Preparation method and application of casein rich in chondroitin sulfate

Abstract

The invention relates to a preparation method and application of casein rich in chondroitin sulfate, and belongs to the technical field of pharmacy. The invention provides a preparation method of casein rich in chondroitin sulfate, which comprises the following steps: (1) dialyzing a sodium caseinate solution, standing after dialyzing, centrifuging and drying to obtain a dry substance; (2) mixing the dried substance with sodium dodecyl sulfate, urea and a calcium chloride solution, dialyzing, and mixing with a chondroitin sulfate solution to obtain a mixed solution 1; and (3) mixing the mixed solution 1 with a cross-linking agent, stirring, dialyzing and drying to obtain the casein rich in chondroitin sulfate. According to the preparation method disclosed by the invention, the chondroitin sulfate and the casein form a stable combination through the cross-linking agent, so that the casein rich in chondroitin sulfate is obtained, the stability of the chondroitin sulfate can be enhanced, the casein can resist high temperature, and meanwhile, the casein is endowed with a special effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a preparation method and application of casein rich in chondroitin sulfate. Background Art

[0002] Chondroitin sulfate (CS), a glycosaminoglycan, is usually present in animal cartilage and is commonly used in health products or drugs. Its main application in medicine is as a drug for treating joint diseases, with the effects of relieving pain and promoting cartilage regeneration. Experimental results show that for patients with coronary atherosclerosis taking 2000 mg of chondroitin sulfate per day, the proportion of acute attacks is only 1.7% (1 / 60). For arteriosclerosis patients in the control group without taking chondroitin sulfate, the acute coronary attack rate is about 27% (16 / 60). In addition, for high-fat diet consumers taking chondroitin sulfate for a long time, the blood fat increase rate seems to be lower than that of the group without taking chondroitin sulfate, and it also has the effect of reducing thrombus formation. However, chondroitin sulfate has low stability after dissolving in water, and its chemical structure (sulfated polysaccharide) is prone to hydrolysis, oxidation or microbial contamination. For example, when pH < 4.0, sulfate groups are easily hydrolyzed and fall off, resulting in structural damage; when the temperature > 40 °C, the hydrolysis rate significantly accelerates and the viscosity decreases; the solubility decreases with high metal ion content, and metal ions such as Fe 3+ and Cu 2+ catalyze oxidation reactions, etc. This also leads to a reduction in the efficacy of orally administered chondroitin sulfate after the action of gastric acid. There is an urgent need to develop chondroitin sulfate with high stability and high activity.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of casein rich in chondroitin sulfate to solve the problems in the prior art that chondroitin sulfate has low stability after dissolving in water and is easily degraded and inactivated under the conditions of low pH, high temperature and high metal ion content.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of casein rich in chondroitin sulfate, comprising the following steps:

[0007] (1) Dialyze the sodium caseinate solution for 3.5 - 4.5 h, take the dialyzed sodium caseinate solution, let it stand for 36 - 60 h, centrifuge to obtain the supernatant, and dry to obtain a dry substance;

[0008] (2) Mix the dried substance obtained in step (1) with sodium dodecyl sulfate, urea, and calcium chloride solution to obtain a mixed solution. Dialyze the mixed solution for 3.5 - 4.5 h, and then mix the dialyzed mixed solution with chondroitin sulfate solution and stir for 55 - 65 min to obtain mixed solution 1;

[0009] (3) Mix the mixed solution 1 obtained in step (2) with a crosslinking agent and stir for 12 - 36 h to obtain a crude liquid. Dialyze the crude liquid for 12 - 36 h, and then dry the dialyzed solution to obtain chondroitin sulfate-rich casein.

[0010] Preferably, the initial concentration of the sodium caseinate solution in step (1) is 45 - 55 mg / mL;

[0011] The method for dialyzing the sodium caseinate solution is to place the sodium caseinate solution in a dialysis bag and place the dialysis bag in calcium chloride solution;

[0012] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa;

[0013] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL.

[0014] Preferably, the temperature for standing in step (1) is 3 - 5 °C;

[0015] The rotation speed for centrifugation is 8000 - 12000 rpm, and the centrifugation time is 5 - 15 min.

[0016] Preferably, the mass ratio of the dried substance to sodium dodecyl sulfate and urea in step (2) is 8 - 12:1 - 3:34 - 38;

[0017] The mass-to-volume ratio of the dried substance to calcium chloride solution is 8 - 12 g:0.8 - 1.2 L;

[0018] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL.

[0019] Preferably, the method for dialyzing the mixed solution in step (2) is to place the mixed solution in a dialysis bag and place the dialysis bag in pure water;

[0020] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa.

[0021] Preferably, the mass-to-volume ratio of the dried substance to chondroitin sulfate solution in step (2) is 8 - 12 g:80 - 120 mL.

[0022] Preferably, the preparation method of the chondroitin sulfate solution in step (2) is to mix chondroitin sulfate with dimethyl sulfoxide;

[0023] The mass ratio of the chondroitin sulfate to dimethyl sulfoxide in the mixture is 1 to 2:8 to 12.

[0024] Preferably, in step (3), the volume ratio of the mixed solution 1 to the crosslinking agent in the mixture is 90 to 110:1;

[0025] The preparation method of the crosslinking agent is to mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and water;

[0026] The mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the N-hydroxysuccinimide is 0.3734 to 0.3934:0.1051 to 0.1251;

[0027] The mass-volume ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the water is 0.3734 to 0.3934 g:0.5 to 1.5 mL.

[0028] Preferably, in step (3), the method for dialyzing the crude liquid is to place the crude liquid in a dialysis bag and place the dialysis bag in pure water;

[0029] The molecular weight cut-off of the dialysis bag is 48 to 52 KDa.

[0030] The present invention also provides the use of the chondroitin sulfate-rich casein prepared by the preparation method in the preparation of health products or drugs for treating joint diseases.

[0031] The present invention has the following technical effects and advantages:

[0032] Casein is the main protein in cow's milk and exists in the form of stable micelles in milk. It forms spherical micelles by α s1 -, α s2 -, β- and κ-casein through calcium phosphate. κ-casein covers the surface of the micelles and gives the micelles a negatively charged surface. α- and β-casein exist in the inner region of the micelles and form a hydrophobic core. The casein micelle structure is stable, so that milk can still remain stable under pasteurization or even ultra-high temperature (>130 °C) sterilization conditions.

[0033] The present invention provides a preparation method of chondroitin sulfate-rich casein, comprising the following steps: (1) Dialyzing a sodium caseinate solution, standing and centrifuging after dialysis, and drying to obtain a dry substance; (2) Mixing the dry substance with sodium dodecyl sulfate, urea, and calcium chloride solution, dialyzing and then mixing with chondroitin sulfate solution to obtain a mixed solution 1; (3) Mixing the mixed solution 1 with a cross-linking agent, stirring, dialyzing, and drying to obtain chondroitin sulfate-rich casein. According to the preparation method of the present invention, chondroitin sulfate and casein spherical micelles are combined through a cross-linking agent, so that chondroitin sulfate and casein spherical micelles form a stable conjugate, obtaining chondroitin sulfate-rich casein, which can enhance the stability of chondroitin sulfate, can withstand high temperatures, and endows casein with special effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 FIG. is a DSC analysis diagram of sodium caseinate, chondroitin sulfate, and chondroitin sulfate-rich casein prepared in Example 1;

[0035] Figure 2 FIG. shows the particle size of sodium caseinate and chondroitin sulfate-rich casein prepared in Example 1 in aqueous solutions with different pH values. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides a preparation method of chondroitin sulfate-rich casein, comprising the following steps:

[0037] (1) Dialyzing the sodium caseinate solution for 3.5 - 4.5 h, taking the dialyzed sodium caseinate solution, standing for 36 - 60 h, centrifuging to take the supernatant, and drying to obtain a dry substance;

[0038] The time for dialyzing the sodium caseinate solution is preferably 4 h;

[0039] The standing time is preferably 42 - 52 h, and more preferably 48 h;

[0040] (2) Mixing the dry substance obtained in step (1) with sodium dodecyl sulfate, urea, and calcium chloride solution to obtain a mixed solution, dialyzing the mixed solution for 3.5 - 4.5 h, mixing the dialyzed mixed solution with chondroitin sulfate solution, and stirring for 55 - 65 min to obtain a mixed solution 1;

[0041] The time for dialyzing the mixed solution is preferably 4 h;

[0042] The mixing of the dry substance obtained in step (1) with sodium dodecyl sulfate, urea, and calcium chloride solution is preferably: mixing the dry substance with calcium chloride solution, heating at 70 °C for 10 min, cooling to room temperature, and then mixing with urea and sodium dodecyl sulfate, and stirring for 30 min;

[0043] The mixing of the dialyzed mixed solution with the chondroitin sulfate solution and the stirring time are preferably 60 min;

[0044] (3) Mix the mixed solution 1 obtained in step (2) with a crosslinking agent, stir for 12 - 36 h to obtain a crude solution, dialyze the crude solution for 12 - 36 h, and dry the dialyzed solution to obtain chondroitin sulfate-rich casein;

[0045] The stirring time is preferably 18 - 28 h, more preferably 24 h;

[0046] The dialysis time of the crude solution is preferably 18 - 28 h, more preferably 24 h.

[0047] In the present invention, in step (1), the initial concentration of the sodium caseinate solution is 45 - 55 mg / mL, preferably 50 mg / mL;

[0048] The method for dialyzing the sodium caseinate solution is to place the sodium caseinate solution in a dialysis bag and place the dialysis bag in a calcium chloride solution;

[0049] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa, preferably 10 KDa;

[0050] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL, preferably 0.5 mmol / mL.

[0051] In the present invention, in step (1), the standing temperature is 3 - 5 °C, preferably 4 °C;

[0052] The rotation speed of the centrifugation is 8000 - 12000 rpm, preferably 10000 rpm, and the centrifugation time is 5 - 15 min, preferably 10 min.

[0053] In the present invention, in step (2), the mass ratio of the dried substance to sodium dodecyl sulfate and urea is 8 - 12:1 - 3:34 - 38, preferably 10:2:36;

[0054] The mass-to-volume ratio of the dried substance to the calcium chloride solution is 8 - 12 g:0.8 - 1.2 L, preferably 10 g:1 L;

[0055] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL, preferably 0.5 mmol / mL.

[0056] In the present invention, in step (2), the method for dialyzing the mixed solution is to place the mixed solution in a dialysis bag and place the dialysis bag in pure water;

[0057] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa, preferably 10 KDa.

[0058] In the present invention, the mass-volume ratio of the dry substance to the chondroitin sulfate solution in step (2) is 8-12 g: 80-120 mL, preferably 10 g: 100 mL.

[0059] In the present invention, the preparation method of the chondroitin sulfate solution in step (2) is to mix chondroitin sulfate with dimethyl sulfoxide;

[0060] The mass ratio of the chondroitin sulfate to dimethyl sulfoxide in the mixing is 1-2: 8-12, preferably 1: 10.

[0061] In the present invention, the volume ratio of the mixed solution 1 to the cross-linking agent in step (3) is 90-110: 1, preferably 100: 1;

[0062] The preparation method of the cross-linking agent is to mix 1-ethyl-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and water;

[0063] The mass ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide to the N-hydroxysuccinimide is 0.3734-0.3934: 0.1051-0.1251, preferably 0.3834: 0.1151;

[0064] The mass-volume ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide to water is 0.3734-0.3934 g: 0.5-1.5 mL, preferably 0.3834 g: 1 mL.

[0065] In the present invention, the method for dialyzing the crude liquid in step (3) is to place the crude liquid in a dialysis bag and place the dialysis bag in pure water;

[0066] The molecular weight cut-off of the dialysis bag is 48-52 KDa, preferably 50 KDa.

[0067] The present invention also provides the application of the chondroitin sulfate-rich casein prepared by the preparation method in the preparation of health products or drugs for treating joint diseases.

[0068] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0069] Example 1

[0070] Preparation of chondroitin sulfate solution: Mix 10 g of chondroitin sulfate with 100 g of dimethyl sulfoxide evenly to obtain a chondroitin sulfate solution for standby.

[0071] Preparation of crosslinking agent: Dissolve 0.3834 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.1151 g of N-hydroxysuccinimide in 1 mL of ultrapure water, stir evenly to obtain the crosslinking agent, and set aside.

[0072] A preparation method of chondroitin sulfate-rich casein, comprising the following steps:

[0073] (1) Place the sodium caseinate solution with an initial concentration of 50 mg / mL in a dialysis bag with a molecular weight cut-off of 10 KDa. After sealing both ends of the bag with clips, place it in a calcium chloride solution with an initial concentration of 0.5 mmol / mL for dialysis for 4 h. Take the dialyzed sodium caseinate solution, let it stand at 4 °C for 48 h, then centrifuge it at 10000 rpm for 10 min, collect the supernatant, and freeze-dry to obtain a dry substance;

[0074] (2) Take 10 g of the dry substance obtained in step (1) and dissolve it in 1 L of calcium chloride solution with an initial concentration of 0.5 mmol / mL. Heat it at 70 °C for 10 min, cool it to room temperature, add 2 g of sodium dodecyl sulfate and 36 g of urea, and stir magnetically at room temperature for 30 min to obtain a mixed solution. Place the mixed solution in a dialysis bag with a molecular weight cut-off of 10 KDa. After sealing both ends of the bag with clips, place it in ultrapure water for dialysis for 4 h. Take the dialyzed mixed solution and mix it with 100 mL of the prepared chondroitin sulfate solution, and stir at room temperature for 60 min to obtain mixed solution 1;

[0075] (3) Add 1 mL of the prepared crosslinking agent to 100 mL of mixed solution 1, stir at room temperature for 24 h to obtain a crude liquid. Place the crude liquid in a dialysis bag with a molecular weight cut-off of 50 KDa. After sealing both ends of the bag with clips, place it in ultrapure water for dialysis for 24 h, change the water 4 times during the period, and take the dialyzed solution and freeze-dry to obtain chondroitin sulfate-rich casein.

[0076] Example 2

[0077] A preparation method of chondroitin sulfate-rich casein, comprising the following steps:

[0078] (1) Place the sodium caseinate solution with an initial concentration of 47 mg / mL in a dialysis bag with a molecular weight cut-off of 10 KDa. After sealing both ends of the bag with clips, place it in a calcium chloride solution with an initial concentration of 0.7 mmol / mL for dialysis for 3.8 h. Take the dialyzed sodium caseinate solution, let it stand at 4 °C for 52 h, then centrifuge it at 10000 rpm for 10 min, collect the supernatant, and freeze-dry to obtain a dry substance;

[0079] (2) Dissolve 10 g of the dry substance obtained in step (1) in 1.2 L of a calcium chloride solution with an initial concentration of 0.5 mmol / mL, heat it at 70 °C for 10 min, add 2 g of sodium dodecyl sulfate and 35 g of urea after cooling to room temperature, stir magnetically at room temperature for 30 min to obtain a mixed solution. Place the mixed solution in a dialysis bag with a molecular weight cut-off of 10 KDa, seal both ends of the bag with clips, and then dialyze it in ultrapure water for 4 h. Take the dialyzed mixed solution and mix it with 100 mL of the chondroitin sulfate solution prepared in Example 1, and stir at room temperature for 60 min to obtain mixed solution 1;

[0080] (3) Add 1 mL of the cross-linking agent prepared in Example 1 to 100 mL of mixed solution 1, stir at room temperature for 26 h to obtain a crude liquid. Place the crude liquid in a dialysis bag with a molecular weight cut-off of 50 KDa, seal both ends of the bag with clips, and then dialyze it in ultrapure water for 22 h, changing the water 4 times during this period. Take the dialyzed solution and freeze-dry it to obtain chondroitin sulfate-rich casein.

[0081] Example 3

[0082] A preparation method of chondroitin sulfate-rich casein, comprising the following steps:

[0083] (1) Place a sodium caseinate solution with an initial concentration of 47 mg / mL in a dialysis bag with a molecular weight cut-off of 10 KDa, seal both ends of the bag with clips, and then dialyze it in a calcium chloride solution with an initial concentration of 0.7 mmol / mL for 3.6 h. Take the dialyzed sodium caseinate solution and let it stand at 4 °C for 48 h, then centrifuge it at 10000 rpm for 10 min, collect the supernatant, and freeze-dry it to obtain a dry substance;

[0084] (2) Dissolve 8 g of the dry substance obtained in step (1) in 1.2 L of a calcium chloride solution with an initial concentration of 0.5 mmol / mL, heat it at 70 °C for 10 min, add 2 g of sodium dodecyl sulfate and 38 g of urea after cooling to room temperature, stir magnetically at room temperature for 30 min to obtain a mixed solution. Place the mixed solution in a dialysis bag with a molecular weight cut-off of 10 KDa, seal both ends of the bag with clips, and then dialyze it in ultrapure water for 4 h. Take the dialyzed mixed solution and mix it with 90 mL of the chondroitin sulfate solution prepared in Example 1, and stir at room temperature for 60 min to obtain mixed solution 1;

[0085] (3) Add 1 mL of the cross-linking agent prepared in Example 1 to 95 mL of mixed solution 1, stir at room temperature for 24 h to obtain a crude liquid. Place the crude liquid in a dialysis bag with a molecular weight cut-off of 50 KDa, seal both ends of the bag with clips, and then dialyze it in ultrapure water for 26 h, changing the water 4 times during this period. Take the dialyzed solution and freeze-dry it to obtain chondroitin sulfate-rich casein.

[0086] Test Example 1

[0087] DSC analysis was performed on untreated sodium caseinate, chondroitin sulfate, and chondroitin sulfate-rich casein prepared in Example 1. The results are as Figure 1 shown. In the figure, the blue CS line represents chondroitin sulfate, the yellow CCM represents sodium caseinate, and the red CS+CCM represents chondroitin sulfate-rich casein prepared in Example 1. The abscissa represents temperature (°C), and the ordinate represents heat flow rate (mW).

[0088] According to Figure 1 it can be seen that sodium caseinate (CCM) shows a sharp heat absorption peak at 150.2 °C, indicating the degradation of casein; chondroitin sulfate (CS) shows a sharp heat absorption peak at 73 °C, indicating the degradation of chondroitin sulfate; chondroitin sulfate-rich casein (CS+CCM) prepared in Example 1 shows a weak endothermic peak at 76 °C and a sharp heat absorption peak at 140 °C - 150 °C, indicating that the thermal stability is significantly improved after chondroitin sulfate binds to casein.

[0089] Test Example 2

[0090] Raw sodium caseinate and chondroitin sulfate-rich casein prepared in Example 1 were respectively dissolved in water, and the pH value was adjusted to 6.7 with 1 mol / L NaOH and HCl solutions. The particle size was measured, and further the particle size of the chondroitin sulfate-rich casein aqueous solution was measured at pH values of 4.0 and 12 respectively. The results are as Figure 2 shown. In the figure, CCM represents sodium caseinate, and CCM-CS represents chondroitin sulfate-rich casein prepared in Example 1.

[0091] According to Figure 2 it can be seen that the particle size of chondroitin sulfate-rich casein prepared in Example 1 is larger than that of sodium caseinate, and its particle size does not change under different pH conditions, indicating that chondroitin sulfate-rich casein prepared in Example 1 has good stability under the conditions of pH 4.0 - 7.0.

[0092] As can be seen from the above embodiments, the present invention provides a preparation method of chondroitin sulfate-rich casein, comprising the following steps: (1) Dialyzing a sodium caseinate solution, standing, centrifuging after dialysis, and drying to obtain a dried substance; (2) Mixing the dried substance with sodium dodecyl sulfate, urea, and calcium chloride solution, dialyzing, and mixing with chondroitin sulfate solution after dialysis to obtain a mixed solution 1; (3) Mixing the mixed solution 1 with a crosslinking agent, stirring, dialyzing, and drying to obtain chondroitin sulfate-rich casein. According to the preparation method of the present invention, chondroitin sulfate and casein spherical micelles are combined through a crosslinking agent, so that chondroitin sulfate and casein spherical micelles form a stable combination, obtaining chondroitin sulfate-rich casein, which can enhance the stability of chondroitin sulfate, can withstand high temperatures, and endow casein with special effects at the same time.

[0093] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of chondroitin sulfate-rich casein, characterized in that, It includes the following steps: (1) Dialyze the sodium caseinate solution for 3.5 - 4.5 h, let the dialyzed sodium caseinate solution stand for 36 - 60 h, centrifuge to obtain the supernatant, and dry to obtain the dry matter; (2) Mix the dry matter obtained in step (1) with sodium dodecyl sulfate, urea, and calcium chloride solution to obtain a mixed solution, dialyze the mixed solution for 3.5 - 4.5 h, mix the dialyzed mixed solution with chondroitin sulfate solution, and stir for 55 - 65 min to obtain mixed solution 1; (3) Mix the mixed solution 1 obtained in step (2) with a crosslinking agent, stir for 12 - 36 h to obtain a crude liquid, dialyze the crude liquid for 12 - 36 h, and dry the dialyzed solution to obtain chondroitin sulfate - rich casein.

2. The preparation method according to claim 1, wherein, In step (1), the initial concentration of the sodium caseinate solution is 45 - 55 mg / mL; The method for dialyzing the sodium caseinate solution is to place the sodium caseinate solution in a dialysis bag and place the dialysis bag in calcium chloride solution; The molecular weight cut - off of the dialysis bag is 8 - 12 KDa; The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL.

3. The preparation method according to claim 1, characterized in that, In step (1), the temperature for standing is 3 - 5 °C; The rotation speed of the centrifuge is 8000 - 12000 rpm, and the centrifugation time is 5 - 15 min.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the dry matter to sodium dodecyl sulfate and urea is 8 - 12:1 - 3:34 - 38; The mass - to - volume ratio of the dry matter to calcium chloride solution is 8 - 12 g:0.8 - 1.2 L; The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL.

5. The preparation method according to claim 1, wherein In step (2), the method for dialyzing the mixed solution is to place the mixed solution in a dialysis bag and place the dialysis bag in pure water; The molecular weight cut - off of the dialysis bag is 8 - 12 KDa.

6. The preparation method according to claim 1, characterized in that, In step (2), the mass - to - volume ratio of the dry matter to chondroitin sulfate solution is 8 - 12 g:80 - 120 mL.

7. The preparation method according to claim 1, wherein In step (2), the preparation method of the chondroitin sulfate solution is to mix chondroitin sulfate with dimethyl sulfoxide; The mass ratio of chondroitin sulfate to dimethyl sulfoxide for mixing is 1 - 2:8 - 12.

8. The preparation method according to claim 1, wherein In step (3), the volume ratio of the mixed solution 1 to the crosslinking agent for mixing is 90 - 110:1; The preparation method of the crosslinking agent is to mix 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide, N - hydroxysuccinimide, and water; The mass ratio of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide to N - hydroxysuccinimide is 0.3734 - 0.3934:0.1051 - 0.1251; The mass - to - volume ratio of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide to water is 0.3734 - 0.3934 g:0.5 - 1.5 mL.

9. The preparation method according to claim 1, characterized in that, In step (3), the method for dialyzing the crude liquid is to place the crude liquid in a dialysis bag and place the dialysis bag in pure water; The molecular weight cut - off of the dialysis bag is 48 - 52 KDa.

10. Use of the chondroitin sulfate - rich casein prepared by the preparation method according to any one of claims 1 - 9 in the preparation of health products or drugs for treating joint diseases.

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