Preparation method and application of hyaluronic acid casein compound

Abstract

The invention relates to a preparation method and application of a hyaluronic acid casein compound, and belongs to the technical field of medicines. The invention provides a preparation method of a hyaluronic acid-casein compound, which comprises the following steps: (1) dialyzing a sodium caseinate solution, standing after dialyzing, centrifuging to take supernate, and drying to obtain a dry substance; (2) mixing the dried substance with a calcium chloride solution, then adding a purification reagent for reaction to obtain a reaction solution, dialyzing the reaction solution, then adding hyaluronic acid, and mixing to obtain a mixed solution 1; and (3) stirring the mixed solution 1 and a cross-linking agent, dialyzing and drying to obtain the hyaluronic acid casein compound. According to the hyaluronic acid-casein compound prepared by the method disclosed by the invention, the hyaluronic acid is loaded into casein micelles, so that the stability of the hyaluronic acid can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and particularly relates to a preparation method and application of a hyaluronic acid casein complex. Background Art

[0002] Hyaluronic acid is a biopolymer widely distributed in the human body, especially in the skin and connective tissues. It is a powerful moisturizer that can enhance the hydration ability of the skin, promote cell proliferation and differentiation, and play an important role in the inflammatory response. Hyaluronic acid is not only used in beauty skin care but also medically used to treat pathological conditions such as atherosclerosis and cancer. However, the poor stability of hyaluronic acid limits its application as a drug, and it is affected by many factors, including temperature, pH value, light, enzymatic hydrolysis, and interaction with other substances. For example, hyaluronic acid exhibits the best stability within the physiological pH range (7.2 - 7.4). Deviating from this range, whether in an acidic or alkaline environment, may lead to the destruction and degradation of the hyaluronic acid molecular structure. For example, in a high-temperature environment, the interaction between hyaluronic acid molecules weakens, changing the molecular structure and then triggering degradation. Research shows that when the temperature exceeds a certain threshold (usually above 60 °C), the degradation rate of hyaluronic acid will increase significantly. For example, light factors such as ultraviolet rays will also have an adverse effect on the stability of hyaluronic acid. Ultraviolet rays will cause hyaluronic acid to undergo a photochemical reaction, resulting in the cleavage and degradation of the molecular chain. Also, hyaluronidase is an enzyme that can degrade hyaluronic acid. In pathological conditions such as inflammation and tumors, the activity of hyaluronidase may increase, leading to a large amount of degradation of hyaluronic acid. In addition, some externally added enzymes may also have a degradation effect on hyaluronic acid. Also, when hyaluronic acid interacts with other substances, its stability may also be affected. For example, metal ions (such as copper, iron, etc.) can catalyze the oxidation reaction of hyaluronic acid, resulting in the cleavage and degradation of the molecular chain. In addition, some chemical substances (such as oxidants, reductants, etc.) will also affect the stability of hyaluronic acid. Therefore, how to improve the stability of hyaluronic acid and enhance its efficacy is an urgent problem to be solved.

[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 a hyaluronic acid casein complex to solve the problem of low stability of hyaluronic acid in the prior art.

[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 a hyaluronic acid casein complex, including 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 46 - 50 h, centrifuge it, take the supernatant, and dry it to obtain a dried substance.

[0008] (2) Mix the dried substance obtained in step (1) with a calcium chloride solution to obtain a mixed solution. Mix a purification reagent and the mixed solution, stir for 25 - 35 min to obtain a reaction solution. Dialyze the reaction solution for 3.5 - 4.5 h to obtain a dialyzed reaction solution. Mix hyaluronic acid with the dialyzed reaction solution, and stir for 55 - 65 min to obtain mixed solution 1.

[0009] (3) Mix mixed solution 1 with a crosslinking agent and stir for 18 - 22 h to obtain a crude liquid. Dialyze the crude liquid for 12 - 36 h, and take the dialyzed solution and dry it to obtain a hyaluronic acid - casein complex.

[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 a 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 6000 - 10000 rpm, and the centrifugation time is 15 - 25 min.

[0016] Preferably, the mass - to - volume ratio of the dried substance to the calcium chloride solution in step (2) is 8 - 12 g:1 L.

[0017] The temperature for mixing the dried substance with the calcium chloride solution is 65 - 75 °C, and the time is 8 - 12 min.

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

[0019] Preferably, the purification reagent in step (2) consists of urea and sodium dodecyl sulfate, and the mass ratio of urea to sodium dodecyl sulfate is 34 - 38:1 - 3.

[0020] The mass - to - volume ratio of the purification reagent to the mixed solution is 36 - 40 g:1 L.

[0021] Preferably, the method of dialyzing the reaction solution in step (2) is to place the reaction solution in a dialysis bag and place the dialysis bag in pure water;

[0022] The molecular weight cut-off of the dialysis bag is 8-12 KDa.

[0023] Preferably, the mass ratio of hyaluronic acid to dry matter in step (2) is 1-3:1.

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

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

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

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

[0028] Preferably, the method of 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;

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

[0030] The present invention provides a hyaluronic acid-casein complex prepared by the preparation method described above, and the hyaluronic acid-casein complex can improve the stability of hyaluronic acid.

[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 and can be used as a carrier to load corresponding substances.

[0033] The present invention provides a method for preparing a hyaluronic acid casein complex, comprising the following steps: (1) Dialyzing a sodium caseinate solution, standing the solution after dialysis, centrifuging to obtain the supernatant, and drying to obtain a dried substance; (2) Mixing the dried substance with a calcium chloride solution, then adding a purification reagent for reaction to obtain a reaction solution, dialyzing the reaction solution, and then adding hyaluronic acid to obtain a mixed solution 1; (3) Stirring, dialyzing, and drying the mixed solution 1 with a crosslinking agent to obtain the hyaluronic acid casein complex. By using the method of the present invention, through the dialysis reaction of the sodium caseinate solution with calcium chloride, and then adding the corresponding purification reagent, the casein is self-assembled to obtain casein micelles, and then hyaluronic acid is loaded into the casein micelles through the action of the crosslinking agent, improving the stability of hyaluronic acid. Detailed implementation mode

[0034] The present invention provides a method for preparing a hyaluronic acid casein complex, comprising the following steps:

[0035] (1) Dialyze the sodium caseinate solution for 3.5 - 4.5 h, take the dialyzed sodium caseinate solution, stand it for 46 - 50 h, centrifuge to obtain the supernatant, and dry to obtain a dried substance;

[0036] The time for dialyzing the sodium caseinate solution is preferably 4 h;

[0037] The standing time is preferably 48 h;

[0038] (2) Mix the dried substance obtained in step (1) with a calcium chloride solution to obtain a mixed solution, mix and stir the purification reagent and the mixed solution for 25 - 35 min to obtain a reaction solution, dialyze the reaction solution for 3.5 - 4.5 h to obtain a dialyzed reaction solution, mix hyaluronic acid with the dialyzed reaction solution, and stir for 55 - 65 min to obtain a mixed solution 1;

[0039] The time for mixing and stirring the purification reagent and the mixed solution is preferably 30 min;

[0040] The time for dialyzing the reaction solution is preferably 4 h;

[0041] The time for mixing and stirring hyaluronic acid with the dialyzed reaction solution is preferably 60 min;

[0042] (3) Mix and stir the mixed solution 1 with a crosslinking agent for 18 - 22 h to obtain a crude liquid, dialyze the crude liquid for 12 - 36 h, and take the dialyzed solution and dry it to obtain the hyaluronic acid casein complex;

[0043] The time for mixing and stirring the mixed solution 1 with the crosslinking agent is preferably 20 h.

[0044] In the present invention, the initial concentration of the sodium caseinate solution in step (1) is 45 - 55 mg / mL, preferably 50 mg / mL;

[0045] 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;

[0046] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa, preferably 10 KDa;

[0047] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL, preferably 0.5 mmol / mL.

[0048] In the present invention, the temperature for standing in step (1) is 3 - 5 °C, preferably 4 °C;

[0049] The rotation speed for centrifugation is 6000 - 10000 rpm, preferably 8000 rpm, and the centrifugation time is 15 - 25 min, preferably 20 min.

[0050] In the present invention, the mass-volume ratio of the dry matter to the calcium chloride solution in step (2) is 8 - 12 g:1 L, preferably 10 g:1 L;

[0051] The temperature for mixing the dry matter with the calcium chloride solution is 65 - 75 °C, preferably 70 °C, and the time is 8 - 12 min, preferably 10 min;

[0052] The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL, preferably 0.5 mmol / mL.

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

[0054] The mass-volume ratio of the purification reagent to the mixed solution is 36 - 40 g:1 L, preferably 38 g:1 L.

[0055] In the present invention, the method for dialyzing the reaction solution in step (2) is to place the reaction solution in a dialysis bag and place the dialysis bag in pure water;

[0056] The molecular weight cut-off of the dialysis bag is 8 - 12 KDa, preferably 10 KDa.

[0057] In the present invention, the mass ratio of hyaluronic acid to the dry matter in step (2) is 1 - 3:1, preferably 2:1.

[0058] In the present invention, the volume ratio of the mixed solution 1 to the crosslinking agent in step (3) is 90 to 110:1, preferably 100:1;

[0059] The preparation method of the crosslinking agent is to mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and water;

[0060] 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, preferably 0.3834:0.1151;

[0061] The mass-to-volume ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to water is 0.3734 to 0.3934 g:0.5 to 1.5 mL, preferably 0.3834 g:1 mL.

[0062] 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;

[0063] The molecular weight cut-off of the dialysis bag is 48 to 52 KDa, preferably 50 KDa.

[0064] The present invention provides a hyaluronic acid casein complex prepared by the described preparation method, and the hyaluronic acid casein complex can improve the stability of hyaluronic acid.

[0065] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Example 1

[0067] 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.

[0068] Preparation of purification reagent: Mix 36 g of urea and 2 g of sodium dodecyl sulfonate evenly.

[0069] A preparation method of a hyaluronic acid casein complex, comprising the following steps:

[0070] (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 and let it stand at 4 °C for 48 h, then centrifuge it at 8000 rpm for 20 min. Collect the supernatant and freeze-dry it to obtain a dry substance;

[0071] (2) Take 10 g of the dry substance obtained in step (1) and dissolve it in 1 L of a calcium chloride solution with an initial concentration of 0.5 mmol / mL. Mix it at 70 °C for 10 min. After cooling to room temperature, add 38 g of the prepared purification reagent and stir it magnetically at room temperature for 30 min to obtain a reaction solution. Place the reaction 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 20 g of hyaluronic acid, and stir it at room temperature for 60 min to obtain mixed solution 1;

[0072] (3) Add 1 mL of the prepared cross-linking agent to 100 mL of mixed solution 1 and stir it at room temperature for 20 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, changing the water 4 times during this period. Take the dialyzed solution and freeze-dry it to obtain the hyaluronic acid casein complex.

[0073] Example 2

[0074] A method for preparing a hyaluronic acid casein complex, comprising the following steps:

[0075] (1) Place the sodium caseinate solution with an initial concentration of 48 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 0.4 mmol / mL for dialysis for 4 h. Take the dialyzed sodium caseinate solution and let it stand at 3 °C for 48 h, then centrifuge it at 10000 rpm for 15 min. Collect the supernatant and freeze-dry it to obtain a dry substance;

[0076] (2) Take 11 g of the dry substance obtained in step (1) and dissolve it in 1 L of a calcium chloride solution with an initial concentration of 0.5 mmol / mL. Mix it at 70 °C for 10 min. After cooling to room temperature, add 40 g of the purification reagent prepared in Example 1 and stir it magnetically at room temperature for 30 min to obtain a reaction solution. Place the reaction 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 22 g of hyaluronic acid, and stir it at room temperature for 60 min to obtain mixed solution 1;

[0077] (3) Add 1 mL of the crosslinking agent prepared in Example 1 to 90 mL of the mixed solution 1, stir at room temperature for 20 h to obtain a crude solution. Place the crude solution 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 24 h, changing the water 4 times during this period. Take the dialyzed solution and freeze-dry it to obtain the hyaluronic acid casein complex.

[0078] Example 3

[0079] A method for preparing a hyaluronic acid casein complex, comprising the following steps:

[0080] (1) Place the sodium caseinate solution with an initial concentration of 55 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 0.5 mmol / mL for 4 h. Take the dialyzed sodium caseinate solution, let it stand at 5 °C for 48 h, then centrifuge it at 6000 rpm for 25 min, collect the supernatant, and freeze-dry it to obtain a dry substance.

[0081] (2) Take 12 g of the dry substance obtained in step (1) and dissolve it in 1 L of a calcium chloride solution with an initial concentration of 0.5 mmol / mL, mix at 70 °C for 10 min, cool to room temperature, add 38 g of the purification reagent prepared in Example 1, and magnetically stir at room temperature for 30 min to obtain a reaction solution. Place the reaction 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 24 g of hyaluronic acid, stir at room temperature for 60 min to obtain mixed solution 1.

[0082] (3) Add 1 mL of the prepared crosslinking agent to 110 mL of the mixed solution 1, stir at room temperature for 20 h to obtain a crude solution. Place the crude solution 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 24 h, changing the water 4 times during this period. Take the dialyzed solution and freeze-dry it to obtain the hyaluronic acid casein complex.

[0083] Test Example 1: Determination of particle size and potential

[0084] Dissolve 1 g of casein and the hyaluronic acid casein complex prepared in Example 1 in 1 L of ultrapure water respectively and mix well to obtain an aqueous sodium caseinate solution and an aqueous hyaluronic acid casein complex solution with a concentration of 1 g / L. Use NANO ZS90 to measure the particle size and Zeta potential of the solution by dynamic light scattering (DLS) technology, and repeat it three times. The results are shown in Table 1.

[0085] Table 1 Particle size and Zeta potential of different substances

[0086] Name of the substance Particle size (nm) Zeta potential (mV) Casein 386.2 -36.24 Casein-hyaluronic acid complex 184.2 -44.25

[0087] As can be seen from Table 1, the particle size of the hyaluronic acid-casein complex is 184.2 nm, which is smaller than that of casein, indicating that the method of the present application successfully enables casein to self-assemble into casein micelles. By comparing the Zeta potential, it can be known that the potential of the hyaluronic acid-casein complex has changed, indicating that hyaluronic acid has been successfully loaded onto casein.

[0088] Test Example 2: Stability determination

[0089] The hyaluronic acid-casein complex prepared in Example 1 was dissolved in 1 L of ultrapure water and mixed evenly to obtain an aqueous solution of hyaluronic acid with a concentration of 1 g / L and an aqueous solution of the hyaluronic acid-casein complex, respectively.

[0090] 1. Temperature stability test

[0091] The aqueous solution of the hyaluronic acid-casein complex was placed in environments with temperatures of 18 °C, 25 °C, 37 °C, and 60 °C, and samples were taken every 7 days for 3 times.

[0092] 2. pH stability test

[0093] The pH of the aqueous solution of the hyaluronic acid-casein complex was adjusted to 3, 7, and 10 using NaOH or HCl, and it was placed at 25 °C. Samples were taken every 7 days for 3 times.

[0094] The particle size of the particles in the samples under different temperature and pH treatments was measured. The results showed that the particle size of the hyaluronic acid-casein complex did not change significantly under different temperature and pH conditions, indicating that the hyaluronic acid-casein complex prepared by the method of the present application has good stability under different temperature and pH conditions.

[0095] As can be seen from the above examples, the present invention provides a method for preparing a hyaluronic acid-casein complex, which includes the following steps: (1) Dialyze the sodium caseinate solution, let it stand still after dialysis, centrifuge to take the supernatant, and dry to obtain a dry substance; (2) Mix the dry substance with a calcium chloride solution, then add a purification reagent for reaction to obtain a reaction solution, dialyze the reaction solution, and then add hyaluronic acid to obtain a mixed solution 1; (3) Stir, dialyze, and dry the mixed solution 1 with a crosslinking agent to obtain the hyaluronic acid-casein complex. By using the method of the present invention, through the dialysis reaction of the sodium caseinate solution with calcium chloride, and then adding the corresponding purification reagent, casein is self-assembled to obtain casein micelles, and then hyaluronic acid is loaded into the casein micelles through the action of the crosslinking agent, improving the stability of hyaluronic acid.

[0096] 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 refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a hyaluronic acid casein complex, characterized in that, It includes the following steps: (1) Dialyze the sodium caseinate solution for 3.5 - 4.5 h. Take the dialyzed sodium caseinate solution, let it stand for 46 - 50 h, centrifuge to obtain the supernatant, and dry to obtain the dried substance; (2) Mix the dried substance obtained in step (1) with the calcium chloride solution to obtain a mixed solution. Mix and stir the purification reagent and the mixed solution for 25 - 35 min to obtain a reaction solution. Dialyze the reaction solution for 3.5 - 4.5 h to obtain the dialyzed reaction solution. Mix hyaluronic acid with the dialyzed reaction solution and stir for 55 - 65 min to obtain mixed solution 1; (3) Mix and stir mixed solution 1 with the cross - linker for 18 - 22 h to obtain a crude solution. Dialyze the crude solution for 12 - 36 h, and take the dialyzed solution and dry it to obtain the hyaluronic acid - casein complex.

2. The preparation method according to claim 1, characterized in that, 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 the 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 centrifugation is 6000 - 10000 rpm, and the centrifugation time is 15 - 25 min.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass - to - volume ratio of the dried substance to the calcium chloride solution for mixing is 8 - 12 g:1 L; The temperature for mixing the dried substance with the calcium chloride solution is 65 - 75 °C, and the time is 8 - 12 min; The initial concentration of the calcium chloride solution is 0.3 - 0.7 mmol / mL.

5. The preparation method according to claim 1, characterized in that, In step (2), the purification reagent consists of urea and sodium dodecyl sulfate, and the mass ratio of urea to sodium dodecyl sulfate is 34 - 38:1 - 3; The mass - to - volume ratio of the purification reagent to the mixed solution for mixing is 36 - 40 g:1 L.

6. The preparation method according to claim 1, wherein, In step (2), the method for dialyzing the reaction solution is to place the reaction 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.

7. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of hyaluronic acid to the dried substance is 1 - 3:

1.

8. The preparation method according to claim 1, characterized in that, In step (3), the volume ratio of mixed solution 1 to the cross - linker for mixing is 90 - 110:1; The preparation method of the cross - linker 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 solution is to place the crude solution 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. The hyaluronic acid casein complex prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The hyaluronic acid - casein complex can improve the stability of hyaluronic acid.