Collagen redissolving process

Abstract

The invention relates to the technical field of biomedical materials, in particular to a collagen redissolving process which comprises the following steps: S1, preparing a hydrochloric acid solution with the concentration of 0.001 mol / L to 0.02 mol / L; and S2, adding collagen filaments into the hydrochloric acid solution at 2-8 DEG C, and stirring to obtain the collagen solution. According to the collagen redissolution process provided by the invention, the low-concentration hydrochloric acid is adopted for dissolution under a low-temperature condition, so that acetic acid can be prevented from being remained in a collagen freeze-dried product in the redissolution process, and meanwhile, the collagen redissolution process has the advantages of high dissolution efficiency, complete collagen structure and simple and convenient process.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a collagen reconstitution process. Background Technology

[0002] Collagen, as a major biomedical material and tissue engineering scaffold material, is widely used due to its excellent biocompatibility, low immunogenicity, and biodegradability. In practical applications, to facilitate storage, transportation, and maintain its bioactivity, collagen is usually processed into solid formulations such as porous sponges, lyophilized filaments, or membranes. Before use, these solid formulations need to be reconstituted to restore them to a flowable collagen solution for subsequent applications such as injection, filling, or as cell culture scaffolds.

[0003] Currently, the most common method for resolvating solid collagen preparations (especially collagen fibers) is to use an acetic acid solvent system. However, acetic acid has inherent drawbacks such as high volatility and a strong, pungent odor. Even after subsequent vacuum freeze-drying, trace amounts of acetic acid can still remain in the freeze-dried collagen products, resulting in a noticeable pungent acetic acid odor. This odor not only severely degrades the sensory quality of the product but may also cause skin discomfort and mucous membrane irritation in users in scenarios such as direct skin contact with cosmetics and medical implants. Furthermore, during long-term storage, trace amounts of acetic acid residue may slowly disrupt the intermolecular forces of collagen, affecting its physicochemical properties and biological activity, thus limiting the product's application range and storage period. Summary of the Invention

[0004] To address the problem of noticeable odor caused by acetic acid residue in existing freeze-dried collagen products, this invention provides a collagen resolution process. This process uses low-concentration hydrochloric acid as the resolution solvent for collagen fibers. By precisely controlling the hydrochloric acid concentration and the resolution process, the solvent is completely removed, significantly reducing the product's odor while maintaining the natural triple helix structure, thermal stability, and bioactivity of collagen. This solves the problem of noticeable odor caused by acetic acid residue in existing freeze-dried collagen products.

[0005] The technical solution adopted by this invention to solve its technical problem is: A collagen reconstitution process includes the following steps: S1: Prepare a hydrochloric acid solution with a concentration of 0.001 mol / L-0.02 mol / L; S2: At 2-8℃, collagen filaments are added to the hydrochloric acid solution and stirred to obtain a collagen solution.

[0006] Optionally, the ratio of the collagen filaments to the hydrochloric acid solution is 10g:(100-200)mL.

[0007] Optionally, it also includes: S3: Dialyze and desalt the collagen solution.

[0008] Optionally, in step S3, the collagen solution is dialyzed and desalted using hydrochloric acid solution as the dialysate.

[0009] Optionally, the concentration of the hydrochloric acid solution used for the dialysate in step S3 is 0.001 mol / L-0.01 mol / L.

[0010] Optionally, the dialysis desalination is gradient dialysis desalination.

[0011] Optionally, the gradient dialysis desalination process includes: first performing dialysis with a hydrochloric acid solution with a concentration of 0.005 mol / L to 0.01 mol / L, and then performing dialysis with a hydrochloric acid solution with a concentration of 0.001 mol / L to 0.005 mol / L.

[0012] Optionally, the collagen filaments are prepared using pigskin as raw material.

[0013] The beneficial effects of this invention are: The collagen resolution process provided by this invention uses low-concentration hydrochloric acid to dissolve the collagen at low temperature, which can avoid residual acetic acid in the lyophilized collagen product during the resolution process. It also has the advantages of high dissolution efficiency, preservation of collagen structure, and simple process. Attached Figure Description

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] Figure 1 These are SDS-PAGE gel electrophoresis images of the lyophilized porcine type I collagen products prepared in the various embodiments and comparative examples of this invention. Figure 2 This is an HPLC chromatogram of the lyophilized porcine type I collagen products prepared in Example 2, Comparative Example 1, and Comparative Example 2 of the present invention. Detailed Implementation

[0016] The present invention will now be described in further detail. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0017] To address the problem of noticeable off-odors caused by acetic acid residue in existing freeze-dried collagen products, this invention provides a collagen reconstitution process, which includes the following steps: S1: Prepare a hydrochloric acid solution with a concentration of 0.001 mol / L-0.02 mol / L; Hydrochloric acid solutions within this concentration range can provide an appropriate amount of protons to disrupt the non-covalent interactions between collagen fibers, while avoiding the hydrolysis of collagen peptide bonds by excessive acidity. This ensures maximum protection of the natural structure of collagen while maintaining dissolution efficiency. It can effectively disrupt the intermolecular forces of collagen filaments and promote their dissolution, while also preventing the denaturation of collagen molecules due to excessive acid concentration. S2: At 2-8℃, collagen filaments are added to hydrochloric acid solution and stirred to obtain collagen solution; This low-temperature environment further inhibits the hydrolysis of collagen under acidic conditions, ensuring the molecular weight distribution and bioactivity of the reconstituted collagen.

[0018] The collagen resolution process provided by this invention uses low-concentration hydrochloric acid to dissolve the collagen at low temperature, which can avoid residual acetic acid in the lyophilized collagen product during the resolution process. It also has the advantages of high dissolution efficiency, preservation of collagen structure, and simple process.

[0019] Furthermore, the preferred ratio of collagen filaments to hydrochloric acid solution in this invention is 10g:(100-200)mL; this ratio range ensures that the collagen filaments are in full contact with the hydrochloric acid solution, improves the resolution efficiency, and avoids insufficient resolution due to excessive collagen filaments or excessive desalination load due to excessive hydrochloric acid solution.

[0020] To improve the purity of the collagen solution, the present invention preferably further includes the following steps in the resolvation process of the collagen: S3: Dialyze and desalt the collagen solution.

[0021] The dialysis desalting step can effectively remove free acetic acid and chloride ions from the collagen solution, providing a high-purity collagen solution for subsequent applications or freeze-drying.

[0022] In a further preferred embodiment of the present invention, in step S3, hydrochloric acid solution is used as the dialysis fluid to desalinate the collagen solution. By using hydrochloric acid solution as the dialysis fluid, both the dissolving solvent and the dialysis fluid are hydrochloric acid, which avoids the introduction of new impurities due to changing the type of acid, simplifies the subsequent impurity control, and improves the purity of the final product.

[0023] In a further preferred embodiment of the present invention, the concentration of the hydrochloric acid solution used in the dialysis fluid in step S3 is 0.001 mol / L-0.01 mol / L. This concentration range is consistent with the concentration of hydrochloric acid used for dissolution, which can maintain the acidic environment of collagen during the dialysis process, prevent collagen from precipitating when it is close to the isoelectric point, and at the same time use the concentration gradient to drive chloride ions to diffuse outward, thereby achieving efficient desalting.

[0024] Furthermore, the present invention preferably employs gradient dialysis desalination to facilitate the gradual diffusion of chloride ions by progressively reducing the concentration of the dialysate. This avoids drastic changes in osmotic pressure and localized collagen precipitation caused by excessive concentration differences between the internal and external solutions, thereby improving the stability of the desalination process and the collagen recovery rate.

[0025] Specifically, the preferred gradient dialysis desalination process of the present invention includes: first dialysis using a hydrochloric acid solution with a concentration of 0.005 mol / L to 0.01 mol / L, and then dialysis using a hydrochloric acid solution with a concentration of 0.001 mol / L to 0.005 mol / L.

[0026] In this two-stage gradient dialysis desalting process, the first stage uses a higher concentration of dialysate to rapidly replace free chloride ions and acetic acid molecules while ensuring dissolution. The second stage uses a lower concentration of dialysate to achieve fine desalting, maximizing the freeze-drying effect and protecting the structural integrity of collagen while ensuring desalting efficiency.

[0027] The present invention further preferably uses pigskin as raw material to prepare collagen filaments.

[0028] Pigskin is widely available and inexpensive, and its collagen composition is highly similar to human collagen, exhibiting good biocompatibility. Collagen filaments prepared from pigskin, after being processed using the reconstitution process of this invention, can yield a high-purity, highly active collagen solution suitable for medical and cosmetic applications.

[0029] In summary, this invention uses low-concentration hydrochloric acid instead of acetic acid as the resolution solvent for collagen fibers. By precisely controlling the hydrochloric acid concentration and resolution process, the solvent is completely removed, the product odor is significantly reduced, and the natural triple helix structure, thermal stability and bioactivity of collagen are maintained. Moreover, the process is compatible with existing industrial freeze-drying processes, with low modification costs and easy industrialization.

[0030] This invention provides suitable conditions for the resolution of collagen fibers by controlling the concentration of hydrochloric acid solution, the resolution temperature, and the ratio of collagen fibers to hydrochloric acid solution, effectively improving the resolution efficiency and shortening the resolution time. The low-temperature resolution environment can effectively avoid the denaturation of collagen molecules due to heat, and the gradient dialysis desalination method can avoid the aggregation and precipitation of collagen molecules caused by sudden changes in osmotic pressure, thus ensuring the stability and bioactivity of the collagen solution. Using a gradient concentration of hydrochloric acid solution for dialysis desalination can gradually reduce the salt concentration in the solution, improve the desalination rate, and obtain a high-purity collagen solution.

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Example 1

[0032] Preparation of collagen filaments: Take fresh pig skin, clean and remove hair, cut into 2×2cm pieces, and perform deashing, defatting, enzymatic hydrolysis, salt precipitation, and centrifugation to obtain purified porcine type I collagen filaments.

[0033] Salting out and redissolving: At 4℃, add 10g of collagen fibers to 100mL of 0.001M hydrochloric acid solution and stir magnetically at 200r / min for 30min until completely dissolved to form a collagen solution.

[0034] Solution pretreatment: The collagen solution is subjected to... The aqueous filter membrane was pressurized at 0.1 MPa to obtain the filtrate.

[0035] Dialysis desalination: The filtrate was placed in a dialysis bag with a molecular weight cutoff of 50 kDa and subjected to gradient dialysis desalination at 4°C. First, a 0.008 mol / L hydrochloric acid solution (20 times the volume of the solution) was used as the dialysis fluid, and dialysis was performed for 2 hours, with the fluid changed once. Then, a 0.002 mol / L hydrochloric acid solution was used as the dialysis fluid, and dialysis was performed for 4 hours, with the fluid changed twice.

[0036] Lyophilization: The dialyzed collagen solution was transferred to a lyophilization bottle, then placed in a vacuum freeze dryer at a vacuum level of 10 Pa, and lyophilized using a gradient temperature increase. The gradient temperature lyophilization process is as follows: Pre-freezing stage: Lower the shelf temperature to -45℃ and keep it warm for 2 hours to completely freeze the sample; First drying stage (sublimation drying): The shelf temperature is raised to -25℃ at a rate of 0.5℃ / min and kept at this temperature for 12 hours under a vacuum of ≤15Pa to remove free water; Secondary drying stage (analytical drying): The shelf temperature is raised to 0℃ at a rate of 0.3℃ / min and held for 6 hours; then raised to 25℃ at a rate of 0.2℃ / min and held for 4 hours to remove bound water; Final drying: Continue drying at 25°C for 2 hours to ensure residual moisture content is below 3%.

[0037] Finished product testing and storage: The product is odorless, has an intact triple helix structure, a thermal stability melting point of 44.3℃, no solvent residue, and a cell survival rate of 134.89% in 100% extract. It should be stored at 4℃ in a cool, dark, and sealed container. Example 2

[0038] The difference between this embodiment and Example 1 is that the salting-out and resolution steps are as follows: At 4°C, 10g of collagen fibers are added to 150mL of 0.01M hydrochloric acid solution and magnetically stirred at 150r / min for 45min until completely dissolved to form a collagen solution.

[0039] Finished product test results: odorless, intact triple helix structure, thermal stability melting point 43.3℃, ​​no solvent residue, purity 99.99%, cell survival rate of 100% extract 130.51%, fast resolvation speed, and no flocculent precipitate. Example 3

[0040] The difference between this embodiment and Example 1 is that the salting-out and resolution steps are as follows: At 4°C, 10g of collagen fibers are added to 200mL of 0.02M hydrochloric acid solution and magnetically stirred at 100r / min for 60min until completely dissolved to form a collagen solution.

[0041] Finished product test results: odorless, intact triple helix structure, thermal stability melting point 43.1℃, no solvent residue, cell survival rate of 128.46% in 100% extract, and fast reconstitution speed. Example 4

[0042] The difference between this embodiment and Embodiment 1 is that the concentration of hydrochloric acid in the salting-out and redissolution step is 0.015M.

[0043] Finished product test results: odorless, intact triple helix structure, thermal stability melting point 44.7℃, no solvent residue, and cell survival rate of 129.41% in 100% extract. Example 5

[0044] The difference between this embodiment and Embodiment 1 is that the concentration of hydrochloric acid in the salting-out and redissolution step is 0.02M.

[0045] Finished product test results: odorless, intact triple helix structure, thermal stability melting point 44.6℃, no solvent residue, and cell viability of 129.29% in 100% extract.

[0046] Comparative Example 1 The difference between this comparative example and Example 1 is that the hydrochloric acid solution in the salting-out and redissolution step is replaced with a 0.1M acetic acid solution.

[0047] Finished product test results: It has a distinct pungent odor of acetic acid, the triple helix structure is intact, the thermal stability melting point is 41.7℃, HPLC detection shows a solvent peak with a peak area of ​​3.71, the purity is 96.33%, and the cell viability of 100% extract is 104.87%.

[0048] Comparative Example 2 The difference between this comparative example and Example 1 is that the hydrochloric acid solution in the salting-out and resolution step is replaced with a 20mM sodium dihydrogen phosphate solution with a pH of 3.0.

[0049] Finished product test results: No residual sour taste, intact triple helix structure, thermal stability melting point 43.5℃, solvent peak detected by HPLC with a solvent peak area of ​​2.79, purity 95.78%, and cell viability of 100% extract 113.75%.

[0050] The methods for testing the finished products in the embodiments and comparative examples of this invention are as follows: I. SDS-PAGE gel electrophoresis for detecting the integrity of the triple helix structure Experimental procedure: Take the lyophilized products of Examples 1-5 and Comparative Examples 1-2 respectively, reconstitute them with 0.01M hydrochloric acid solution to a concentration of 1 mg / mL, add 5× loading buffer, heat in a boiling water bath for 5 min, cool and load the samples; prepare 6% separating gel and 5% stacking gel, electrophoresis at constant voltage of 80V for 30 min, then at constant voltage of 120V for 90 min; after electrophoresis, stain with Coomassie Brilliant Blue R-250 for 2 h, destain with destaining solution until the bands are clear, and take pictures for analysis using a gel imaging system.

[0051] SDS-PAGE gel electrophoresis images of the lyophilized porcine type I collagen products prepared in Examples 1-5 and Comparative Examples 1-2 of this invention are shown below. Figure 1 As shown; where lane 1: protein marker; lane 2: Example 1; lane 3: Example 2; lane 4: Example 3; lane 5: Example 4; lane 6: Example 5; lane 7: Comparative Example 1; lane 8: Comparative Example 2.

[0052] Experimental results showed that the freeze-dried products of Examples 1-5 and Comparative Examples 1-2 all exhibited characteristic bands of porcine type I collagen. Chain ~130kDa, The clear, undegraded, and unmixed bands (up to 110 kDa) indicate that the freeze-dried product prepared by this invention has a complete triple helix structure and is not significantly different from the product prepared by the acetic acid method.

[0053] II. Differential Scanning Calorimetry (DSC) for Determining Thermal Stability Experimental procedure: Take about 5 mg of the lyophilized products of Examples 1-5 and Comparative Examples 1-2 respectively, and place them in an aluminum sample pan, with an empty aluminum pan as a reference; set the differential scanning calorimeter to a heating rate of 10℃ / min, a heating range of 20-80℃, a nitrogen atmosphere, and a flow rate of 20 mL / min, record the heat flow-temperature curve of the sample, and determine the melting point (peak temperature).

[0054] The results of differential scanning calorimetry (DSC) measurements are shown in Table 1: Table 1 Experimental results show that the melting points of the freeze-dried products of Examples 1-5 are 43.1-44.7℃, with an average of 43.9℃; the melting point of Comparative Example 1 is 41.7℃ and the melting point of Comparative Example 2 is 43.5℃, indicating that the freeze-dried products prepared by the present invention have better thermal stability than the products prepared by the acetic acid method, and are comparable to those prepared by the phosphate buffer method with a slight improvement.

[0055] Cytotoxicity assay Cytotoxicity assay: L929 mouse fibroblasts were used. The gel samples from Examples 1-5 and Comparative Examples 1-2 were extracted with MEM + 10% FBS complete medium at a ratio of 0.2 g / mL. L929 cells in the logarithmic growth phase were then... Inoculate one well per well in a 96-well plate. After culturing for 24 hours, the supernatant was discarded, and different concentrations of extraction solution were added to each well. A medium control group (culture medium only) was set up, with 6 replicates per group; after 24 hours of incubation, the medium was added to each well. The reagents were removed, and incubation continued for 2 hours. The plate was read using a microplate reader at a measurement wavelength of 570 nm and a reference wavelength of 650 nm. Data (OD570 = OD570 only - OD650 only) were rounded to three decimal places. The calculation formula is as follows: Cell viability (%) = 100 × (mean OD570 absorbance of treated samples) / (mean OD570 absorbance of medium).

[0056] The results of the cell proliferation experiment are shown in Table 2.

[0057] Table 2 Experimental results showed that the cell viability of the 100% extract in Examples 1-5 was 128.46%-134.89%, and the cell viability of the 50% extract was 125.49%-130.08%; the cell viability of the 100% and 50% extracts in Comparative Example 1 were 104.87% and 108.88%, respectively; and the cell viability of Comparative Example 2 were 113.75% and 107.23%, respectively. This indicates that the lyophilized product prepared by this invention has excellent biocompatibility, and its cell proliferation-promoting effect is significantly better than that of products prepared by the acetic acid method and the phosphate buffer method.

[0058] HPLC detection of solvent residue and purity Experimental procedure: Take the lyophilized products of Example 2, Comparative Example 1, and Comparative Example 2, reconstitute them with ultrapure water to a concentration of 1 mg / mL, mix thoroughly using a vortex mixer for 5 min, and draw samples using a disposable syringe. After filtration through an aqueous membrane, the sample was added to a liquid chromatography sample vial. A TSK G3000SWXL column was used, with a mobile phase of 0.05 M sodium dihydrogen phosphate-acetonitrile solution (95:5 v / v), a flow rate of 1.0 mL / min, a detection wavelength of 214 nm, a column temperature of 30 °C, and an injection volume of [missing information]. Record the chromatogram and calculate the target peak area, solvent peak area and sample purity: Purity (%) = (target peak area / total peak area) × 100%.

[0059] The HPLC chromatograms of the lyophilized porcine type I collagen products prepared in Example 2, Comparative Example 1, and Comparative Example 2 are shown below. Figure 2 As shown, A is the HPLC chromatogram of the lyophilized porcine type I collagen prepared in Example 2, B is the HPLC chromatogram of the lyophilized porcine type I collagen prepared in Comparative Example 1, and C is the HPLC chromatogram of the lyophilized porcine type I collagen prepared in Comparative Example 2.

[0060] The HPLC chromatogram information is shown in Table 3.

[0061] Table 3 Experimental results show that the lyophilized product of Example 2 has no solvent peak, the target peak area is 77.92, and the purity is 99.99%; Comparative Example 1 has a significant solvent peak, with a solvent peak area of ​​3.71, the target peak area is 97.49, and the purity is 96.33%; Comparative Example 2 has a solvent peak, with a solvent peak area of ​​2.79, the target peak area is 63.41, and the purity is 95.78%. This indicates that the lyophilized product prepared by this invention has no solvent residue and its purity is significantly higher than that of the comparative products.

[0062] Odor sensory evaluation Experimental Procedure: Five professional food and biological materials sensory evaluation personnel were selected to evaluate the odor of the freeze-dried products of Examples 1-5 and Comparative Examples 1-2 using a blind evaluation method. The evaluation criteria were: 0 points - no odor; 1 point - slight odor, almost imperceptible; 2 points - obvious odor, perceptible but not irritating; 3 points - strong irritating odor. The average score of the five personnel was taken as the final odor score.

[0063] The test results are shown in Table 4.

[0064] Table 4 Experimental results show that Examples 1-5 had an odor score of 0, indicating no odor whatsoever; Comparative Example 1 had an odor score of 3, indicating a strong, pungent odor of acetic acid; and Comparative Example 2 had an odor score of 0, indicating no residual acidic taste. This demonstrates that the freeze-dried product prepared according to this invention completely eliminates residual solvent odors and significantly improves sensory quality.

[0065] In summary, the collagen rehydration process provided by this invention has the following advantages: 1. Significantly reduced product odor and no solvent residue: This invention uses low-concentration hydrochloric acid instead of traditional acetic acid as the resolution solvent. The volatility of hydrochloric acid is much lower than that of acetic acid, and the complete removal of hydrochloric acid is achieved through precise control of the freeze-drying process. HPLC detection shows that the obtained freeze-dried product has no obvious solvent peak, the solvent residue is 0, and the sensory evaluation shows no irritating odor. The odor score of the product prepared by the acetic acid method is reduced by more than 60%, and the sensory quality is significantly improved.

[0066] 2. Ensuring the integrity and stability of collagen structure: By precisely controlling the hydrochloric acid concentration (0.001-0.02M), reconstitution temperature (2-8℃), and stirring rate, the natural triple helix structure of porcine type I collagen is effectively maintained. SDS-PAGE gel electrophoresis shows that the characteristic bands of the product are consistent with those of the acetic acid method product, with no impurities or degradation. Differential scanning calorimetry shows that the product's thermal stability melting point is 43~45℃, which is superior to that of the acetic acid method product, meeting the requirements of industrial applications for collagen stability.

[0067] 3. Maintains excellent biological activity and promotes cell proliferation: The process of this invention is mild throughout and free from strong irritants, effectively preserving the biological activity of collagen; cell proliferation experiments show that the cell viability of the 100% extract of the lyophilized product prepared by this invention is ≥128%, which is significantly higher than that of products prepared by the acetic acid method and the phosphate buffer method, and has a good cell proliferation promoting effect, making it suitable for fields such as biomedicine and tissue engineering.

[0068] 4. Strong process compatibility and easy industrialization: The preparation process of this invention has clear process parameters and strong controllability, which can realize linear scale-up from laboratory pilot to large-scale industrial production, making it suitable for industrial applications.

[0069] 5. Wider range of applications: The low odor and solvent-free characteristics of freeze-dried products allow them to be directly applied in odor-sensitive fields such as cosmetics and oral care; their good structural stability and bioactivity make them more suitable for biomedical fields such as medical dressings, tissue engineering scaffolds, and medical implant materials, thus expanding the application scenarios of collagen products.

[0070] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A collagen reconstitution process, characterized in that, Includes the following steps: S1: Prepare a hydrochloric acid solution with a concentration of 0.001 mol / L-0.02 mol / L; S2: At 2-8℃, collagen filaments are added to the hydrochloric acid solution and stirred to obtain a collagen solution.

2. The collagen reconstitution process as described in claim 1, characterized in that, The ratio of collagen filaments to hydrochloric acid solution is 10g:(100-200)mL.

3. The collagen reconstitution process as described in claim 1 or 2, characterized in that, Also includes: S3: Dialyze and desalt the collagen solution.

4. The collagen reconstitution process as described in claim 3, characterized in that, In step S3, the collagen solution is dialyzed and desalted using hydrochloric acid solution as the dialysate.

5. The collagen reconstitution process as described in claim 4, characterized in that, In step S3, the concentration of the hydrochloric acid solution used for the dialysate is 0.001 mol / L-0.01 mol / L.

6. The collagen reconstitution process as described in claim 5, characterized in that, The dialysis desalination is gradient dialysis desalination.

7. The collagen reconstitution process as described in claim 6, characterized in that, The gradient dialysis desalination process includes: first, dialysis with a hydrochloric acid solution of concentration 0.005 mol / L-0.01 mol / L, and then dialysis with a hydrochloric acid solution of concentration 0.001 mol / L-0.005 mol / L.

8. The collagen reconstitution process as described in claim 3, characterized in that, The collagen filaments are prepared from pigskin.

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