A method for determining the degree of self-assembly of natural collagen

Abstract

The present invention belongs to the field of detection, and discloses a method for determining the self-assembly degree of natural collagen, which includes dissolving Silole to obtain a Silole solution; mixing the Silole solution and the collagen solution to be tested to obtain a mixture, and performing a primary incubation; after the primary incubation is completed, dialyzing to remove free small molecules to obtain a collagen / Silole complex solution, and measuring the initial fluorescence intensity F0 of the collagen / Silole complex solution; incubating the collagen / Silole complex solution for self-assembly, and measuring the fluorescence intensity F1 of the collagen / Silole complex solution; calculating the self-assembly degree of natural collagen according to F0 and F1. Compared with the prior art, (1) the detection method provided by the present invention has the advantages of simple operation (without a cumbersome sample treatment process) and high sensitivity (able to detect the self-assembly of low-concentration natural collagen below 0.1 mg / mL); (2) it can be used for monitoring the self-assembly of type I collagen from different sources, with a wide monitoring range.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for determining the self-assembly degree of natural collagen. Background Art

[0002] Collagen is one of the most abundant and widely distributed protein species in multicellular organisms. It has a characteristic (Gly-X-Y)n sequence pattern and presents a unique triple-helix structure. As the main component of the extracellular matrix, collagen plays a crucial role in the development, migration, shaping, and function of body tissues. At the same time, due to the excellent biocompatibility, low immunogenicity, and biodegradability of collagen, it has been widely used in the fields of tissue engineering, clinical medicine, biomaterials, etc.

[0003] Self-assembly is one of the most important molecular behaviors of natural collagen. In vivo, collagen forms collagen fibers through self-assembly and serves as the main component of the extracellular matrix in connective tissues, thereby providing structural and mechanical support for cells and also providing a three-dimensional microenvironment for cell proliferation, adhesion, migration, differentiation, etc. In vitro, natural collagen can also self-assemble into collagen fibers / collagen gels under certain conditions such as concentration, pH, and temperature. A large number of studies have shown that self-assembly is one of the most important means to improve the thermal stability, enzyme resistance, and biomechanical properties of natural collagen. Moreover, the collagen fibers / collagen gels formed by in vitro self-assembly have been widely used in the fields of scaffold materials for tissue engineering, artificial organs, and tissue repair. Therefore, it is very important to construct a simple, sensitive, and efficient method for monitoring the self-assembly of natural collagen, which will provide theoretical guidance for the construction of collagen fibers / collagen gels with excellent performance.

[0004] CN115112904A discloses a method for detecting the integrity of the triple-helix structure of collagen by using collagen peptide-coated metal nanoclusters and disulfide nanomaterials. The method includes: 1) obtaining collagen peptide-coated metal nanoclusters; 2) mixing the collagen peptide-coated metal nanoclusters with a disulfide solution; 3) mixing the mixture obtained in step 2) with a collagen standard and a collagen sample to be tested respectively, and measuring the maximum fluorescence intensity in the range of 360 nm - 550 nm at an excitation wavelength of 340 nm, which are respectively denoted as F 标 、F A , and comparing with the fluorescence intensity F0 of the blank system. The integrity of the triple-helix structure in the sample = (F A - F0) / (F 标 - F0)*100%. The detection method provided by the present invention has the advantages of simple operation, high sensitivity, short detection time, etc.; it can be used for detecting the integrity of the triple-helix structure of almost all type I collagen on the market, and has a wide detection range.

[0005] CN110426356A discloses a natural collagen / nano - gold composite and a method for synchronously monitoring the self - assembly of natural collagen. 1) Dissolve natural collagen in an acetic acid aqueous solution to obtain a natural collagen solution; 2) Mix and blend the natural collagen solution with a chloroauric acid solution; 3) Mix and blend a reducing agent solution with the solution obtained in step 2); 4) Dialyze the solution obtained in step 3) to obtain a collagen / nano - gold composite before self - assembly, and measure its absorbance at 515 - 525 nm, denoted as A0; 5) Self - assemble the solution obtained in step 4), and measure the absorbance of the collagen / nano - gold composite after self - assembly at 515 - 525 nm, denoted as A1; 6) When A1:A0 > 1, it is determined that the self - assembly is completed; when A1:A0 ≤ 1, continue the self - assembly. The monitoring method provided by the present invention is simple to operate, low in cost, and can present multiple information on the self - assembly of collagen.

[0006] CN109884153A discloses a new method for determining the integrity of the triple - helix structure of type I collagen, belonging to the field of biological detection technology. Its 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 into small - molecular - weight peptide segments by proteases. SDS - PAGE can be used for visual analysis of the decomposed peptide segments. The more decomposed peptide segments there are, the worse the integrity of the triple - helix structure of the collagen sample; conversely, if there are no small - molecular - weight peptide segments that are not decomposed in SDS - PAGE, it indicates that the integrity of the triple - helix structure of the collagen sample is better. The present invention can also quantitatively detect dimer proteins in a collagen sample through a gel electrophoresis imaging system or a thin - layer scanning system, thereby calculating the retention rate of the triple - helix structure of the collagen sample, making up for the deficiency of qualitative detection. By using this method to judge the integrity of the triple - helix structure of collagen, the results have good reproducibility, strong specificity, are simple to operate, and can be used for the detection of collagen products.

[0007] CN116735506A discloses a method for detecting the triple - helix ratio of collagen. The method for detecting the triple - helix ratio of collagen in the present invention includes: mixing a triple - helix collagen standard product and a collagen monomer standard product in different ratios to obtain collagen standard products with different triple - helix ratios, and using the Sirius red staining method to detect the absorbance values of the collagen standard products with different triple - helix ratios, and making a standard curve based on the triple - helix ratio and the absorbance value. This method can achieve rapid, low - cost quantification of the triple - helix ratio of collagen, has high specificity, accuracy, and good repeatability, and can be widely applied to the collagen industry.

[0008] CN116888143A discloses and provides a method for determining the collagen content by using collagen hybridization peptides. The present disclosure provides a method for quantifying the amount of disrupted collagen (such as triple helix-disrupted collagen) and / or total collagen. The present disclosure provides a method for quantifying the amount of disrupted collagen (such as triple helix-disrupted collagen) as a part of the total collagen.

[0009] The detection method of the recombinant collagen triple helix structure in CN116840160A. The method includes the following steps: pretreatment step: dissolving the recombinant collagen in a solvent and standing to obtain a collagen solution to be detected; circular dichroism detection step: performing circular dichroism detection on the collagen solution to be detected, and determining whether the recombinant collagen has a triple helix structure according to the circular dichroism spectrum. This method is based on circular dichroism technology, can effectively detect the triple helix structure of recombinant collagen produced by microbial fermentation, and has significant advantages such as rapid, simple operation and short duration. At the same time, the present invention effectively improves the detection accuracy of the triple helix structure by optimizing the pretreatment conditions and circular dichroism detection conditions of the recombinant collagen.

[0010] Generally speaking, the main methods for monitoring the self-assembly of native collagen are as follows: (1) Turbidity method: During the self-assembly of native collagen, as collagen monomers gradually and orderly assemble to form collagen fibers, the turbidity of the solution gradually increases, and three typical stages, namely the lag phase, the growth phase and the plateau phase, are formed. The turbidity method is simple to operate, but its sensitivity is relatively low, and it can only be used to monitor the middle and late stages (growth phase and plateau phase) of self-assembly. The microscopic changes in the early stage (lag phase) of self-assembly cannot be monitored by the turbidity method. (2) Microscopy techniques: Since the self-assembly of collagen forms collagen fibers with a 3D network structure, a fibrous structure with specific dimensions and alternating light and dark periodic striations can be observed through scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. However, microscopy techniques require large-scale instrument equipment, the sample preparation cost is relatively high, the sample preparation is cumbersome, and the testing time is relatively long. (3) Metal nanoparticle technology: Utilize the unique optical properties of nanoparticles to monitor the self-assembly of collagen. This technology is simple to operate, but the metal nanoparticles are expensive. At the same time, the above technologies can only be used to monitor the self-assembly of collagen at a relatively high concentration (>0.1 mg / mL), and there is still a lack of simple and efficient technical means for monitoring the self-assembly of low-concentration collagen. Summary of the Invention

[0011] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a method for determining the degree of self-assembly of native collagen.

[0012] The technical solution adopted by the present invention is:

[0013] A method for determining the self-assembly degree of type I collagen, comprising the following steps:

[0014] Dissolve a silole compound with aggregation-induced emission characteristics to obtain a silole compound solution;

[0015] Dissolve the collagen to be tested to obtain a collagen solution to be tested;

[0016] Mix the silole compound solution and the collagen solution to be tested to obtain a mixture, and perform the first incubation;

[0017] After the first incubation, dialyze to remove free small molecules to obtain a collagen / Silole complex solution, and measure the initial fluorescence intensity F0 of the collagen / Silole complex solution;

[0018] Incubate the collagen / Silole complex solution for self-assembly, and measure the fluorescence intensity F1 of the collagen / Silole complex solution;

[0019] Calculate the self-assembly degree of native collagen according to F0 and F1.

[0020] In some examples of the method, the general structural formula of the silole compound is In the formula, each substituent is as follows:

[0021] R1 = CH3, C6H5, or

[0022] R2 = CH3, C6H5,

[0023] R3 = H or

[0024] R4 = H or

[0025] X is Cl, Br or I. Preferably, R3 = R4.

[0026] Particularly, the silole compound is

[0027] In some examples of the method, the temperature of the first incubation does not exceed 15 °C.

[0028] In some examples of the method, the incubation time of the first incubation is 1-4 hours.

[0029] In some examples of the method, the concentration of the silole compound in the mixture is 0.25-1 μmol / L.

[0030] In some method examples, the concentration of collagen in the mixed solution is 0.05 - 1 mg / mL.

[0031] In some method examples, the temperature for incubating self-assembly is 25 - 35 °C.

[0032] In some method examples, the solvent for silole compounds is water, ethanol, or an aqueous solution of ethanol.

[0033] In some method examples, the dialysis solution used during dialysis is a phosphate buffer solution.

[0034] In some method examples, the temperature during dialysis does not exceed 15 °C.

[0035] In some method examples, the cut-off molecular weight for dialysis is 3000 - 20000 Da.

[0036] In some method examples, the solvent for the collagen solution to be measured is an aqueous acetic acid solution with a concentration of 0.1 - 0.5 mol / L.

[0037] In some method examples, the larger the F1 / F0 value, the higher the degree of self-assembly of type I collagen.

[0038] The above features can be arbitrarily combined without conflict.

[0039] The beneficial effects of the present invention are:

[0040] Compared with the prior art, (1) the detection method provided by the present invention has advantages such as simple operation (without a cumbersome sample processing process) and high sensitivity (able to detect the self-assembly of low-concentration natural collagen below 0.1 mg / mL); (2) it can be used for monitoring the self-assembly of type I collagen from different sources, with a wide monitoring range. Description of the Drawings

[0041] Figure 1 It is the circular dichroism spectra of collagen and the collagen / Silole complex for Example 1. Collagen shows a positive peak and a negative peak at 221 nm and 197 nm respectively, indicating that natural collagen has a triple helix structure. Compared with pure collagen, the circular dichroism spectrum of the collagen / Silole complex shows no obvious change, indicating that the introduction of Silole does not change the triple helix structure of collagen, thus ensuring that the collagen / Silole complex still has self-assembly performance.

[0042] Figure 2Fluorescence spectra of the collagen / Silole complex before and after self-assembly at a low concentration (0.05 mg / mL) in Example 1. Before the self-assembly of native collagen, the fluorescence intensity of the complex was low; after self-assembly, the fluorescence intensity of the complex increased significantly.

[0043] Figure 3 For Comparative Example 1, the fluorescence spectra of the collagen complex before and after self-assembly showed no obvious change in fluorescence intensity. Detailed implementation manners

[0044] A method for determining the degree of self-assembly of type I collagen includes the following steps:

[0045] Dissolve a silole compound with aggregation-induced emission characteristics to obtain a silole compound solution;

[0046] Dissolve the collagen to be tested to obtain a collagen solution to be tested;

[0047] Mix the silole compound solution and the collagen solution to be tested to obtain a mixture, and perform the first incubation;

[0048] After the first incubation, dialyze to remove free small molecules to obtain a collagen / Silole complex solution, and measure the initial fluorescence intensity F0 of the collagen / Silole complex solution;

[0049] Incubate the collagen / Silole complex solution for self-assembly, and measure the fluorescence intensity F1 of the collagen / Silole complex solution;

[0050] Calculate the degree of self-assembly of native collagen based on F0 and F1.

[0051] Research shows that Silole does not affect the self-assembly of collagen. After redissolving and reassembling the collagen to be tested, its degree of self-assembly is basically the same as that of the original collagen.

[0052] In some examples of the method, the general structural formula of the silole compound is In the formula, each substituent is as follows:

[0053] R1 = CH3, C6H5, or

[0054] R2 = CH3, C6H5,

[0055] R3 = H or

[0056] R4 = H or

[0057] X is Cl, Br or I. Preferably, R3 = R4.

[0058] These silole compounds have good solubility in water and / or ethanol solution, can effectively bind well with the dissolved single-stranded collagen, and can tolerate dialysis.

[0059] In particular, the silole compounds are

[0060] The temperature of the primary incubation is controlled below the self-assembly starting temperature of collagen, which is beneficial to reducing the detection error. In some examples of the method, the temperature of the primary incubation does not exceed 15 °C.

[0061] The incubation time of the primary incubation is preferably such that the silole compounds are fully adsorbed on the collagen. In some examples of the method, the incubation time of the primary incubation is 1 - 4 hours. Research shows that an incubation time of 1 - 4 hours can ensure the full adsorption of the silole compounds, which is beneficial to obtaining more accurate detection results.

[0062] In some examples of the method, the concentration of the silole compounds in the mixture is 0.25 - 1 μmol / L. This concentration can fully meet the detection requirements, and at the same time, it avoids the waste of the silole compounds, in view of reducing the detection cost.

[0063] In some examples of the method, the concentration of collagen in the mixture is 0.05 - 1 mg / mL.

[0064] In some examples of the method, the temperature of the incubation for self-assembly is 25 - 35 °C.

[0065] In some examples of the method, the solvent of the silole compounds is water, ethanol, or an aqueous solution of ethanol.

[0066] The dialysis solution as long as it does not affect the adsorption of the silole compounds to collagen and does not cause collagen denaturation is fine. In some examples of the method, the dialysis solution used during dialysis is a phosphate buffer solution.

[0067] The temperature during dialysis should be such that collagen does not undergo self-assembly. In some examples of the method, the temperature during dialysis does not exceed 15 °C.

[0068] During dialysis, it is sufficient to effectively remove the unadsorbed silole compounds and retain the collagen molecules. In some examples of the method, the cut-off molecular weight for dialysis is 3000 - 20000 Da. This can meet the requirements and has a certain efficiency.

[0069] The solvent for dissolving the collagen to be measured should be able to unwind the triple-helix structure of collagen without causing collagen denaturation. In some examples of the method, the solvent of the collagen solution to be measured is an aqueous acetic acid solution with a concentration of 0.1 - 0.5 mol / L.

[0070] In some examples of the method, the larger the F1 / F0 value, the higher the self-assembly degree of type I collagen.

[0071] The above features can be combined arbitrarily without conflict.

[0072] Aggregation-Induced Emission (AIE) technology is a unique luminescence phenomenon first proposed by the team of Academician Ben Zhong Tang of The Hong Kong University of Science and Technology in 2001. AIE refers to a class of molecules that do not emit light or emit weakly in solution but emit significantly stronger light after aggregation. This luminescence phenomenon is opposite to the phenomenon of traditional fluorescent materials that emit weaker light or quenching (Aggregation-Caused Quenching, ACQ) when aggregated. Materials based on AIE technology show broad application prospects in many fields due to their unique luminescence characteristics and mechanisms.

[0073] There are many types of AIE materials that have been developed. Due to the different properties of AIE materials, their application fields are also different (see: Liu Huan, Xie Yulong. Research progress of organic materials with aggregation-induced emission properties [J]. Functional Materials, 2023, 54(5): 5075 - 5085.; Yang Mingdi, Chen Guangmei, Zhou Hongping, etc. Research progress of aggregation-induced emission materials [J]. 2022(3)). In the field of biological detection, aggregation-induced emission is affected by various factors such as solvents and analytes. In most cases, satisfactory detection results cannot be obtained based on AIE technology.

[0074] The molecules of polyaryl-substituted siloles have a special three-dimensional spatial structure. Taking Hexaphenylsilole (HPS) as an example, the five-membered ring inside HPS serves as the core, and the six benzene rings on the periphery form a helical configuration. Since the dihedral angle between the benzene rings on the periphery and the central five-membered ring is approximately 60°, which is different from the situation without steric hindrance in the traditional case, due to the influence of the helical structure, the middle C-C bond shows more of the properties of a single bond, that is, it is more likely to rotate and twist. Therefore, in a dilute solution, the benzene rings on the periphery can rotate very freely, thereby promoting non-radiative decay and inhibiting the formation of excitons into photons, resulting in fluorescence quenching. When the molecules aggregate, the rotational motion of the benzene rings on the periphery is restricted, thereby reducing the non-radiative decay caused by rotation, and thus enhancing the aggregation-induced emission. The inventors unexpectedly found that Silole can bind well to type I collagen without affecting the self-assembly of collagen, forming a natural collagen (single-chain) / Silole complex. For the natural collagen / Silole complex, before the self-assembly of natural collagen, Silole is in a solution state with a very low fluorescence intensity; when the natural collagen self-assembles, Silole aggregates along with the self-assembly of collagen, resulting in an enhanced fluorescence intensity. Based on this, the inventors developed a new method for monitoring the self-assembly of natural collagen.

[0075] The technical solution of the present invention will be further described below in conjunction with embodiments. In the following examples, the structural formula of Silole-1 is The structural formula of Silole-2 is The structural formula of Silole-3 is

[0076] Example 1

[0077] Weigh an appropriate amount of bovine Achilles tendon collagen sample, prepare 5 mL of a sample with a concentration of 0.05 mg / mL using 0.5 mol / L acetic acid, add 0.25 mL of a 20 μmol / L Silole-2 aqueous solution, incubate at low temperature for 4 hours, then dialyze with a PBS buffer solution with a pH of 7.4 (the molecular weight cut-off of the dialysis bag is 10,000 Daltons), change the dialysis solution every 3 h, and dialyze for 48 hours to obtain a bovine Achilles tendon collagen / Silole-2 complex. Take 2 mL of the complex and incubate it in an incubator at 35 °C for 2.5 hours, and measure the fluorescence spectra of the complex before and after self-assembly using a fluorescence spectrophotometer.

[0078] Example 2

[0079] Weigh an appropriate amount of porcine skin collagen sample, and prepare 5 mL of a sample with a concentration of 0.1 mg / mL using 0.1 mol / L acetic acid. Add 0.5 mL of 10 μmol / L Silole-1 aqueous solution, incubate at low temperature for 3 hours, then dialyze with a PBS buffer solution with a pH of 8.0 (the molecular weight cut-off of the dialysis bag is 10,000 Daltons), change the dialysis fluid every 3 h, and dialyze for 36 hours to obtain a porcine skin collagen / Silole-1 complex. Take 2 mL of the complex and incubate it in an incubator at 35 °C for 3 hours, and test the fluorescence spectra of the complex before and after self-assembly using a fluorescence spectrophotometer.

[0080] Example 3

[0081] Weigh an appropriate amount of grass carp skin collagen sample, and prepare 5 mL of a sample with a concentration of 0.5 mg / mL using 0.5 mol / L acetic acid. Add 0.5 mL of 10 μmol / L Silole-2 aqueous solution, incubate at low temperature for 3 hours, then dialyze with a PBS buffer solution with a pH of 8.0 (the molecular weight cut-off of the dialysis bag is 10,000 Daltons), change the dialysis fluid every 3 h, and dialyze for 48 hours to obtain a grass carp skin collagen / Silole-2 complex. Take 2 mL of the complex and incubate it in an incubator at 30 °C for 2.5 hours, and test the fluorescence spectra of the complex before and after self-assembly using a fluorescence spectrophotometer.

[0082] Example 4

[0083] Weigh an appropriate amount of bovine Achilles tendon collagen sample, and prepare 5 mL of a sample with a concentration of 0.1 mg / mL using 0.5 mol / L acetic acid. Add 0.5 mL of 10 μmol / L Silole-3 aqueous solution, incubate at low temperature for 4 hours, then dialyze with a PBS buffer solution with a pH of 7.6 (the molecular weight cut-off of the dialysis bag is 10,000 Daltons), change the dialysis fluid every 3 h, and dialyze for 48 hours to obtain a bovine Achilles tendon collagen / Silole-3 complex. Take 2 mL of the complex and incubate it in an incubator at 35 °C for 3 hours, and test the fluorescence spectra of the complex before and after self-assembly using a fluorescence spectrophotometer.

[0084] Figure 1 It is the circular dichroism spectra of bovine Achilles tendon collagen and bovine Achilles tendon collagen / Silole-2 complex in Example 1. Bovine Achilles tendon collagen shows a positive peak and a negative peak at 221 nm and 197 nm respectively, indicating that it has a complete triple helix structure. Compared with the individual bovine Achilles tendon collagen, the circular dichroism spectrum of the bovine Achilles tendon collagen / Silole-2 complex has no obvious change, indicating that the introduction of Silole-2 does not change the triple helix structure of bovine Achilles tendon collagen, thus ensuring that the collagen / Silole-2 complex still has self-assembly performance.

[0085] Figure 2 Fluorescence spectra of the bovine Achilles tendon collagen / Silole-2 complex before and after self-assembly at a low concentration (0.05 mg / mL) in Example 1. Before the bovine Achilles tendon collagen was assembled, the fluorescence intensity of the complex was low; after self-assembly, the fluorescence intensity of the complex increased significantly.

[0086] Comparative Example 1

[0087] The aggregation luminescent substance used was TPE-(COOH)4, and its structural formula was

[0088] An appropriate amount of bovine Achilles tendon collagen sample was weighed and made into 5 mL of a sample with a concentration of 0.05 mg / mL using 0.5 mol / L acetic acid. 0.25 mL of a 20 μmol / L aqueous solution of TPE-(COOH)4 was added, and after incubating at low temperature for 4 hours, dialysis was carried out using a PBS buffer solution with a pH of 7.4 (the molecular weight cut-off of the dialysis bag was 10,000 Daltons), and the dialysis solution was changed every 3 h. After dialysis for 48 hours, a bovine Achilles tendon collagen / TPE-(COOH)4 complex was obtained. 2 mL of the complex was incubated in an incubator at 35 °C for 2.5 hours, and the fluorescence spectra of the complex before and after self-assembly were measured by a fluorescence spectrophotometer. The fluorescence intensity did not change significantly ( Figure 3 ).

[0089] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for determining the degree of self-assembly of type I collagen, characterized in that, It includes the following steps: Dissolve the silole compound with aggregation-induced emission characteristics to obtain a silole compound solution; Dissolve the collagen to be tested to obtain a collagen solution to be tested; Mix the silole compound solution and the collagen solution to be tested to obtain a mixture. The concentration of the silole compound in the mixture is 0.25 - 1 μmol / L, and perform the primary incubation; After the primary incubation is completed, dialyze to remove free small molecules to obtain a collagen / Silole complex solution, and measure the initial fluorescence intensity F0 of the collagen / Silole complex solution; Incubate the collagen / Silole complex solution for self-assembly, and measure the fluorescence intensity F1 of the collagen / Silole complex solution; Calculate the self-assembly degree of native collagen according to F0 and F1; The structural formula of the silole compound is 2. The method according to claim 1, characterized in that, The temperature of the primary incubation does not exceed 15°C and / or the incubation time is 1 - 4 hours.

3. The method according to claim 1, characterized in that, The concentration of collagen in the mixture is 0.05 - 1 mg / mL.

4. The method according to claim 1, characterized in that The temperature of the incubation for self-assembly is 25 - 35°C.

5. The method according to claim 1, wherein The solvent of the silole compound is water, ethanol, or an aqueous solution of ethanol.

6. The method according to claim 1, characterized in that, The dialysis solution used during dialysis is a phosphate buffer solution and / or the temperature during dialysis does not exceed 15°C.

7. The method according to claim 1, wherein The solvent of the collagen solution to be tested is an aqueous acetic acid solution with a concentration of 0.1 - 0.5 mol / L.

8. The method according to claim 1, characterized in that The larger the F1 / F0 value, the higher the self-assembly degree of type I collagen.

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