 minutes, and then cooling to room temperature, filtering, and obtaining a filtrate; (4) The filtrate is first subjected to microfiltration; then adsorbed using a macroporous resin chromatography column, and eluted using a sodium chloride solution, and the eluate is collected; (5) concentrating the eluate under reduced pressure, adding ethanol to the obtained concentrate, allowing the concentrate to stand, and filtering to obtain a precipitate; (6) adding sodium chloride solution to the precipitate 1, mixing, subjecting the obtained mixed solution to enzymatic hydrolysis with chondroitin sulfate lyase, and then subjecting the obtained mixed solution to enzyme inactivation treatment, and filtering to obtain filtrate 2; subjecting the filtrate 2 to multiple microwave irradiation treatments, and simultaneously subjecting the filtrate 2 to ultrasonic treatment each time to obtain a treated solution; (7) ultrafiltration the treated liquid using an ultrafiltration membrane to obtain an ultrafiltrate; (8) adjusting the pH of the ultrafiltrate with hydrochloric acid, adding ethanol, stirring, letting stand, and then filtering to obtain a second precipitate; (9) Dehydrating the second precipitate with ethanol, filtering and drying to obtain the sodium chondroitin sulfate.

2. The preparation process of high-purity low molecular weight sodium chondroitin sulfate according to claim 1, characterized in that: In step (1), the pretreated cartilage raw material is obtained by the following method: washing the cartilage raw material, heating and boiling, removing the floating oil, taking out and washing, to obtain the pretreated cartilage raw material; The mesh size of the sieving is 60-100 mesh; During the liquid nitrogen immersion treatment, the immersion time is 20-40 minutes.

3. The preparation process of high-purity low molecular weight sodium chondroitin sulfate according to claim 1, characterized in that: In step (2), the amount of water added is 5-6 times the mass of the finely ground cartilage material; The soaking time is 3-5 hours, and the boiling time is 30-60 minutes; the amount of sodium chloride added is 2.5-4% of the mass of the cartilage fine powder.

4. The process for preparing high-purity low molecular weight sodium chondroitin sulfate according to claim 1, characterized in that: In step (4), the microfiltration is performed using a 0.2 μm microfiltration component; The macroporous resin in the macroporous resin chromatography column is macroporous resin D315; the usage amount of the macroporous resin is 2.5-3.5 times the mass of the cartilage fine powder; the mass fraction of the sodium chloride solution is 10-15%, the mass ratio of the sodium chloride solution to the macroporous resin is 3-4:1, and the elution time is 2.5-3.5h.

5. The process for preparing high-purity low molecular weight sodium chondroitin sulfate according to claim 1, characterized in that: Step (5) specifically comprises the following steps: concentrating the eluate under reduced pressure at 60-70° C. to make the obtained concentrate 30-35% of the volume of the eluate, then adding ethanol with a volume fraction of more than 95% in an amount of 8-10 times the volume of the concentrate, letting it stand for 3-5 hours, and then filtering to obtain a precipitate.

6. The process for preparing high-purity low molecular weight sodium chondroitin sulfate according to claim 1, characterized in that: In step (6), a sodium chloride solution in an amount 10-12 times the mass of the precipitate is added to the precipitate; the mass fraction of the sodium chloride solution is 2.5-3%; The obtained mixed solution is subjected to chondroitin sulfate lyase enzymatic hydrolysis, comprising the following steps: adjusting the pH value of the obtained mixed solution to 6.5-7.0 with hydrochloric acid, maintaining the temperature between 35-38° C., adding chondroitin sulfate lyase in an amount of 0.05-0.1% by weight of the cartilage fine powder, and performing enzymatic hydrolysis at a constant temperature for 2-3 hours; In step (6), the power of the microwave radiation treatment is 1000-1500W; the time of each microwave radiation treatment is 3-5min, and the interval time between two adjacent microwave radiation treatments is 1-2min; the number of microwave radiation treatments is 8-12 times; and ultrasonic treatment is performed simultaneously during each microwave radiation treatment, and the power of the ultrasonic treatment is 500-1000W.

7. The process for preparing high-purity low-molecular-weight sodium chondroitin sulfate according to any one of claims 1 to 6, characterized in that: In step (7), the molecular weight cut-off of the ultrafiltration membrane is 1000-5000Da.

8. The process for preparing high-purity low-molecular-weight sodium chondroitin sulfate according to any one of claims 1 to 6, characterized in that: In step (8), the ultrafiltrate is adjusted to a pH value of 5.8-6.0 with hydrochloric acid; ethanol is added so that the volume fraction of ethanol in the resulting solution is 85-90%, the stirring time is 1-1.5 hours, and the standing time is 7-10 hours; Step (9) specifically comprises the following steps: soaking the precipitate 2 in 92-95% ethanol by volume for 2 times, each time for 2-3 hours, filtering and then washing with anhydrous ethanol, and then vacuum drying at 50-55° C. to obtain sodium chondroitin sulfate.

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