A method for detecting active collagen content using self-assembly properties

Abstract

The present invention belongs to the technical field of natural active collagen, and specifically relates to a method for detecting the content of active collagen using self-assembly properties. The present invention utilizes the unique self-assembly properties of active collagen with a complete triple helical structure, induces the active collagen to self-assemble into collagen fibers using PBS buffer, and simultaneously uses trypsin to further enzymatically hydrolyze the denatured collagen into small molecules. The precipitated collagen fibers and the denatured polypeptide solution are then separated by ultrafiltration and centrifugation. By detecting the content of collagen in the precipitate, the content of active collagen in the collagen can be simply and quickly quantitatively analyzed. The detection method of the present invention can be used to detect the content of active collagen in various dosage forms of collagen products such as collagen solutions, dressings, sponges, powders, and gel preparations, and has a wide range of applications.

Description

Technical Field

[0001] The invention belongs to the technical field of natural active collagen, and particularly relates to a method for detecting the content of active collagen by utilizing self-assembly performance. Background Art

[0002] Patent CN108659117A discloses a method for quantitatively detecting the content of collagen triple helix structure. This method utilizes the principle that collagen with an intact triple helix structure cannot be hydrolyzed by trypsin, while collagen with a damaged / partially damaged triple helix structure can be hydrolyzed by trypsin. By measuring the change in hydroxyproline content in the collagen solution before and after trypsin hydrolysis, the content of intact collagen triple helix structure is calculated. However, this method has the disadvantage that it requires prolonged enzymatic hydrolysis of the sample using trypsin, making it applicable only to the detection of collagen solutions. Furthermore, factors such as trichloroacetic acid concentration, temperature, and ionic strength can affect the test results, making rapid and accurate detection impossible.

[0003] Patent CN109884153A discloses a method for quantitatively detecting the triple helical structure of a collagen sample by utilizing the fact that collagen with an intact triple helical structure is resistant to proteases, while collagen with a lost or partially lost triple helical structure is easily decomposed by proteases into small peptides. The decomposed peptides are then visualized and analyzed by SDS-PAGE electrophoresis. However, this method has disadvantages in that the detection process is complex and can only detect the retention rate of the triple helical structure in a solution, not other forms of collagen products. Furthermore, the detection results are easily affected by the sensitivity of the electrophoretic bands and the resolution of the gel.

[0004] Therefore, existing methods for measuring collagen activity primarily rely on protease sensitivity. These methods are characterized by long testing cycles, cumbersome procedures, and easily affected by test conditions. This makes them difficult to apply to real-time, online qualitative and quantitative testing of collagen and related products during production, transportation, and storage. Furthermore, existing methods are only suitable for measuring triple helix content in collagen solutions and are incapable of measuring the active collagen content in other collagen formulations, such as sponges, gels, and powders. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the existing technology and provide a method for detecting the content of active collagen by utilizing self-assembly properties, so as to effectively achieve the advantages of shortened cycle, simple operation, reduced detection cost, stable detection results, etc., and can be used for the detection of active collagen content in other dosage forms of collagen products such as sponges, gels, and powders.

[0006] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0007] In a first aspect, the present invention provides a method for detecting the content of active collagen, comprising the following steps:

[0008] S1. Dissolve or dilute the sample to be tested with pure water or an acidic solution to obtain a sample solution to be tested;

[0009] S2, adding trypsin digestion solution and PBS buffer to the sample solution to be tested, adjusting the pH and then performing self-assembly to obtain a self-assembly solution;

[0010] S3, subjecting the self-assembly solution to a first ultrafiltration centrifugation, retaining the supernatant and precipitate, placing the supernatant in an ultrafiltration tube filter element for a second ultrafiltration centrifugation, removing the filter element and placing it upside down on the precipitate for a third ultrafiltration centrifugation, mixing the precipitate with the concentrated solution in the filter element to obtain self-assembly collagen;

[0011] S4. Determine the content of self-assembled collagen and the collagen content of the sample to be tested before self-assembly by using the Folin-phenol method, and calculate the ratio of the content of self-assembled collagen to the content of collagen of the sample to be tested before self-assembly to obtain the content of active collagen in the sample to be tested;

[0012] In step S2, the final concentration of the total protein in the self-assembly solution is 0.5-1 mg / mL; the mass ratio of the trypsin to the total protein in the self-assembly solution is 1:(10-20);

[0013] In step S3, the specific conditions of the first ultrafiltration centrifugation are: centrifugation at 10,000-20,000 g for 15-30 min; the specific conditions of the second ultrafiltration centrifugation are: centrifugation at 7,500-14,000 g for 20-30 min; and the specific conditions of the third ultrafiltration centrifugation are: centrifugation at 7,500-14,000 g for 3-5 min.

[0014] The present invention utilizes the unique self-assembly property of active collagen with a complete triple helix structure, induces the active collagen to self-assemble into collagen fibers using PBS buffer, and simultaneously uses trypsin to further enzymatically hydrolyze the denatured collagen into small molecules. The precipitated collagen fibers and the denatured polypeptide solution are then separated by ultrafiltration and centrifugation. By detecting the collagen content in the precipitate, the content of active collagen in the collagen can be simply and quickly quantitatively analyzed.

[0015] Experimental research found that the present invention utilizes the synergistic effect of collagen's self-assembly properties and protein sensitivity to greatly improve the specificity and accuracy of active collagen detection, shorten the detection cycle, simplify the operation, stabilize the detection results, and reduce the detection cost. It can achieve rapid and accurate detection of active collagen and can be used for real-time online qualitative or quantitative detection of collagen solutions and related products during production, transportation and storage.

[0016] Among them, the role of trypsin is to further enzymatically hydrolyze denatured collagen into small molecules, which is beneficial to the separation of denatured collagen and active collagen, and improves the accuracy of detection; the role of PBS buffer is to provide suitable pH and ionic strength to induce active collagen to self-assemble to form collagen fibers. Through experimental research, it was found that when the sample solution to be tested, trypsin and PBS buffer are precisely controlled within the above-mentioned preferred ratio and concentration range, the synergistic effect of the self-assembly performance and protein sensitivity of active collagen can be effectively utilized, thereby achieving effective separation of active collagen and inactive collagen, and improving the accurate qualitative and quantitative detection of active collagen in the sample to be tested.

[0017] Furthermore, when ultrafiltration centrifuging the self-assembled collagen fibers to effectively separate active and inactive collagen, precise control of the filter element's molecular cutoff and centrifugation conditions is required to ensure maximum retention of active collagen fibers, thereby improving the accuracy of subsequent testing. Furthermore, the present invention further improves the retention of active collagen by centrifuging the self-assembled solution and then filtering the supernatant through the filter element again.

[0018] More preferably, the final concentration of the protein in the self-assembly solution is 0.75-1 mg / mL; and the mass ratio of the trypsin to the total protein in the self-assembly solution is 1:10.

[0019] Preferably, in step S1, pure water or an acidic solution is used to dissolve or dilute the sample to be tested to a collagen concentration of 1-10 mg / mL.

[0020] More preferably, the sample to be tested is diluted to a concentration of 3-4 mg / mL.

[0021] The dilution concentration of the test sample also affects the accuracy of the final active collagen quantitative analysis. If the sample concentration is too low, low concentrations of active collagen will not be detected, thereby reducing the detection sensitivity. If the sample concentration is too high, the measurement results will be saturated or the error will increase. Moreover, too high a collagen concentration will limit the effect of trypsin, making it unable to fully enzymatically hydrolyze denatured collagen, affecting the final quantitative results. Therefore, using the above-defined test sample dilution concentration to detect the active collagen content in the sample can further improve the accuracy of the test results.

[0022] Preferably, in step S1, the acidic solution includes a hydrochloric acid solution or an acetic acid solution, the concentration of the hydrochloric acid solution is 0.001-0.01 mol / L, and the mass concentration of the acetic acid solution is 0.1-0.5%.

[0023] Preferably, in step S2, the pH is adjusted to 6.5-7.5, and the specific conditions for the self-assembly are: self-assembly at 28-32° C. for 1-48 hours.

[0024] pH value, temperature and time have a significant impact on the self-assembly process of active collagen and the final detection effect. Collagen will exhibit different charge states under different pH environments, which will affect its interaction with the surrounding solution molecules and, in turn, its ability to self-assemble into collagen fibers; the time and temperature of self-assembly will directly affect the folding and polymerization of collagen, thereby affecting the formation of collagen fibers and, in turn, the accuracy of the detection. Experimental research has found that using the above-mentioned preferred range of conditions for self-assembly can effectively ensure that active collagen self-assembles and forms stable collagen fibers, thereby ensuring efficient quantitative analysis of active collagen.

[0025] More preferably, in step S2, the pH is adjusted to 7, and the specific conditions of the self-assembly are: self-assembly at 30° C. for 3-16 hours.

[0026] Through experimental research, it was found that when the above-mentioned optimal parameter conditions are used for self-assembly, the active collagen in different dosage forms can reach the self-assembly plateau within 3-16 hours, ensuring to the greatest extent that the self-assembly of the active collagen in the test sample is complete and stable, achieving the optimal self-assembly state, thereby ensuring the accuracy of the test results, and effectively shortening the test time, effectively improving the experimental efficiency without sacrificing the accuracy of the results.

[0027] Preferably, in step S2, the trypsin digestion solution is prepared by weighing trypsin and adding it to PBS buffer.

[0028] Preferably, in step S2, the concentration of the PBS buffer is 0.005-0.015 mol / L.

[0029] More preferably, the concentration of the PBS buffer is 0.01 mol / L.

[0030] Experimental research has found that the concentration of PBS buffer used during self-assembly also has a significant impact on the detection effect. If the concentration of PBS buffer is too low, it will lead to incomplete collagen self-assembly, affecting the sensitivity and accuracy of the detection; if the concentration is too high, the increase in ionic strength will affect the self-assembly process, and then affect the separation of the precipitate, resulting in large errors in the detection value. Therefore, when the PBS buffer is used at the above optimal concentration for the self-assembly reaction, the self-assembly efficiency and the accuracy of the results can be effectively guaranteed by regulating the ionic strength and pH.

[0031] Preferably, in step S3, the molecular cutoff of the ultrafiltration centrifuge tube is 50-300 KDa.

[0032] More preferably, the molecular cut-off of the ultrafiltration centrifuge tube is 100 KDa.

[0033] Preferably, the sample to be tested is a collagen product, and the dosage forms of the collagen product include solution, dressing, sponge, powder and gel preparation.

[0034] Existing methods for detecting active collagen primarily focus on the triple helix structure in collagen solutions, which is effective for collagen solution products. However, for collagen products in different dosage forms, such as sponges, gels, and powders, the complex collagen structure makes it difficult to accurately determine the active collagen content using conventional methods. The detection method provided by the present invention has broad applicability, not only accurately detecting the active ingredient in collagen solutions but also effectively applying it to a variety of collagen product dosage forms, including dressings, sponges, powders, and gels.

[0035] In a second aspect, the present invention provides the use of the method for detecting the active collagen content in detecting the active collagen content of a collagen product.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention innovatively utilizes the synergistic effect of the self-assembly performance of active collagen and protein sensitivity to achieve the separation of active collagen and inactive collagen. By quantifying the self-assembly ability of active collagen, it solves the problems of the existing detection methods such as long cycle, cumbersome operation, high detection cost and unstable detection results;

[0038] (2) The detection method of the present invention has a wide range of applicability and can be used to accurately detect the content of active collagen in various dosage forms of collagen products such as collagen solutions, dressings, sponges, powders, and gel preparations, both qualitatively and quantitatively. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a picture of self-assembled collagen fibers obtained during the process of detecting the active collagen content in the collagen solution in Example 1, wherein: Figure 1 A is the solution after the first centrifugation after self-assembly, with collagen fiber precipitation at the bottom; Figure 1 B is the collagen fibers precipitated after the first centrifugation after self-assembly; Figure 1 C is the collagen fiber finally obtained after three ultrafiltration and centrifugation;

[0040] Figure 2 This is a schematic diagram of the collagen sponge sample in Example 4;

[0041] Figure 3This is a schematic diagram of the collagen powder sample in Example 5;

[0042] Figure 4 This is a schematic diagram of the collagen freeze-dried powder sample in Example 6;

[0043] Figure 5 Schematic diagram of the collagen gel sample in Example 7. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the following examples are provided to further illustrate the present invention in detail. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the present invention.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0046] The nominal titer of the trypsin described in the Examples and Comparative Examples is ≥3000 U / mg. The preparation method of the trypsin digestion solution is as follows: weigh trypsin and add it to 0.01 M PBS buffer to prepare 3000 U / mL trypsin digestion solution.

[0047] The detection method of the Folin-phenol method described in the Examples and Comparative Examples was carried out with reference to the second method of Folin-phenol method (Lowry method) in the General Chapter 0731 of Volume IV of the Chinese Pharmacopoeia, Determination of Protein Content.

[0048] Example 1

[0049] This embodiment provides a method for detecting the content of active collagen in a collagen solution, comprising the following steps:

[0050] S1. Preparation of test solution: dilute the test sample to 3 mg / mL with purified water and mix well for later use;

[0051] S2. Self-assembly: Take 250 μL of the test solution, add 675 μL of 0.01 M PBS buffer and 75 μL of 3000 U / mL trypsin digestion solution (the final concentration of total protein in the mixed solution is 0.75 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is 1:10), then adjust the pH value to about 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes at the same time; then, draw 500 μL of the mixture into the outer tube of a 1.5 mL 100 KDa ultrafiltration centrifuge tube, transfer it to a constant temperature incubator at 30°C for self-assembly for 3 hours;

[0052] S3. Separation of self-assembled collagen fibers:

[0053] 1. Centrifuge the self-assembled solution at room temperature and 20,000 g for 15 minutes to separate and retain the precipitate and supernatant;

[0054] 2. Add all the above supernatant to a pre-cleaned 1.5mL 100kDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature at 14,000g for 20 minutes. Finally, remove the filter element and place it upside down in the above precipitate. Centrifuge at room temperature at 14,000g for 3 minutes to transfer the retained concentrate from the ultrafiltration inner tube to the outer tube.

[0055] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0056] Example 2

[0057] This embodiment provides a method for detecting the content of active collagen in a collagen solution, comprising the following steps:

[0058] S1. Preparation of test solution: dilute the test sample to 1 mg / mL with purified water and mix well for later use;

[0059] S2. Self-assembly: Take 500 μL of the test solution, add 475 μL of 0.01 M PBS buffer and 25 μL of 3000 U / mL trypsin digestion solution (the final concentration of total protein in the mixed solution is 0.5 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is 1:20), then adjust the pH value to about 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes at the same time; then, draw 500 μL of the mixture into the outer tube of a 1.5 mL 100 KDa ultrafiltration centrifuge tube, transfer to a constant temperature incubator at 28°C for self-assembly for 1 hour;

[0060] S3. Separation of self-assembled collagen fibers:

[0061] 1. Centrifuge the self-assembled solution at room temperature and 10,000 g for 30 min to separate and retain the precipitate and supernatant;

[0062] 2. Add all the supernatant to a pre-cleaned 1.5 mL 100 kDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature and 7500 g for 30 minutes. Finally, remove the filter element and place it upside down in the precipitate. Centrifuge at room temperature and 7500 g for 5 minutes to transfer the retained concentrate from the inner ultrafiltration tube to the outer ultrafiltration tube.

[0063] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0064] Example 3

[0065] This embodiment provides a method for detecting the content of active collagen in a collagen solution, comprising the following steps:

[0066] S1. Preparation of test solution: dilute the test sample to 10 mg / mL with purified water and mix well for later use;

[0067] S2. Self-assembly: Take 100 μL of the test solution, add 850 μL of 0.01 M PBS buffer and 50 μL of 3000 U / mL trypsin digestion solution (the final concentration of total protein in the mixed solution is 1 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is 1:20), then adjust the pH value to about 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes at the same time; then, draw 500 μL of the mixture into the outer tube of a 1.5 mL 100 KDa ultrafiltration centrifuge tube, transfer to a constant temperature incubator set to 32°C for self-assembly for 48 hours;

[0068] S3. Separation of self-assembled collagen fibers:

[0069] 1. Centrifuge the self-assembled solution at room temperature and 20,000 g for 15 minutes to separate and retain the precipitate and supernatant;

[0070] 2. Add all the above supernatant to a pre-cleaned 1.5mL 100kDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature at 14,000g for 20 minutes. Finally, remove the filter element and place it upside down in the above precipitate. Centrifuge at room temperature at 14,000g for 3 minutes to transfer the retained concentrate from the ultrafiltration inner tube to the outer tube.

[0071] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0072] Example 4

[0073] This embodiment provides a method for detecting the content of active collagen in a collagen sponge, comprising the following steps:

[0074] S1. Preparation of test solution: Dissolve the test sample to 4 mg / mL in 0.1% acetic acid solution and set aside.

[0075] S2. Self-assembly: Take 250 μL of the test sample solution, add 675 μL of 0.01 M PBS buffer and 75 μL of 3000 U / mL trypsin digestion solution (the final total protein concentration in the mixed solution is 1 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is approximately 1:13). Adjust the pH to approximately 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes. Then, pipette 500 μL of this mixture into the outer tube of a 1.5 mL 100 kDa ultrafiltration centrifuge tube and transfer it to an electrically heated water bath maintained at 30°C for self-assembly for 16 hours.

[0076] S3. Separation of self-assembled collagen fibers:

[0077] 1. Centrifuge the self-assembled solution at room temperature and 14,000 g for 15 minutes to separate and retain the precipitate and supernatant;

[0078] 2. Add all the supernatant from the above to a pre-cleaned 1.5 mL 100 kDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature at 14,000 g for 20 minutes. Finally, remove the filter element and place it upside down in the above precipitate. Centrifuge at room temperature at 10,000 g for 5 minutes to transfer the retained concentrate from the inner ultrafiltration tube to the outer ultrafiltration tube.

[0079] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0080] Example 5

[0081] This embodiment provides a method for detecting the content of active collagen in collagen powder, comprising the following steps:

[0082] S1. Preparation of test solution: Dissolve the test sample to 4 mg / mL in 0.1% acetic acid solution and set aside.

[0083] S2. Self-assembly: Take 250 μL of the test sample solution, add 675 μL of 0.01 M PBS buffer and 75 μL of 3000 U / mL trypsin digestion solution (the final total protein concentration in the mixed solution is 1 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is approximately 1:13). Adjust the pH to approximately 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes. Then, pipette 500 μL of this mixture into the outer tube of a 1.5 mL 100 kDa ultrafiltration centrifuge tube and transfer it to an electrically heated water bath maintained at 30°C for self-assembly for 16 hours.

[0084] S3. Separation of self-assembled collagen fibers:

[0085] 1. Centrifuge the self-assembled solution at room temperature and 12000g for 30 minutes to separate and retain the precipitate and supernatant;

[0086] 2. Add all the above supernatant to a pre-cleaned 1.5mL 100KDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature and 10,000g for 30 minutes. Finally, remove the filter element and place it upside down in the above precipitate. Centrifuge at room temperature and 10,000g for 5 minutes to transfer the retained concentrate from the ultrafiltration inner tube to the outer tube.

[0087] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0088] Example 6

[0089] This embodiment provides a method for detecting the content of active collagen in collagen freeze-dried powder, comprising the following steps:

[0090] S1. Preparation of test solution: Dissolve the test sample to 4 mg / mL in 0.1% acetic acid solution and set aside.

[0091] S2. Self-assembly: Take 250 μL of the test sample solution, add 675 μL of 0.01 M PBS buffer and 75 μL of 3000 U / mL trypsin digestion solution (the final total protein concentration in the mixed solution is 1 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is approximately 1:13). Adjust the pH to approximately 7.0 with 1 mol / L sodium hydroxide solution, mix thoroughly, and prepare two parallel tubes. Then, pipette 500 μL of this mixture into the outer tube of a 1.5 mL 100 kDa ultrafiltration centrifuge tube and transfer it to an electrically heated water bath maintained at 30°C for self-assembly for 16 hours.

[0092] S3. Separation of self-assembled collagen fibers:

[0093] 1. Centrifuge the self-assembled solution at room temperature and 20,000 g for 20 minutes to separate and retain the precipitate and supernatant;

[0094] 2. Add all the above supernatant to a pre-cleaned 1.5mL 100KDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature and 12000g for 30 minutes. Finally, remove the filter element and invert it into the above precipitate. Centrifuge at room temperature and 12000g for 3 minutes to transfer the retained concentrate from the ultrafiltration inner tube to the outer tube.

[0095] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0096] Example 7

[0097] This embodiment provides a method for detecting the content of active collagen in collagen gel, comprising the following steps:

[0098] S1. Preparation of test solution: dilute the test sample to 4 mg / mL with 0.1% acetic acid solution and set aside;

[0099] S2. Self-assembly: Take 250 μL of the test solution, add 675 μL of 0.01 M PBS buffer and 75 μL of 3000 U / mL trypsin digestion solution (the final concentration of total protein in the mixed solution is 1 mg / mL, and the mass ratio of trypsin to total protein in the mixed solution is about 1:13), and then adjust the pH value to about 7.0 with 1 mol / L sodium hydroxide solution. Mix thoroughly and prepare two parallel tubes at the same time; then, draw 500 μL of the mixture into the outer tube of a 1.5 mL 100 KDa ultrafiltration centrifuge tube and transfer it to a constant temperature incubator set to 30°C for self-assembly for 16 hours.

[0100] S3. Separation of self-assembled collagen fibers:

[0101] 1. Centrifuge the self-assembled solution at room temperature and 12000g for 30 minutes to separate and retain the precipitate and supernatant;

[0102] 2. Add all the above supernatant to a pre-cleaned 1.5mL 100KDa ultrafiltration centrifuge tube (outer tube + filter element) and centrifuge at room temperature and 10,000g for 30 minutes. Finally, remove the filter element and place it upside down in the above precipitate. Centrifuge at room temperature and 10,000g for 5 minutes to transfer the retained concentrate from the ultrafiltration inner tube to the outer tube.

[0103] S4. Detect the collagen content of the test sample before and after self-assembly using the Folin phenol method, and calculate the ratio of the self-assembled collagen content to the collagen content of the test sample before self-assembly to obtain the content of active collagen in the test sample.

[0104] Comparative Example 1

[0105] This comparative example provides a method for detecting the content of active collagen in a collagen solution, which differs from Example 1 only in that:

[0106] After separating the self-assembled collagen fibers, the self-assembled solution was centrifuged at room temperature and 20,000 g for 15 minutes to separate the precipitate, which was directly used as the self-assembled collagen. The supernatant was not subjected to ultrafiltration and centrifugation. The rest of the operation process was the same as in Example 1.

[0107] Comparative Example 2

[0108] This comparative example provides a method for detecting the content of active collagen in a collagen solution, which differs from Example 1 only in that:

[0109] After separating the self-assembled collagen fibers, the filter element was taken out and inverted on the sediment for a third ultrafiltration centrifugation. The specific conditions of the centrifugation were centrifugation at 1000 g for 2 minutes. The rest of the operation process was the same as in Example 1.

[0110] Comparative Example 3

[0111] This comparative example provides a method for detecting the content of active collagen in a collagen solution. The difference between this method and Example 1 is that the mass ratio of trypsin to total protein in the mixed solution is 1:5. The specific process is as follows:

[0112] During the self-assembly process, 250 μL of the test solution was added to 600 μL of 0.01 M PBS buffer and 150 μL of 3000 U / mL trypsin digestion solution, so that the mass ratio of trypsin to total protein in the mixed solution was 1:5. The mixture was thoroughly mixed and two parallel tubes were prepared. The remaining operation process was the same as in Example 1.

[0113] Comparative Example 4

[0114] This comparative example provides a method for detecting the content of active collagen in a collagen solution. The difference between this method and Example 1 is that the mass ratio of trypsin to total protein in the mixed solution is 1:25. The specific process is as follows:

[0115] During the self-assembly process, 250 μL of the test solution was added to 720 μL of 0.01 M PBS buffer and 30 μL of 3000 U / mL trypsin digestion solution, so that the mass ratio of trypsin to total protein in the mixed solution was 1:25. The mixture was thoroughly mixed and two parallel tubes were prepared. The remaining operation process was the same as in Example 1.

[0116] Comparative Example 5

[0117] This comparative example provides a method for detecting the content of active collagen in a collagen solution, which differs from Example 1 only in that Tris buffer is used instead of PBS buffer for the self-assembly reaction. The rest of the operation process is the same as Example 1.

[0118] Effect Examples

[0119] The results of the determination of the active collagen content in the test samples obtained by the detection methods described in Examples 1-7 and Comparative Examples 1-5 are shown in Table 1.

[0120] Table 1 Active collagen content

[0121]

[0122] The results in Table 1 show that the detection method of the present invention can effectively detect the active collagen content in collagen products of different dosage forms, and the test results are relatively stable and have good reproducibility. When the same collagen product was tested in Examples 1-3, the test results of Examples 1 and 3 were relatively better, and there was no significant difference between the test results of the two. However, the technical solution described in Example 1 can effectively shorten the detection time, shortening the self-assembly time from 48h to 3h, achieving higher detection efficiency. When Comparative Examples 1-3 and 5 were tested for the same collagen solution, the detection results of active collagen were significantly reduced compared with Example 1; the relative standard deviation of the active collagen content obtained by the comparative example 4 reached 11.30%, and the error range (±10.21%) was extremely large, that is, the test results had poor repeatability and low credibility; the above results show that the detection process defined in the technical solution of the present invention, the conditions and parameters involved, and the choice of buffer have an important influence on the accuracy of the test results. It is necessary to accurately control the parameters within the optimal range of the present invention to maximize the accuracy, reproducibility and detection efficiency of the test.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting active collagen content, characterized in that: The following steps are involved: S1. Dissolve or dilute the sample to be tested with pure water or an acidic solution to obtain a sample solution to be tested; S2, adding trypsin digestion solution and PBS buffer to the sample solution to be tested, adjusting the pH and then performing self-assembly to obtain a self-assembly solution; S3, subjecting the self-assembly solution to a first ultrafiltration centrifugation, retaining the supernatant and precipitate, placing the supernatant in an ultrafiltration tube filter element for a second ultrafiltration centrifugation, removing the filter element and placing it upside down on the precipitate for a third ultrafiltration centrifugation, mixing the precipitate with the concentrated solution in the filter element to obtain self-assembly collagen; S4. Determine the content of self-assembled collagen and the collagen content of the sample to be tested before self-assembly by using the Folin-phenol method, and calculate the ratio of the content of self-assembled collagen to the content of collagen of the sample to be tested before self-assembly to obtain the content of active collagen in the sample to be tested; In step S2, the final concentration of the total protein in the self-assembly solution is 0.5-1 mg / mL; the mass ratio of the trypsin to the total protein in the self-assembly solution is 1:(10-20); In step S3, the specific conditions of the first ultrafiltration centrifugation are: centrifugation at 10,000-20,000 g for 15-30 min; the specific conditions of the second ultrafiltration centrifugation are: centrifugation at 7,500-14,000 g for 20-30 min; the specific conditions of the third ultrafiltration centrifugation are: centrifugation at 7,500-14,000 g for 3-5 min; In step S2, the specific conditions of the self-assembly are: self-assembly at 28-32° C. for 1-48 hours; and adjusting the pH to 7.

2. The method for detecting active collagen content according to claim 1, wherein: In step S1, the acidic solution includes a hydrochloric acid solution or an acetic acid solution. The concentration of the hydrochloric acid solution is 0.001-0.01 mol / L, and the mass concentration of the acetic acid solution is 0.1-0.5%.

3. The method for detecting active collagen content according to claim 1, wherein: In step S1, the collagen concentration of the sample solution to be tested is 1-10 mg / mL.

4. The method for detecting active collagen content according to claim 1, wherein: In step S2, the trypsin digestion solution is prepared by weighing trypsin and adding it to PBS buffer; The concentration of the PBS buffer is 0.005-0.015 mol / L.

5. The method for detecting active collagen content according to claim 1, wherein: In step S3, the molecular cutoff of the ultrafiltration tube filter element is 50-300KDa.

6. The method for detecting active collagen content according to claim 1, wherein: The sample to be tested is a collagen product, and the dosage forms of the collagen product include solution, dressing, sponge, powder and gel preparation.

7. Use of the method for detecting the active collagen content according to any one of claims 1 to 6 in detecting the active collagen content of a collagen product.

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