Method for reducing viscosity of soluble non-denatured II-type collagen solution and improving dispersity of soluble non-denatured II-type collagen solution

Abstract

The invention discloses a method for reducing the viscosity of a soluble non-denatured II-type collagen solution and improving the dispersity of the soluble non-denatured II-type collagen solution. The viscosity of the soluble non-denatured II-type collagen solution is reduced by more than 75% and the dispersity is improved by more than 60% by utilizing the ion acting force of salt ions and the effect of causing isoelectric point deviation, the appearance of the solution is clearer and more transparent, and the processing and application of products in the field of functional foods are facilitated.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility. Background Art

[0002] Collagen is a fibrous protein characterized by its triple helix structure and is widely found in animals. Its core function is to maintain the elasticity and structural stability of tissues. It is an indispensable protein component in the connective tissue of vertebrates, supporting and protecting organs, mitigating impact, providing nutrition, and providing cell adhesion. So far, more than 29 types of collagen encoded by different genes have been found on the earth. Collagen is one of the most abundant proteins in organisms, and its classification is mainly based on the amino acid sequence and chain composition. Different types of collagen play different roles in organisms. Among them, type II collagen is mainly distributed in tissues such as cartilage and vitreous body. This specific distribution makes it play an important role in the structure and function of these tissues. At the same time, type II collagen, with its unique structural and functional characteristics, not only plays a key role in organisms, but also shows broad application prospects in the fields of medicine, food, cosmetics, etc.

[0003] Compared with traditional methods such as surgical treatment, non-steroidal anti-inflammatory drug treatment, Chinese medicine treatment, and functional foods such as glucosamine (1500 mg / d) and chondroitin sulfate (1200 mg / d), non-denatured type II collagen can achieve better therapeutic effects at a lower dose (40 mg / d) without side effects. Therefore, non-denatured type II collagen has shown significant biological therapeutic advantages in the prevention and treatment of osteoarthritis and has broad application prospects.

[0004] Soluble non-denatured type II collagen has the non-helical regions at both ends of the collagen chain removed by certain means (usually enzymatic hydrolysis). At this point, the collagen still maintains a complete triple helical structure, but the covalent cross-linking has been destroyed, prompting the release of collagen monomers from the collagen fibers and allowing them to be well dissolved or dispersed in a solution system under certain conditions. However, soluble non-denatured type II collagen has poor dispersibility in most solution environments. Even if it is well dispersed, its special molecular structure leads to extremely high viscosity, which affects its processing and application. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility.

[0006] The purpose of the present invention is achieved through the following technical solutions: A method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility comprises the following steps: (1) adding soluble non-denatured type II collagen to the solution and mixing to obtain a protein suspension; (2) adding chloride salt to water, mixing well, and cooling sufficiently to obtain a pre-cooled chloride salt solution; (3) Mixing the protein suspension obtained in step (1) with the pre-cooled chloride solution obtained in step (2), adjusting the pH, and obtaining a soluble non-denatured type II collagen solution.

[0007] The soluble non-denatured type II collagen described in step (1) is a protein of animal origin; preferably, it is a protein extracted from chicken sternal cartilage.

[0008] The solution in step (1) is an HCl solution; preferably a 0.01 mol / L HCl solution.

[0009] The concentration of soluble non-denatured type II collagen in the protein suspension of step (1) is 5 to 20 mg / mL.

[0010] The chloride salt in step (2) is at least one of NaCl, KCl, CaCl2, MgCl2 and NH4Cl; preferably CaCl2.

[0011] The concentration of the chloride solution in step (2) is 0.5 to 2.0 mol / L, preferably 1.0 mol / L.

[0012] The sufficient cooling in step (2) is cooling to 0-8°C, preferably cooling to 4°C.

[0013] The pH adjustment in step (3) is to adjust the pH to 3.5-7.0.

[0014] The final concentration of the soluble non-denatured type II collagen in the soluble non-denatured type II collagen solution in step (3) is 5 to 20 mg / mL, preferably 5 mg / mL.

[0015] The final concentration of chloride salt in the soluble non-denatured type II collagen solution in step (3) is 0.02-0.5 mol / L.

[0016] The above method for reducing the viscosity of the soluble non-denatured type II collagen solution and improving its dispersibility is used in processing the soluble non-denatured type II collagen.

[0017] Application of the above method for reducing the viscosity of soluble non-denatured type II collagen solution and improving its dispersibility in the preparation of food or health products.

[0018] Compared with the prior art, the present invention has the following advantages and effects: The invention discloses a method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility. The invention utilizes the ionic force of salt ions and the effect of causing isoelectric point shift to reduce the viscosity of the soluble non-denatured type II collagen solution by more than 75%, improve the dispersibility by more than 60%, and make the solution clearer and more transparent, which is beneficial to the processing and application of products in the field of functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the viscosity measurement results of soluble non-denatured type II collagen mixed with CaCl2 solution in the implementation of the present invention.

[0020] Figure 2 Schematic diagram of the dispersibility measurement results of soluble non-denatured type II collagen mixed with CaCl2 solution in the implementation of the present invention.

[0021] Figure 3 Schematic diagram of the appearance of soluble non-denatured type II collagen after mixing with CaCl2 solution in the implementation of the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0023] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0024] Example 1 A method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility comprises the following steps: (1) Preparation of soluble non-denatured type II collagen: Cut the chicken sternal cartilage into pieces, wash them thoroughly to remove the film and surface grease, add deionized water, homogenize them with a homogenizer, add NaCl solution to remove impurities, centrifuge to obtain the precipitate, and wash the salt solution remaining on the surface of the precipitate. Soak the precipitate in HCl solution, add pepsin for enzymatic hydrolysis, centrifuge to collect the supernatant, collect the precipitate at neutral low temperature and dialyze, freeze-dry and pulverize the dialyzed sample to obtain soluble non-denatured type II collagen powder (SCII).

[0025] (2) Dispersing the soluble non-denatured type II collagen prepared in step (1) at a concentration of 10 mg / mL in a 0.01 mol / L hydrochloric acid solution, stirring the solution with a magnetic stirrer at 4°C overnight, and adjusting the pH to 3.5; (3) Prepare 1.0 mol / L CaCl2 solution and cool it to 4°C; (4) The soluble non-denatured type II collagen suspension obtained in step (1) was fully mixed with the CaCl2 solution obtained in step (2), the final concentration of the non-denatured type II collagen was 5 mg / mL, the CaCl2 concentration was 0.12 mol / L, and the pH of the final solution was adjusted to 3.5 to obtain protein solution A.

[0026] The prepared protein sample solution was added to the HAAKE-MARS III rheometer to test its viscosity. The platform temperature was set to 25 °C, the sample volume was 1 mL, the equilibrium time was 2 min, and the sample was recorded from 1 to 150 s. -1 The viscosity curve of rate shear is taken as 30 s -1 The apparent viscosity was taken as the final measured value. At the same time, the sample solution was added to a UV-visible spectrophotometer to detect its absorbance at 313 nm, and then all samples were centrifuged at 8000 rpm for 15 min at 20°C to determine the protein content in the supernatant and calculate its dispersion degree. The calculation formula for dispersion was protein content in the supernatant / total protein content*100%.

[0027] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution A is 47.21 mPa, the dispersion is 79.16%, and the solution is clear and transparent without visible white precipitate, which proves that protein solution A achieves good solubility and dispersion.

[0028] Example 2 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the pH was adjusted to 5.5 in steps (2) and (4). Finally, a protein solution B was obtained and tested by the method of Example 1.

[0029] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution B is 34.04 mPa, the dispersion is 76.36%, and the solution is clear and transparent without visible white precipitate, which proves that protein solution B achieves good solubility and dispersion.

[0030] Example 3 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the pH was adjusted to 7.0 in steps (2) and (4), to obtain a protein solution C, which was then tested by the method of Example 1.

[0031] The experimental results are as follows Figures 1 to 3As shown, the viscosity of protein solution C is 39.46 mPa, the dispersion is 82.64%, and the solution is clear and transparent without visible white precipitate, which proves that protein solution C achieves good solubility and dispersion.

[0032] Comparative Example 1 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the pH was adjusted to 2.0 in steps (2) and (4), to obtain a protein solution I, which was then tested by the method of Example 1.

[0033] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution I is 191.75 mPa, the dispersion is 55.44%, the solution is turbid, and a white precipitate is obvious after standing, which proves that protein solution I cannot achieve good dissolution and dispersion.

[0034] Comparative Example 2 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the solution prepared in step (3) was not precooled, to obtain a protein solution II, which was then tested by the method of Example 1.

[0035] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution II is 41.00 mPa, the dispersion is 12.51%, the solution is turbid, and white precipitation is obvious after standing, which proves that protein solution II cannot achieve good dissolution and dispersion.

[0036] Comparative Example 3 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the pH was adjusted to 11.0 in steps (2) and (4), to obtain a protein solution III, which was then tested by the method of Example 1.

[0037] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution III is 133.04 mPa, the dispersion is 35.46%, the solution is turbid, and white precipitation is obvious after standing, which proves that protein solution III cannot achieve good dissolution and dispersion.

[0038] Comparative Example 4 Soluble non-denatured type II collagen was prepared by the method of Example 1 and mixed with a CaCl2 solution to prepare a protein solution, except that the concentration of CaCl2 in the solution obtained by mixing in step (4) was 0.8 mol / L, and a protein solution IV was obtained. The protein solution was then tested by the method of Example 1.

[0039] The experimental results are as follows Figures 1 to 3 As shown, the viscosity of protein solution IV is 62.44 mPa, the dispersion is 37.31%, the solution is turbid, and white precipitation is obvious after standing, which proves that protein solution IV cannot achieve good dissolution and dispersion.

[0040] Table 1 Test results of protein solutions prepared by different methods Group Appearance Viscosity Dispersion Example 1 clarify 47.21 mPa 79.16% Example 2 clarify 34.04 mPa 76.36% Example 3 clarify 39.46 mPa 82.64% Comparative Example 1 turbid 191.75 mPa 55.44% Comparative Example 2 turbid 41.00 mPa 12.51% Comparative Example 3 turbid 133.04 mPa 35.46% Comparative Example 4 turbid 62.44 mPa 37.31% The above experimental results show that the protein solutions prepared in Examples 1 to 3 achieve good dissolution, and the experimental data prove that their viscosity is significantly reduced, while the protein solutions prepared in the comparative examples all show that they cannot be completely dissolved, their viscosity is also high, and their dispersion is uneven. The protein solution prepared in the embodiment of the present invention has broad application prospects in the preparation of related food and drug fields, and does not require the introduction of toxic and harmful solvents, and is very suitable for the application of soluble non-denatured type II collagen in production and processing.

[0041] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility, characterized in that The steps include: (1) adding soluble non-denatured type II collagen to the solution and mixing to obtain a protein suspension; (2) adding chloride salt to water, mixing well, and cooling sufficiently to obtain a pre-cooled chloride salt solution; (3) Mixing the protein suspension obtained in step (1) with the pre-cooled chloride solution obtained in step (2), adjusting the pH, and obtaining a soluble non-denatured type II collagen solution.

2. The method according to claim 1, characterized in that: The solution in step (1) is HCl solution; The concentration of soluble non-denatured type II collagen in the protein suspension of step (1) is 5 to 20 mg / mL.

3. The method according to claim 1, characterized in that: The chloride salt in step (2) is at least one of NaCl, KCl, CaCl2, MgCl2 and NH4Cl.

4. The method according to claim 1, characterized in that: The concentration of the chloride solution in step (2) is 0.5 to 2.0 mol / L; The sufficient cooling in step (2) is cooling to 0-8°C.

5. The method according to claim 1, characterized in that: The pH adjustment in step (3) is to adjust the pH to 3.5-7.

0.

6. The method according to claim 1, characterized in that: The final concentration of the soluble non-denatured type II collagen in the soluble non-denatured type II collagen solution in step (3) is 5 to 20 mg / mL; The final concentration of chloride salt in the soluble non-denatured type II collagen solution in step (3) is 0.02-0.5 mol / L.

7. Use of the method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility according to any one of claims 1 to 6 in processing soluble non-denatured type II collagen.

8. Use of the method for reducing the viscosity of a soluble non-denatured type II collagen solution and improving its dispersibility according to any one of claims 1 to 6 in the preparation of food or health products.

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