A method for detecting the integrity of the triple helix structure of active collagen

Abstract

The present invention relates to the technical field of protein structure detection, and in particular to a method for detecting the integrity of the triple helix structure of active collagen, comprising obtaining an active collagen detection liquid to be detected, and performing centrifugation to obtain an initial structure detection liquid; obtaining an upper layer liquid and a bottom layer liquid of the initial structure detection liquid, performing byproduct detection and recording the detection result; determining the byproduct interference parameter of the initial structure detection liquid to determine the type of abnormal protein structure tendency; removing the upper layer liquid of the initial structure detection liquid to obtain a triple helix structure analysis liquid, and determining the analysis method of the triple helix structure analysis liquid according to the type of abnormal protein structure tendency of the initial structure detection liquid. The present invention can adaptively select the detection method, accurately evaluate the integrity of the triple helix structure of the active collagen, ensure the reliability of the detection and avoid product waste at the same time, provide an effective monitoring means for the collagen purification process, and improve the purification efficiency and product quality of the collagen.

Description

Technical Field

[0001] The present invention relates to the technical field of protein structure detection, and in particular to a method for detecting the integrity of an active collagen triple helix structure. Background Art

[0002] Existing technologies for detecting protein triple helix structures, such as SDS-PAGE gel electrophoresis and atomic force microscopy (AFM), usually rely on specific chemical or physical properties of proteins. These methods are complex, costly, and time-consuming, and may damage the samples, affecting their natural state and function. In addition, some technologies have high requirements for sample purity and concentration and are not suitable for the detection of complex samples or trace samples. These limitations reduce the applicability and accuracy of the detection, and restrict the real-time monitoring and rapid evaluation of dynamic changes in protein structure.

[0003] Chinese Patent Publication No.: CN109884153B discloses a new method for determining the integrity of the triple helix structure of type I collagen. The basic principle is that collagen with a complete triple helix structure is resistant to enzymatic hydrolysis by proteases, while collagen with a lost or partially lost triple helix structure is easily decomposed by proteases into small molecular weight peptides. The decomposed peptides can be visualized and analyzed by SDS-PAGE. The more decomposed peptides there are, the worse the integrity of the triple helix structure of the collagen sample is. On the contrary, if there are no decomposed small molecule peptides in SDS-PAGE, it indicates that the integrity of the triple helix structure of the collagen sample is good. The dimer protein in the collagen sample is quantitatively detected by a gel electrophoresis imaging system or a thin layer scanning system, so as to calculate the retention rate of the triple helix structure of the collagen sample. It can be seen that the new method for determining the integrity of the triple helix structure of type I collagen has the following problems: the content of the collagen with a destroyed structure is determined only based on the polypeptides in the collagen, and the influence of polypeptides from other sources on the detection results is not considered. Summary of the invention

[0004] To this end, the present invention provides a method for detecting the integrity of the triple helix structure of active collagen, so as to overcome the problem that the existing technology only determines the content of collagen with a destroyed structure based on the polypeptides in the collagen, without considering the influence of polypeptides from other sources on the detection results.

[0005] To achieve the above object, the present invention provides a method for detecting the integrity of the triple helix structure of active collagen, comprising:

[0006] Obtaining active collagen test solution to be tested, performing preliminary centrifugation to obtain initial structure test solution;

[0007] Obtaining the upper layer liquid and the bottom layer liquid of the initial structure detection liquid, performing by-product detection, and recording the by-product detection results respectively;

[0008] Determine the byproduct interference parameter of the initial structure detection solution according to each byproduct detection result, and determine the protein structure abnormality tendency type of the initial structure detection solution according to the byproduct interference yield;

[0009] The upper layer of the initial structure detection liquid is removed to obtain a triple helix structure analysis liquid, and the analysis method of the triple helix structure analysis liquid is determined according to the protein structure abnormality tendency type of the initial structure detection liquid, including:

[0010] Obtaining a collagen sample solution, performing an enzymatic hydrolysis test on the triple helix structure analysis solution, and determining the abnormal amount of the triple helix structure of collagen in the active collagen test solution according to the enzymatic hydrolysis test result, so as to determine whether there is an abnormality in the active collagen extraction process;

[0011] Alternatively, a collagen sample solution is obtained, a spectral test is performed on the triple helix structure analysis solution, and the triple helix collagen content is determined according to the spectral test result to determine whether the structural integrity of the active collagen detection solution meets the standard.

[0012] Further, the by-product detection includes,

[0013] The upper layer liquid and the bottom layer liquid are sampled respectively, and recorded as upper layer sample and bottom layer sample;

[0014] detecting the polypeptide concentration in the supernatant and detecting the polymer viscosity in the bottom layer sample;

[0015] Among them, by-products include peptides and collagen polymers.

[0016] Furthermore, the byproduct interference parameter of the initial structure detection solution is determined according to each byproduct detection result. The byproduct interference parameter is determined according to formula (1):

[0017] ,

[0018] In formula (1), H is the byproduct interference parameter, m1 is the peptide concentration, μ1 is the polymer viscosity, m0 is the standard peptide concentration, and μ01 is the standard polymer viscosity.

[0019] Further, determining the type of protein structural abnormality tendency of the initial structure detection solution according to the by-product interference yield includes:

[0020] If the by-product interference parameter is greater than or equal to the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure strong abnormality tendency type;

[0021] If the by-product interference parameter is less than the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure weak abnormality tendency type.

[0022] Furthermore, the analysis method of the collagen structure is determined according to the type of abnormal protein structure tendency of the initial structure detection liquid, including:

[0023] If the protein structure abnormal tendency type is a weak protein structure abnormal tendency type, a collagen sample solution is obtained, a spectral test is performed on the triple helix structure analysis solution, and the triple helix structure collagen content is determined according to the spectral test result to determine whether the structural integrity of the active collagen test solution meets the standard;

[0024] If the protein structure abnormality tendency type is a protein structure strong abnormality tendency type, a collagen sample solution is obtained, and the triple helix structure analysis solution is subjected to enzymatic hydrolysis detection. The amount of triple helix structure abnormality of collagen in the active collagen detection solution is determined based on the enzymatic hydrolysis detection result to determine whether there is an abnormality in the active collagen extraction process.

[0025] Further, performing a spectral test on the triple helix structure analysis solution includes:

[0026] Using a spectrometer to measure the triple helix structure analysis liquid several times;

[0027] A spectrum is drawn according to the average value of each detection and preprocessed to obtain a spectrum analysis graph;

[0028] The content of triple helix collagen is determined based on the curve corresponding to the triple helix collagen in the spectrum analysis diagram.

[0029] Further, the structure integrity of the active collagen test solution is determined to be in compliance with the standard according to the content of the triple helix structure collagen, including:

[0030] If the triple helix structure collagen content is greater than or equal to the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution meets the standard;

[0031] If the triple helix structure collagen content is less than the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution does not meet the standard.

[0032] Further, performing enzymatic detection on the triple helix structure analysis solution includes:

[0033] Adjusting the pH value of the triple helix structure analysis solution to a preset pH range;

[0034] Adding a preset mass of enzyme to the triple helix structure analysis solution after adjusting the pH value to obtain a system solution to be enzymatically hydrolyzed;

[0035] Adding a precipitation solution to the enzymatically hydrolyzed system solution and centrifuging the solution to obtain a collagen precipitate solution;

[0036] The content of hydroxyproline in the supernatant of the collagen precipitation solution was determined and recorded as the enzymatic test result.

[0037] Furthermore, the amount of abnormal triple helix structure of collagen in the active collagen test solution is determined according to the enzymatic test result. The amount of abnormal triple helix structure is determined according to formula (2):

[0038] ,

[0039] In formula (2), Q is the abnormal amount of triple helix structure, c1 is the hydroxyproline content corresponding to the enzymatic test result, c2 is the hydroxyproline content of the collagen sample solution, k is the ratio of hydroxyproline to collagen, and V is the volume of the collagen sample solution.

[0040] Further, determining whether there is an abnormality in the active collagen extraction process according to the abnormal amount of the triple helix structure includes:

[0041] If the abnormal amount of the triple helix structure of collagen in the active collagen test solution is greater than the abnormal amount of the standard structure, it is determined that there is an abnormality in the active collagen extraction process;

[0042] If the abnormal amount of the triple helix structure of collagen in the active collagen detection solution is less than or equal to the abnormal amount of the standard structure, it is determined that there is no abnormality in the active collagen extraction process.

[0043] Compared with the prior art, the beneficial effect of the present invention is that the initial structure detection liquid is obtained by preliminary centrifugation, and the by-product interference parameter is determined by by-product detection, so as to judge the abnormal tendency type of the protein structure. Based on this type, the corresponding analysis method is selected, and the content of triple helix structure collagen is determined by spectral testing, or the abnormal amount of triple helix structure of collagen is determined by enzymatic detection. The present invention can accurately evaluate the triple helix structure integrity of active collagen, and adaptively select non-destructive or destructive detection methods, which not only ensures the reliability of detection, but also avoids product waste, improves detection efficiency and accuracy, and in addition, through real-time monitoring and data analysis, provides an effective monitoring means for the collagen purification process, helps to optimize production conditions, improve collagen purification efficiency and product quality, and ensure the stability and functionality of active collagen.

[0044] Furthermore, in the present invention, the active collagen test solution may have various manifestations after centrifugation. For example, collagen with a destroyed triple helix structure becomes a polymer and sinks to the bottom, while the destroyed polypeptide is in the upper liquid. On the contrary, collagen with a complete triple helix structure is relatively stable, and the viscosity of the collagen polymer is higher than that of the collagen. Therefore, the by-product interference parameter of the initial structure test solution is calculated to characterize the tendency of the triple helix structure to be incomplete in the active collagen test solution, thereby providing data support for the subsequent determination of the type of abnormal protein structure tendency, facilitating the subsequent adaptive selection of further analysis methods for the collagen structure, and improving the accuracy of structural integrity detection.

[0045] Furthermore, in the present invention, the analysis method for subsequent analysis of the integrity of the triple helix structure of collagen is determined according to the type of abnormal protein structure tendency. In actual situations, the active collagen test solution with small by-product interference parameters is smaller than the collagen with destroyed structure. Therefore, in view of the above situation, spectral detection can be used to determine whether the content of structurally intact collagen in the active collagen test solution meets the requirements through a non-destructive detection method, thereby avoiding product waste while ensuring reliability.

[0046] Furthermore, in the present invention, destructive testing is performed on solutions with large by-product interference parameters. In actual situations, the active collagen detection solution with large by-product interference parameters has its structure destroyed to produce various by-products with a high collagen content. Therefore, selecting destructive testing can determine whether there are abnormalities in the preparation process of the active collagen detection solution, thereby adjusting the production process and ensuring the structural integrity of the extracted collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a flow chart of a method for detecting the integrity of the triple helix structure of active collagen according to an embodiment of the present invention;

[0048] Figure 2 A logic diagram for determining the type of abnormal protein structure tendency according to an embodiment of the present invention;

[0049] Figure 3 A logic diagram for determining whether the structural integrity of an active collagen test solution meets the standard according to an embodiment of the present invention;

[0050] Figure 4 This is a logic diagram for determining whether there is an abnormality in the active collagen extraction process according to an embodiment of the present invention. DETAILED DESCRIPTION

[0051] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0052] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0053] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is merely for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0054] See also Figure 1 As shown, it is a flow chart of a method for detecting the integrity of the triple helix structure of active collagen according to an embodiment of the present invention; the present invention provides a method for detecting the integrity of the triple helix structure of active collagen, comprising:

[0055] Step S1, obtaining an active collagen test solution to be tested, and performing preliminary centrifugation to obtain an initial structure test solution;

[0056] Step S2, obtaining the upper layer liquid and the bottom layer liquid of the initial structure detection liquid, performing by-product detection, and recording the by-product detection results respectively;

[0057] Step S3, determining the byproduct interference parameter of the initial structure detection solution according to each byproduct detection result, and determining the protein structure abnormality tendency type of the initial structure detection solution according to the byproduct interference yield;

[0058] Step S4, removing the upper layer of the initial structure detection solution to obtain a triple helix structure analysis solution, and determining an analysis method for the triple helix structure analysis solution according to the protein structure abnormality tendency type of the initial structure detection solution, including:

[0059] Obtaining a collagen sample solution, performing an enzymatic hydrolysis test on the triple helix structure analysis solution, and determining the abnormal amount of the triple helix structure of collagen in the active collagen test solution according to the enzymatic hydrolysis test result, so as to determine whether there is an abnormality in the active collagen extraction process;

[0060] Alternatively, a collagen sample solution is obtained, a spectral test is performed on the triple helix structure analysis solution, and the triple helix structure collagen content is determined according to the spectral test result to determine whether the structural integrity of the active collagen detection solution meets the standard.

[0061] In practice, the device for performing the centrifugal operation is preferably a centrifugal extractor, and the centrifugal speed in the preliminary centrifugation is selected within the range of [300g, 3000g], where g is the acceleration due to gravity.

[0062] The present invention obtains the initial structure detection liquid through preliminary centrifugation, and determines the by-product interference parameter through by-product detection, so as to judge the abnormal tendency type of the protein structure. Based on this type, the corresponding analysis method is selected, and the triple helix collagen content is determined by spectral testing, or the triple helix structure abnormality of collagen is determined by enzymatic detection. The present invention can accurately evaluate the triple helix structural integrity of active collagen, and adaptively select non-destructive or destructive detection methods, which not only ensures the reliability of the detection, but also avoids product waste, improves the detection efficiency and accuracy, and in addition, through real-time monitoring and data analysis, provides an effective monitoring means for the collagen purification process, helps to optimize production conditions, improves the purification efficiency and product quality of collagen, and ensures the stability and functionality of active collagen.

[0063] See also Figure 2 As shown, it is a logic diagram for determining the type of abnormal protein structure tendency according to an embodiment of the present invention, and the byproduct detection includes:

[0064] The upper layer liquid and the bottom layer liquid are sampled respectively, and recorded as upper layer sample and bottom layer sample;

[0065] detecting the polypeptide concentration in the supernatant and detecting the polymer viscosity in the bottom layer sample;

[0066] Among them, by-products include peptides and collagen polymers.

[0067] In practice, the polypeptide concentration can be determined by analytical methods, such as UV spectroscopy, amino acid analysis, or collagen quantification methods;

[0068] The viscosity of polymers can be measured by a viscometer, generally a rotational viscometer is used. A rotational viscometer can measure the viscosity of a liquid by a rotating rotor.

[0069] It is understandable that the destruction of the triple helix structure of collagen may produce polypeptides and collagen polymers, and centrifugation can allow the polypeptides with small mass to be separated into the upper liquid, while the collagen with a destroyed triple helix structure will form collagen polymers during the centrifugation process due to its unstable structure. The collagen polymers are at the bottom after centrifugation because their mass is greater than that of the collagen with a complete triple helix structure.

[0070] Specifically, the byproduct interference parameter of the initial structure detection solution is determined according to each byproduct detection result. The byproduct interference parameter is determined according to formula (1):

[0071] ,

[0072] In formula (1), H is the byproduct interference parameter, m1 is the peptide concentration, μ1 is the polymer viscosity, m0 is the standard peptide concentration, and μ01 is the standard polymer viscosity.

[0073] In implementation, the standard polypeptide concentration and the standard polymer concentration are determined based on the byproduct detection results of the qualified collagen solution.

[0074] Collagen itself is a larger molecule, while polymers are composed of multiple collagen molecules to form a more complex structure. The viscosity of polymers is usually higher than that of single collagen molecules. The higher the degree of cross-linking in the collagen polymer, the more complex the network structure it forms and the higher the viscosity.

[0075] Specifically, determining the type of protein structural abnormality tendency of the initial structure detection solution according to the by-product interference yield includes:

[0076] If the by-product interference parameter is greater than or equal to the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure strong abnormality tendency type;

[0077] If the by-product interference parameter is less than the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure weak abnormality tendency type.

[0078] In practice, the standard by-product interference parameter is selected in the interval [1.03,1.2].

[0079] In the present invention, the active collagen test liquid may have various manifestations after centrifugation. For example, collagen with a destroyed triple helix structure becomes a polymer and sinks to the bottom, while the destroyed polypeptide is in the upper liquid. On the contrary, collagen with a complete triple helix structure is relatively stable, and the viscosity of the collagen polymer is higher than that of the collagen. Therefore, the by-product interference parameter of the initial structure test liquid is calculated to characterize the tendency of the incomplete triple helix structure in the active collagen test liquid, thereby providing data support for the subsequent determination of the type of abnormal protein structure tendency, facilitating the subsequent adaptive selection of further analysis methods for the collagen structure, and improving the accuracy of structural integrity detection.

[0080] Specifically, the analysis method of the collagen structure is determined according to the type of abnormal protein structure tendency of the initial structure detection liquid, including:

[0081] If the protein structure abnormal tendency type is a weak protein structure abnormal tendency type, a collagen sample solution is obtained, a spectral test is performed on the triple helix structure analysis solution, and the triple helix structure collagen content is determined according to the spectral test result to determine whether the structural integrity of the active collagen test solution meets the standard;

[0082] If the protein structure abnormality tendency type is a protein structure strong abnormality tendency type, a collagen sample solution is obtained, and the triple helix structure analysis solution is subjected to enzymatic hydrolysis detection. The amount of triple helix structure abnormality of collagen in the active collagen detection solution is determined based on the enzymatic hydrolysis detection result to determine whether there is an abnormality in the active collagen extraction process.

[0083] See also Figure 3 As shown, it is a logic diagram for determining whether the structural integrity of the active collagen detection solution meets the standard according to an embodiment of the present invention, and the spectrum test of the triple helix structure analysis solution includes:

[0084] Using a spectrometer to measure the triple helix structure analysis liquid several times;

[0085] A spectrum is drawn according to the average value of each detection and preprocessed to obtain a spectrum analysis graph;

[0086] The content of triple helix collagen is determined based on the curve corresponding to the triple helix collagen in the spectrum analysis diagram.

[0087] In implementation, the spectrum tester is a circular dichroism spectrometer equipped with an ultraviolet-visible spectrum range (190-250 nm) so as to cover the wavelength range of collagen characteristics; the optical path length of the optical path cell during the measurement process is 1 mm or 0.1 mm;

[0088] During the measurement process, the triple helix structure analysis liquid is loaded into the optical path cell, and the CD measurement number is not less than 3 times, preferably 5 times; after the measurement, a spectrum is obtained, and the spectrum is a CD spectrum that will show the difference in light absorption, usually in the range of 190 to 250 nm.

[0089] The spectrum was preprocessed, including smoothing to reduce noise; the signal of the blank sample was subtracted to obtain the net CD signal, and the spectrum analysis graph was obtained.

[0090] During the test, ensure that the concentration of collagen is between 0.1 and 1 mg / mL.

[0091] Because triple helical collagen usually shows a significant negative peak at 205 nm, the content of triple helical collagen can be obtained by integrating the intensity of the 205 nm peak in the spectrum analysis graph.

[0092] Specifically, the content of triple helix collagen determines whether the structural integrity of the active collagen test solution meets the standards, including:

[0093] If the triple helix structure collagen content is greater than or equal to the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution meets the standard;

[0094] If the triple helix structure collagen content is less than the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution does not meet the standard.

[0095] In implementation, the content of standard triple helix structure collagen is determined based on qualified collagen samples by performing spectral testing on the qualified collagen samples.

[0096] In the present invention, the analysis method for subsequent analysis of the integrity of the triple helix structure of collagen is determined according to the type of abnormal protein structure tendency. In actual situations, the active collagen test solution with small by-product interference parameters is less than the collagen with destroyed structure. Therefore, in view of the above situation, spectral detection can be used to determine whether the content of structurally intact collagen in the active collagen test solution meets the requirements through a non-destructive detection method, thereby avoiding product waste while ensuring reliability.

[0097] See also Figure 4 As shown, it is a logic diagram for determining whether there is an abnormality in the active collagen extraction process according to an embodiment of the present invention. The enzymatic detection of the triple helix structure analysis solution includes:

[0098] Adjusting the pH value of the triple helix structure analysis solution to a preset pH range;

[0099] Adding a preset mass of enzyme to the triple helix structure analysis solution after adjusting the pH value to obtain a system solution to be enzymatically hydrolyzed;

[0100] Adding a precipitation solution to the enzymatically hydrolyzed system solution and centrifuging the solution to obtain a collagen precipitate solution;

[0101] The content of hydroxyproline in the supernatant of the collagen precipitation solution was determined and recorded as the enzymatic test result.

[0102] In the implementation, the preset pH interval is [3.6, 3.7];

[0103] The enzyme is preferably trypsin, and the mass ratio of trypsin to total protein in the sample solution is 1:20 to 1:100;

[0104] The enzymatic hydrolysis system solution is placed under constant temperature oscillation conditions for enzymatic hydrolysis, the enzymatic hydrolysis temperature is controlled at 15-30°C, the oscillation speed is 50-120rpm, and the enzymatic hydrolysis time is 16-24 hours;

[0105] The precipitation solution is a trichloroacetic acid solution with a mass concentration of 10% to 30%. The centrifugation parameters are 4°C, 5000rpm and 10 minutes. Collagen with a complete triple helix structure is precipitated in the collagen precipitation solution, while collagen with a destroyed triple helix structure is dissolved in the supernatant.

[0106] The concentration of the collagen sample solution is the same as that of the collagen in the initial structure detection solution. The same enzymatic hydrolysis test is used on the collagen sample solution to obtain the hydroxyproline content of the sample solution, which is recorded as the sample reference result.

[0107] It is understood that collagen with a complete triple helix structure cannot be enzymatically digested by trypsin, while collagen with a destroyed or partially destroyed triple helix structure can be enzymatically digested into polypeptides by trypsin. By measuring the change in the content of hydroxyproline in collagen before and after enzymatic digestion, the content of collagen with a complete triple helix structure in collagen can be quantitatively analyzed.

[0108] Specifically, the amount of abnormal triple helix structure of collagen in the active collagen test solution is determined according to the enzymatic test result. The amount of abnormal triple helix structure is determined according to formula (2):

[0109] ,

[0110] In formula (2), Q is the abnormal amount of triple helix structure, c1 is the hydroxyproline content corresponding to the enzymatic test result, c2 is the hydroxyproline content of the collagen sample solution, k is the ratio of hydroxyproline to collagen, and V is the volume of the collagen sample solution.

[0111] In practice, hydroxyproline is a characteristic amino acid in collagen, and its proportion in collagen is relatively stable, with k generally being 0.14 (ie, 14 grams of hydroxyproline are contained in every 100 grams of collagen).

[0112] Specifically, determining whether there is an abnormality in the active collagen extraction process according to the abnormal amount of the triple helix structure includes:

[0113] If the abnormal amount of the triple helix structure of collagen in the active collagen test solution is greater than the abnormal amount of the standard structure, it is determined that there is an abnormality in the active collagen extraction process;

[0114] If the abnormal amount of the triple helix structure of collagen in the active collagen detection solution is less than or equal to the abnormal amount of the standard structure, it is determined that there is no abnormality in the active collagen extraction process.

[0115] In implementation, the standard structural abnormality amount is 0.05 to 0.1 times the content of abnormal triple helical collagen in the collagen sample solution.

[0116] In the present invention, destructive testing is performed on solutions with large by-product interference parameters. In actual situations, the active collagen detection solution with large by-product interference parameters has its structure destroyed to produce various by-products with a high collagen content. Therefore, destructive testing is selected to determine whether there are abnormalities in the preparation process of the active collagen detection solution, thereby adjusting the production process and ensuring the structural integrity of the extracted collagen.

[0117] If the active collagen triple helix structure integrity detection method of the present invention is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of the technical solution can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including a number of instructions to enable a computer device (which can be a personal computer, server, or network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk and other media that can store program codes.

[0118] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the integrity of the triple helix structure of active collagen, characterized in that: include: Obtaining active collagen test solution to be tested, performing preliminary centrifugation to obtain initial structure test solution; Obtaining the upper layer liquid and the bottom layer liquid of the initial structure detection liquid, performing by-product detection, and recording the by-product detection results respectively; Determine the byproduct interference parameter of the initial structure detection solution according to the polypeptide concentration and the collagen polymer viscosity in each of the byproduct detection results, and determine the protein structure abnormality tendency type of the initial structure detection solution according to the byproduct interference parameter, include, If the by-product interference parameter is greater than or equal to the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure strong abnormality tendency type; If the by-product interference parameter is less than the standard by-product interference parameter, the protein structure abnormality tendency type of the initial structure detection solution is a protein structure weak abnormality tendency type; The upper layer of the initial structure detection liquid is removed to obtain a triple helix structure analysis liquid, and the analysis method of the triple helix structure analysis liquid is determined according to the protein structure abnormality tendency type of the initial structure detection liquid, including: If the protein structure abnormal tendency type is a protein structure strong abnormal tendency type, obtaining a collagen sample solution, performing an enzymatic hydrolysis test on the triple helix structure analysis solution, and determining the amount of triple helix structure abnormality of the collagen in the active collagen test solution according to the enzymatic hydrolysis test result to determine whether there is an abnormality in the active collagen extraction process; If the protein structure abnormal tendency type is a weak protein structure abnormal tendency type, obtain a collagen sample solution, perform a spectral test on the triple helix structure analysis solution, and determine the triple helix structure collagen content based on the spectral test results to determine whether the structural integrity of the active collagen test solution meets the standard.

2. The method for detecting the integrity of the triple helix structure of active collagen according to claim 1, characterized in that: The by-product detection includes, The upper layer liquid and the bottom layer liquid are sampled respectively, and recorded as upper layer sample and bottom layer sample; detecting the polypeptide concentration in the upper layer sample and detecting the polymer viscosity in the bottom layer sample; Among them, by-products include peptides and collagen polymers.

3. The method for detecting the integrity of the active collagen triple helix structure according to claim 2, characterized in that: The byproduct interference parameter of the initial structure detection solution is determined according to each byproduct detection result. The byproduct interference parameter is determined according to formula (1): ， In formula (1), H is the byproduct interference parameter, m1 is the peptide concentration, μ1 is the polymer viscosity, m0 is the standard peptide concentration, and μ01 is the standard polymer viscosity.

4. The method for detecting the integrity of the triple helix structure of active collagen according to claim 3, characterized in that: The spectrum test of the triple helix structure analysis liquid includes: Using a spectrometer to measure the triple helix structure analysis liquid several times; A spectrum is drawn according to the average value of each detection and preprocessed to obtain a spectrum analysis graph; The content of triple helix collagen is determined based on the curve corresponding to the triple helix collagen in the spectrum analysis diagram.

5. The method for detecting the integrity of the active collagen triple helix structure according to claim 4, characterized in that: The content of triple helix collagen determines whether the structural integrity of the active collagen test solution meets the standards, including: If the triple helix structure collagen content is greater than or equal to the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution meets the standard; If the triple helix structure collagen content is less than the standard triple helix structure collagen content, it is determined that the structural integrity of the active collagen test solution does not meet the standard.

6. The method for detecting the integrity of the triple helix structure of active collagen according to claim 3, characterized in that: The enzymatic detection of the triple helix structure analysis solution comprises: Adjusting the pH value of the triple helix structure analysis solution to a preset pH range; Adding a preset mass of enzyme to the triple helix structure analysis solution after adjusting the pH value to obtain a system solution to be enzymatically hydrolyzed; Adding a precipitation solution to the enzymatically hydrolyzed system solution and centrifuging the solution to obtain a collagen precipitate solution; The content of hydroxyproline in the supernatant of the collagen precipitation solution was determined and recorded as the enzymatic test result.

7. The method for detecting the integrity of the triple helix structure of active collagen according to claim 6, characterized in that: The abnormal amount of triple helix structure of collagen in the active collagen test solution is determined according to the enzymatic test result. The abnormal amount of triple helix structure is determined according to formula (2): , In formula (2), Q is the abnormal amount of triple helix structure, c1 is the hydroxyproline content corresponding to the enzymatic test result, c2 is the hydroxyproline content of the collagen sample solution, k is the ratio of hydroxyproline to collagen, and V is the volume of the collagen sample solution.

8. The method for detecting the integrity of the triple helix structure of active collagen according to claim 7, characterized in that: Determining whether there is an abnormality in the active collagen extraction process according to the abnormal amount of the triple helix structure includes: If the abnormal amount of the triple helix structure of collagen in the active collagen test solution is greater than the abnormal amount of the standard structure, it is determined that there is an abnormality in the active collagen extraction process; If the abnormal amount of the triple helix structure of collagen in the active collagen detection solution is less than or equal to the abnormal amount of the standard structure, it is determined that there is no abnormality in the active collagen extraction process.

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