A method for detecting the proportion of collagen triple helix

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a detection method of collagen triple helix proportion. The detection method of collagen triple helix proportion comprises the following steps: mixing a triple helix collagen standard and a collagen monomer standard according to different proportions to obtain collagen standards with different triple helix proportions, performing absorbance detection on the collagen standards with different triple helix proportions by using a Sirius red staining method, and preparing a standard curve according to the triple helix proportion and the absorbance. The method can realize rapid, low-cost and quantitative detection of the collagen triple helix proportion, has high specificity and accuracy, good repeatability, and can be widely applied to the collagen industry.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for detecting the triple helix ratio of collagen. Background Technology

[0002] Collagen is a biological macromolecule and the most abundant and widely distributed functional protein in mammals, accounting for 25%–30% of total protein. As the most abundant protein in the extracellular matrix, collagen is the main structural protein in connective tissue and almost all thin-walled interstitial tissues, playing a role in stabilizing tissues and organs and maintaining their structural integrity. As a natural biological macromolecule, collagen has good biocompatibility, biodegradability, and absorbability, and promotes the formation of cells such as epithelial cells, making it widely used in medicine, beauty, cosmetics, and health products.

[0003] There are many types of collagen, with common types being type I, type II, type III, type V, and type XI. All members of the collagen family share a common characteristic: a right-handed triple helix structure composed of three α-chains. These three chains can be identical (homotrimer) or different (heterostrimmer). Within the molecule, each of the three α-chains forms an extended left-handed helix, and these three chains then supercoil around the central axis in a right-handed manner to form the triple helix structure. The function of a protein is determined by its structure; only collagen with a complete and stable triple helix structure can exert its full efficacy.

[0004] Collagen has wide applications in the medical aesthetics and skincare industries. Currently, there are two main sources of collagen on the market: natural extraction, primarily through acid, alkali, or enzymatic methods to extract collagen from animal skin or bones, but due to limited availability, supply is far below demand; and recombinant human collagen produced through fermentation using yeast or other expression systems. However, the structural characteristics and bioactivity of recombinant collagen, especially in terms of hydroxylation and triple-helix structure, still lag significantly behind natural collagen. Furthermore, the triple-helix structure of collagen is highly susceptible to processing conditions and other external factors, such as temperature, pressure, and chemical reagents introduced during processing. These factors can disrupt the triple-helix structure and affect its bioactivity. Therefore, how to induce triple-helix formation in recombinant collagen and how to improve the triple-helix ratio have become key research areas for scientific institutions and biotechnology companies. The triple-helix ratio is a crucial indicator in the construction, fermentation, and purification of recombinant collagen production strains. Therefore, establishing a simple, rapid, and low-cost method for detecting the triple-helix ratio in collagen products is essential.

[0005] Currently available methods for collagen detection mainly include:

[0006] 1. Circular dichroism spectroscopy: This is a qualitative detection method for the triple helix structure of collagen. Its characteristics are: a strong negative absorption at about 198 nm and a relatively weak positive absorption at about 220 nm. The intensity of the absorption can reflect the degree of retention of the triple helix structure of collagen.

[0007] 2. Infrared spectroscopy: This is a qualitative detection method for the triple helix structure of collagen. It observes the changes in the secondary structure of the protein by observing the absorption reaction of specific bond energies.

[0008] While the two methods mentioned above can detect the triple helix structure of collagen, they cannot determine the specific content; they are only qualitative methods and cannot quantitatively detect the proportion of triple helix structures. Furthermore, both methods require sophisticated equipment and are costly. Both analytical methods have limitations: circular dichroism spectroscopy can only analyze clear solutions within a narrow range, therefore, single characterization methods often cannot comprehensively and systematically resolve the effect of temperature on collagen structure; infrared spectroscopy detects changes in dipole moments generated during molecular vibrations and is sensitive to polar groups. Therefore, neither of these methods currently enables rapid and accurate quantification of triple helix components in collagen.

[0009] 3. Detection of hydroxyproline content in enzymatic hydrolysate: This is a quantitative detection method for the triple helix structure of collagen. It utilizes the principle that collagen with a complete triple helix structure cannot be hydrolyzed by trypsin, while collagen with a completely or partially destroyed triple helix structure can be hydrolyzed by trypsin. By detecting the change in the content of hydroxyproline, a characteristic amino acid of collagen, in the collagen solution system before and after trypsin hydrolysis, the content of collagen with a complete triple helix structure can be calculated.

[0010] The detection methods described above, which utilize trypsin or pepsin to disrupt the triple helix structure of collagen, also have drawbacks compared to methods that do not require disruption of the triple helix structure. First, the enzyme digestion conditions vary for different proteins; for example, pH, time, and protease ratios all require specific experimental determination. Inappropriate conditions may lead to incomplete digestion, resulting in quantitative errors. Furthermore, the detection of the content of hydroxyproline, a characteristic amino acid of collagen in the solution system, requires trichloroacetic acid precipitation. Factors such as trichloroacetic acid concentration, temperature, and ionic strength all affect collagen precipitation. That is, different trichloroacetic acid concentrations, temperatures, and ionic strengths may lead to different precipitates and precipitation effects. Additionally, it remains uncertain whether incomplete collagen triple helix structures are retained in the precipitate and whether complete collagen triple helix structures exist in the supernatant.

[0011] 4. Detection of dimer protein content in the enzymatic hydrolysate using a gel electrophoresis imaging system or thin-layer chromatography system: This is a quantitative detection method for collagen, allowing calculation of the retention rate of the triple helix structure in collagen samples. The basic principle is that collagen with an intact triple helix structure is resistant to enzymatic digestion by proteases, while collagen with a lost or partially lost triple helix structure is easily broken down into small-molecule peptides by proteases. SDS-PAGE allows for visual analysis of these broken-down peptides; the more broken-down peptides, the worse the integrity of the triple helix structure of the collagen sample; conversely, if no broken-down small-molecule peptides are observed in SDS-PAGE, the collagen sample's triple helix structure is considered to be relatively intact. However, this method is highly subjective, heavily influenced by the gel properties of the SDS-PAGE gel, staining, and destaining time. Furthermore, the repeatability of results when using a gel electrophoresis imaging system or thin-layer chromatography system is poor, making it impossible to guarantee the accuracy of each quantification.

[0012] 5. Detection of Collagen Triple Helix Structure Using Disulfide Nanomaterials: This method utilizes the principle that disulfide nanomaterials can accurately detect the triple helix structure of collagen. The steps are as follows: A disulfide nanomaterial solution is added to a probe peptide solution to form a first system; the disulfide nanomaterial solution is then added to a mixture of the probe peptide solution and the test peptide solution to form a second system; the fluorescence intensity of the first and second systems is detected and determined to determine whether a collagen triple helix structure has formed between the probe peptide and the test peptide in the second system. The concentration of the probe peptide in the first system is equal to that in the second system, and the probe peptide is a collagen peptide with a specific sequence modified with fluorescent molecules. This method has the following drawbacks: First, the preparation of the fluorescent probe is complex; second, this method detects the triple helix structure formed by the probe peptide and the test peptide, rather than detecting the pre-existing triple helix structure of collagen in the system, leading to inconsistencies in accuracy; finally, collagen solutions have high viscosity, and this method is difficult to use to determine the content of triple helix structures in high-viscosity collagen systems.

[0013] 6. Sirius Red Staining Method: Sirius red is a strong acidic anionic dye. Each molecule contains six sulfonic acid groups, which readily bind tightly to the basic amino acid groups of collagen, resulting in staining that is non-fading and highly specific. Utilizing the principle of specific binding between Sirius red and collagen, a red complex is obtained after washing with NaOH and centrifugation. The collagen content is determined by measuring the absorbance at a specific wavelength of 540-560 nm. Patent application CN103776778A uses acetic acid solution to prepare a collagen solution, stains it with Sirius red, centrifuges it, and measures the absorbance of the supernatant to plot a standard curve and quantify the collagen content. However, this method can only determine the content of 100% triple-helix collagen in animal tissues and cannot accurately determine the proportion of triple-helix structures in collagen containing a mixture of triple-helix and monomeric structures (such as recombinant collagen).

[0014] In conclusion, there is an urgent need to establish a simple, rapid, and low-cost method for detecting the triple helix ratio in collagen products. Summary of the Invention

[0015] This invention provides a method for detecting the triple helix ratio of collagen.

[0016] Specifically, the present invention provides the following technical solutions:

[0017] This invention provides a method for detecting the triple helix ratio of collagen. The method includes: mixing triple helix collagen standards and collagen monomer standards in different proportions to obtain collagen standards with different triple helix ratios; detecting the absorbance of the collagen standards with different triple helix ratios using Sirius red staining; and constructing a standard curve based on the triple helix ratio and absorbance.

[0018] Collagen products obtained by expressing recombinant collagen in host cells typically contain not only triple-helix collagen but also collagen monomers. Therefore, it is necessary to detect the proportion of triple-helix collagen in the collagen product to optimize and regulate host cells and the collagen production process. Although Sirius red staining can rapidly quantify collagen, this method is limited to the quantification of 100% triple-helix collagen and cannot quantify the proportion of triple-helix collagen in recombinant collagen products. This invention develops a method for detecting the proportion of triple-helix collagen in collagen based on Sirius red staining. This invention discovers that after staining a mixture of triple-helix collagen and collagen monomers with Sirius red, the absorbance at a wavelength of 540-560 nm shows a significant correlation with the proportion of triple-helix collagen. Based on this discovery, a method for detecting the proportion of triple-helix collagen was developed.

[0019] However, currently, collagen monomers from various animal sources are difficult to obtain commercially or are expensive. Therefore, this invention attempts to prepare collagen monomer standards using 100% triple-helix collagen standards. In the field of detection, the quality and accuracy of the content of standards are key factors affecting the accuracy of detection results. In the early stages of research and development, this invention found that denaturing 100% triple-helix collagen standards through heat treatment, causing the triple-helix structure to unwind into collagen monomers, often leads to a significant deviation between the detected results of the triple-helix ratio in the mixed standard of triple-helix collagen and collagen monomers and the actual results, and the linear correlation between the triple-helix ratio and the absorbance value after staining is poor. Through continuous experimentation, this invention unexpectedly discovered that treating triple-helix collagen standards at pH 6-8 and 65-100℃ yields stable collagen monomers. Sirius red staining experiments showed that after collagen unwinds from 100% triple helix to monomers, its binding affinity to Sirius red dye significantly decreases. However, the concentration of collagen monomers obtained under the above conditions exhibits a good correlation with staining absorbance, and it significantly improves the linear correlation between the triple-helix ratio and staining absorbance, thus enhancing the accuracy of triple-helix ratio detection results. Sirius red staining experiments demonstrate that, given a constant collagen concentration, the triple-helix ratio is linearly positively correlated with the absorbance after staining. Therefore, by establishing a standard curve, the proportion of triple-helix structures in collagen can be quantitatively detected.

[0020] Preferably, the preparation method of the collagen monomer standard includes: treating the triple helix collagen standard under pH 6-8 and 65-100℃ conditions.

[0021] Preferably, the processing time is 10-30 minutes.

[0022] In some embodiments of the present invention, the triple helix collagen standard is treated at pH 6-8 and 90-100℃ for 8-15 minutes to obtain the collagen monomer standard.

[0023] If the pH of the triple helix collagen standard is not within the range of 6-8, the pH of the triple helix collagen standard solution can be adjusted to 6-8 using NaOH solution or acidic solution before heat treatment.

[0024] In the above method, the concentrations of collagen standards with different triple helix ratios are the same. When the concentrations of collagen standards with different triple helix ratios are the same, the triple helix ratio shows a good correlation with the absorbance after Sirius red staining.

[0025] This invention does not impose any special restrictions on the concentration of collagen standards with different triple helix ratios.

[0026] In some embodiments of the present invention, the concentration of collagen standards with different triple helix ratios is 0.5-2 mg / mL.

[0027] Preferably, the triple helix collagen standard is a 100% triple helix collagen standard.

[0028] Preferably, the triple helix ratio of the collagen standards with different triple helix ratios is 0-100%.

[0029] For setting the concentration of collagen standards with different triple helix ratios, those skilled in the art can do so according to the general rules for preparing standard curves or the detection requirements. For example, collagen monomer standards, collagen standards with triple helix ratios of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% triple helix collagen standards can be set with a 10% gradient; or collagen monomer standards, collagen standards with triple helix ratios of 20%, 40%, 60%, 80%, and 100% triple helix collagen standards can be set with a 20% gradient.

[0030] To construct a standard curve, the triple helix ratio can be used as the horizontal axis and the absorbance value as the vertical axis, and the regression equation formula for the standard curve can be obtained.

[0031] The detection method described above also includes: detecting the absorbance of the sample to be tested using the Sirius red staining method, and calculating the triple helix ratio of the sample to be tested based on the standard curve.

[0032] The concentration of the sample to be tested is the same as the concentration of the collagen standards with different triple helix ratios.

[0033] In the detection methods described above, the staining solution and washing solution used in the Sirius red staining method contain surfactants.

[0034] Adding surfactants to Sirius red dyeing and washing solutions can reduce the non-specific binding of Sirius red dye to collagen and improve the accuracy of detection.

[0035] The surfactant can be an ionic surfactant or a nonionic surfactant. Optional surfactants include Triton X-100, fatty acid glycerides, polysorbates, etc.

[0036] Preferably, the surfactant is Triton X-100; the volume percentage of Triton X-100 in the dyeing solution and washing solution is 0.08-0.15%.

[0037] Preferably, the staining solution used in the Sirius red staining method comprises the following components: 0.8-1.2 g / L Sirius red, 12-13 g / L picric acid, and 0.08-0.15% Triton X-100.

[0038] The washing solution used in the Sirius red staining method comprises the following components: 0.4-0.6M acetic acid, 140-160mM sodium chloride, and 0.08-0.15% Triton X-100.

[0039] Preferably, the Sirius red staining method includes: mixing a standard or test sample with a staining solution for staining, collecting the precipitate and washing the precipitate with a washing solution, dissolving the washed precipitate in a reconstitution solution, and detecting the absorbance value at a wavelength of 540-560 nm.

[0040] Preferably, the staining time is 25-35 minutes. After staining, the precipitate is separated by centrifugation.

[0041] The complex solution is an alkaline solution. Preferably, it is a sodium hydroxide solution. More preferably, it is a 0.4-0.6M sodium hydroxide solution.

[0042] In the detection methods described above, the sample to be tested can be recombinant collagen or collagen extracted from animal tissue.

[0043] The core principle of the detection method of this invention is that triple-helix collagen and monomeric collagen have different binding efficiencies with Sirius red dye. Under a fixed concentration, the staining absorbance is linearly positively correlated with the triple-helix ratio in the collagen. Therefore, this invention does not have special limitations on the type of collagen; it can be human or any animal-derived collagen. As long as the collagen sample contains a triple-helix structure, differences in staining efficiency will appear under the heat treatment conditions of this invention, allowing for detection using the method of this invention. The sample can be used as a standard or a test sample. The standard can be natural collagen extracted from animal materials. The higher the similarity between the standard and the test sample in terms of animal origin, the better the accuracy of the detection. In some embodiments of this invention, the method for detecting the triple-helix ratio of collagen includes the following steps:

[0044] 1. Preparation of triple helix collagen standard: Weigh 100% triple helix collagen and dissolve it in 0.2-0.5M acetic acid aqueous solution to obtain triple helix collagen standard;

[0045] 2. Preparation of collagen monomer standards: Triple helical collagen standards were prepared according to the method in step (1). The triple helical collagen standards were heat-treated at pH 6-8 and 65-100℃ for 10-30 min to obtain collagen monomer standards.

[0046] 3. Preparation of collagen standards with different triple helix ratios: Triple helix collagen standards and collagen monomer standards are mixed in different ratios to obtain collagen standards with the same concentration but containing different triple helix ratios (0-100%).

[0047] 4. Sample preparation: Dissolve the sample to be tested in water. The concentration of the sample solution is the same as the concentration of collagen standards with different triple helix ratios.

[0048] 5. Sirius red staining method for detection:

[0049] (1) Staining: Collagen standards with different triple helix ratios and the samples to be tested are mixed with staining solution, thoroughly mixed, stained for 25-35 min under shaking at 50-100 rpm, and then centrifuged to separate the precipitate.

[0050] The staining solution includes the following components: 0.8-1.2 g / L Sirius Red, 12-13 g / L Picric Acid, and 0.08-0.15% Triton X-100;

[0051] (2) Washing: Wash the precipitate separated in step (1) with pre-cooled washing liquid, centrifuge to discard the supernatant and remove unbound dye;

[0052] The washing solution comprises the following components: 0.4-0.6M acetic acid, 140-160mM sodium chloride, and 0.08-0.15% Triton X-100;

[0053] (3) Combined with the release and detection of dye: The precipitate after washing in step (2) is mixed with the complex solution to dissolve the dye in the complex precipitate, and the absorbance is detected at a wavelength of 550-560nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] The complex solution is a 0.4-0.6M sodium hydroxide solution;

[0055] (4) Standard curve plotting and protein quantification: Plot a standard curve based on the absorbance values ​​of collagen standards with different triple helix ratios and their corresponding triple helix ratios. Substitute the absorbance value of the sample to be tested into the standard curve to obtain the triple helix ratio in the sample to be tested.

[0056] The present invention also provides a method for producing recombinant collagen, the method comprising: culturing host cells capable of expressing recombinant collagen, and detecting the triple helix ratio of recombinant collagen in the culture medium using the collagen triple helix ratio detection method described above.

[0057] The beneficial effects of this invention are as follows: The method for detecting the triple helix ratio of collagen provided by this invention has at least the following advantages:

[0058] 1. Low cost and readily available raw materials: The Sirius red staining solution used in this method is inexpensive and readily available. The equipment required for the detection process (such as ultraviolet spectrophotometer or ELISA reader, metal bath, etc.) are all conventional instruments, with low cost and no need to rely on complex and expensive equipment such as circular dichroism spectroscopy or infrared spectroscopy.

[0059] 2. Simple and fast operation: The detection method of this invention has high specificity and does not require complex pretreatment of collagen samples. If the collagen is purified by tissue extraction, it only needs to be dissolved in an aqueous solution for detection. If the collagen is obtained by bio-fermentation, it only needs to be broken down and centrifuged. There is no need to perform other complex pretreatment and purification steps on the sample. At the same time, it does not involve the complex operation of probe preparation, which greatly saves detection time and manpower and material resources, and can realize rapid detection of collagen triple helix ratio.

[0060] 3. High accuracy and good repeatability: In the detection method of the present invention, the triple helix ratio of collagen and the absorbance value after staining have a good linear correlation within the range of 0-100%, the detection results are highly accurate and are not affected by subjective factors, and have good repeatability.

[0061] 4. Applicable to the detection of various types of collagen, with a wide range of applications: The detection method of this invention can be applied to all collagen containing a triple helix structure, without being limited by the collagen sequence and peptide chain length. It can be applied to various collagen samples extracted naturally or fermented and recombined, and is not affected by the high viscosity of the collagen system. It can be widely used in the collagen industry. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0063] Figure 1 This is the standard curve of triple-helix collagen in Example 1 of the present invention.

[0064] Figure 2 This is the standard curve of collagen monomers in Example 2 of the present invention (protein concentration of 1 mg / mL, pH 6).

[0065] Figure 3 This is the standard curve of collagen monomers in Example 3 of the present invention (protein concentration of 1 mg / mL, pH 8).

[0066] Figure 4This is the standard curve of collagen triple helix ratio in Example 4 of the present invention (protein concentration is 1 mg / mL, pH 6).

[0067] Figure 5 This is the standard curve of collagen triple helix ratio in Example 5 of the present invention (protein concentration 1 mg / mL, pH 8).

[0068] Figure 6 This is the standard curve of collagen triple helix ratio in Example 7 of the present invention (protein concentration 1 mg / mL, pH 6).

[0069] Figure 7 This is the standard curve of collagen triple helix ratio in Example 7 of the present invention (protein concentration is 1 mg / mL, pH 8). Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0071] Example 1: Determination of Triple Helical Collagen Content by Sirius Red Staining Method

[0072] 1. Principle

[0073] Sirius red is a strong acid dye that readily binds to the basic groups in collagen molecules, resulting in strong adsorption. Utilizing the principle of the specific binding of Sirius red to collagen, a red complex is obtained after washing with NaOH and centrifugation. The collagen content is determined by measuring the absorbance under specific absorption wavelengths of 540-560 nm.

[0074] Naturally extracted porcine collagen sponge is a highly cross-linked triple-helix collagen, which can be used as a standard for 100% triple-helix collagen. Due to the high degree of cross-linking and low solubility of natural porcine collagen, it can only be decross-linked and dissolved in an acidic environment. Therefore, in the preparation of porcine collagen solutions, it is generally necessary to dissolve the porcine collagen sponge in 0.5M acetic acid.

[0075] 2. Detection Method

[0076] (1) Sample processing

[0077] Weigh out porcine collagen sponge and dissolve it in 0.5M acetic acid solution to a final concentration of 1 mg / mL, which will be used as a triple-helix collagen standard. Take 0, 25, 50, and 100 μL of the triple-helix collagen standard prepared above, and add 0.5M acetic acid to each sample volume to 100 μL to prepare standard solutions with concentration gradients of 0, 250, 500, and 1000 μg / mL. Prepare three parallel samples for each concentration.

[0078] Dissolve or dilute the sample to be tested with neutral pH distilled water or buffer solution to prepare three parallel samples, 100 μL per tube.

[0079] (2) Staining

[0080] Add 1000 μL of staining solution (1 g / L Sirius Red dye, saturated picric acid, 0.1% Triton X-100) to each tube of standard and test sample, mix thoroughly, and then place on a horizontal shaker and shake slowly (50-100 rpm) for 30 min. During this process, the dye and collagen form a complex precipitate, which separates from the soluble dye in the staining solution. Centrifuge all samples at 13000 × g for 10 min, ensuring that all precipitates are centrifuged to the bottom of the tube.

[0081] (3) Washing

[0082] Add 750 μL of pre-cooled washing buffer (0.5 M acetic acid, 150 mM sodium chloride, 0.1% Triton X-100) to each tube, gently rinse the precipitate with a pipette, centrifuge at 13000 × g for 10 min, discard the supernatant, and remove unbound dye.

[0083] (4) Combining dye release and detection

[0084] Add 1000 μL of reconstitution solution (0.5 M sodium hydroxide) to each tube to dissolve the dye in the complex precipitate again. Transfer 300 μL from each tube into a 96-well plate and detect the absorption light at 556 nm using a microplate reader.

[0085] (5) Plotting the standard curve of triple helix collagen and quantifying the protein

[0086] Based on the absorbance values ​​of porcine collagen standards (triple-helix collagen standards) at different concentrations, a standard curve was plotted (to ensure parallelism and accuracy, the standard curve needs to be re-plotted for each experiment), such as... Figure 1 As shown. Substituting the absorbance value of the sample to be tested into the standard curve, the concentration of 100% triple helix collagen sample can be obtained.

[0087] Example 2: Determination of collagen monomer content using Sirius red staining method

[0088] This embodiment provides a method for determining collagen monomer content using Sirius red staining. The method involves converting triple-helix collagen standards into collagen monomers through heat treatment under specific conditions, followed by determination of the collagen monomer content using Sirius red staining. The steps are as follows:

[0089] 1. Sample preparation:

[0090] Weigh out porcine collagen sponge and dissolve it in 0.5M acetic acid solution to a final concentration of 1 mg / mL. Then, use 2M NaOH solution to adjust the pH of the collagen solution to 6, so that the final concentration is 1 mg / mL. Then, heat at 100℃ for 10 min and cool to room temperature to obtain collagen monomer standards. Take 0, 25, 50, and 100 μL of collagen monomer standards respectively, and add 0.5M acetic acid to make up the volume of each sample to 100 μL to prepare standard solutions with concentration gradients of 0, 250, 500, and 1000 μg / mL. Prepare three parallel samples for each concentration.

[0091] Dissolve or dilute the sample to be tested with distilled water or buffer solution of neutral pH, and prepare three replicates, 100 μL per tube.

[0092] 2. Staining

[0093] Same as Example 1.

[0094] 3. Washing

[0095] Same as Example 1.

[0096] 4. Combining dye release and detection

[0097] Same as Example 1.

[0098] 5. Plotting the standard curve of collagen monomers

[0099] A standard curve was plotted based on the absorbance values ​​of collagen monomer standards at different concentrations, such as... Figure 2 As shown. Substitute the absorbance value of the sample to be tested into the standard curve (to ensure parallelism and accuracy of the experiment, the standard curve needs to be redrawn for each experiment) to obtain the content of collagen monomers in the sample to be tested.

[0100] Example 3: Determination of collagen monomer content using Sirius red staining method

[0101] This embodiment provides a method for determining collagen monomer content using Sirius red staining. The method involves converting triple-helix collagen standards into collagen monomers through heat treatment under specific conditions, followed by determination of the collagen monomer content using Sirius red staining. The steps are as follows:

[0102] 1. Sample preparation:

[0103] Weigh out porcine collagen sponge and dissolve it in 0.5M acetic acid solution to a final concentration of 1 mg / mL. Then, use 2M NaOH solution to adjust the pH of the collagen solution to 8, so that the final concentration is 1 mg / mL. Then, heat at 100℃ for 10 min and cool to room temperature to obtain collagen monomer standards. Take 0, 25, 50, and 100 μL of collagen monomer standards respectively, and use 0.5M acetic acid to make up the volume of each sample to 100 μL to prepare standard solutions with concentration gradients of 0, 250, 500, and 1000 μg / mL. Prepare three parallel samples for each concentration.

[0104] Dissolve or dilute the sample to be tested with distilled water or buffer solution of neutral pH, and prepare three replicates, 100 μL per tube.

[0105] 2. Staining

[0106] Same as Example 1.

[0107] 3. Washing

[0108] Same as Example 1.

[0109] 4. Combining dye release and detection

[0110] Same as Example 1.

[0111] 5. Plotting the standard curve of collagen monomers

[0112] A standard curve was plotted based on the absorbance values ​​of collagen monomer standards at different concentrations, such as... Figure 3 As shown. Substitute the absorbance value of the sample to be tested into the standard curve (to ensure parallelism and accuracy of the experiment, the standard curve needs to be redrawn for each experiment) to obtain the content of collagen monomers in the sample to be tested.

[0113] Example 4: Method for determining the triple helix ratio of collagen

[0114] Based on the results of Examples 1-3 above, it is evident that the absorbance values ​​obtained after staining with Sirius red for triple-helix collagen standards and collagen monomer standards of the same concentration differ. This difference is fundamentally determined by the ratio of triple-helix to monomer components in the sample itself; that is, the higher the proportion of triple-helix, the greater the difference in absorbance value between the sample and the monomer. Therefore, the above method can be used to quickly identify the presence of triple-helix structures and quantitatively detect the proportion of triple-helix structures in collagen samples.

[0115] This embodiment provides a method for detecting the triple helix ratio of collagen, the steps of which are as follows:

[0116] 1. Sample preparation

[0117] The sample to be tested was dissolved in distilled water, and the pH of the sample was adjusted to 6 using 2M NaOH solution to obtain a final concentration of 1 mg / mL.

[0118] 2. Preparation of Standards

[0119] Weigh out porcine collagen sponge and dissolve it in 0.5M acetic acid solution to a final concentration of 1 mg / mL, which serves as a 100% triple-helix collagen standard. Adjust the pH of the 100% triple-helix collagen standard to 6 using 2M NaOH solution, and then heat at 100℃ for 10 min to obtain a collagen monomer standard. By mixing the 100% triple-helix collagen standard and the collagen monomer standard in different proportions, collagen standards with different triple-helix ratios can be prepared, with a total protein concentration of 1 mg / mL for all mixtures.

[0120] 3. Plotting the standard curve

[0121] Collagen standards with different triple helix ratios and a concentration of 1 mg / mL were stained with Sirius red (staining, washing, dye release, and detection steps were the same as in Example 1), and absorbance values ​​were measured. A triple helix ratio standard curve was plotted based on the triple helix ratio and absorbance values ​​of the collagen standards with different triple helix ratios (to ensure experimental parallelism and accuracy, the standard curve needed to be re-plotted for each experiment). Figure 4 As shown.

[0122] 4. Determination of the sample to be tested

[0123] The sample to be tested is stained with Sirius red (the staining, washing, binding dye release and detection steps are the same as in Example 1), the absorbance value is detected, and the absorbance value of the sample to be tested is substituted into the standard curve to obtain the triple helix ratio of the sample to be tested.

[0124] Example 5: Method for Determining the Triple Helix Ratio of Collagen

[0125] This embodiment provides a method for detecting the triple helix ratio of collagen, the steps of which are as follows:

[0126] 1. Sample preparation

[0127] The sample to be tested was dissolved in distilled water, and the pH of the sample was adjusted to 8 using 2M NaOH solution to obtain a final concentration of 1 mg / mL.

[0128] 2. Preparation of Standards

[0129] Weigh out porcine collagen sponge and dissolve it in 0.5M acetic acid solution to a final concentration of 1 mg / mL, which serves as a 100% triple-helix collagen standard. Adjust the pH of the 100% triple-helix collagen standard to 8 using 2M NaOH solution, and then heat at 100℃ for 10 min to obtain a collagen monomer standard. By mixing the 100% triple-helix collagen standard and the collagen monomer standard in different proportions, collagen standards with different triple-helix ratios can be prepared, all with a total protein concentration of 1 mg / mL.

[0130] 3. Plotting the standard curve

[0131] Collagen standards with different triple helix ratios and a concentration of 1 mg / mL were stained with Sirius red (staining, washing, dye release, and detection steps were the same as in Example 1), and absorbance values ​​were measured. A triple helix ratio standard curve was plotted based on the triple helix ratio and absorbance values ​​of the collagen standards with different triple helix ratios (to ensure experimental parallelism and accuracy, the standard curve needed to be re-plotted for each experiment). Figure 5 As shown.

[0132] 4. Determination of the sample to be tested

[0133] The sample to be tested is stained with Sirius red (the staining, washing, binding dye release and detection steps are the same as in Example 1), the absorbance value is detected, and the absorbance value of the sample to be tested is substituted into the standard curve to obtain the triple helix ratio of the sample to be tested.

[0134] Furthermore, it has been verified that by adjusting the heat treatment conditions in step 2 of the detection method in this embodiment to 65°C for 30 minutes, while keeping the other steps unchanged, a triple helix ratio standard curve can still be plotted based on the triple helix ratio and absorbance of collagen standards with different triple helix ratios. The correlation between these curves and the actual results is consistent with... Figure 5 The standard curve is similar.

[0135] Example 6

[0136] This embodiment provides a method for detecting the triple helix ratio of collagen, which differs from the method in Example 4 only in that: in step 2, 100% triple helix collagen standard is not pH adjusted (i.e., pH is 2.7), and is heated at 100°C for 10 minutes to serve as a collagen monomer standard. The results show that the correlation (i.e., linear relationship) between the triple helix ratio and absorbance value in the standard curve plotted using this method is poor.

[0137] This embodiment also provides a method for detecting the triple helix ratio of collagen, which differs from the method in Example 4 only in that: in step 2, 100% triple helix collagen standard is adjusted to pH 3.7 using 2M NaOH solution, heated at 100°C for 10 min, and then used as a collagen monomer standard. The results show that the correlation (i.e., linear relationship) between the triple helix ratio and absorbance value in the standard curve plotted using this method is poor.

[0138] This embodiment also provides a method for detecting the triple helix ratio of collagen, which differs from the method in Example 4 only in that: in step 2, 100% triple helix collagen standard is adjusted to pH 12 using 2M NaOH solution, heated at 100°C for 10 min, and then used as a collagen monomer standard. The results show that the correlation (i.e., linear relationship) between the triple helix ratio and absorbance value in the standard curve plotted using this method is poor.

[0139] Example 7 Application of the detection method

[0140] The triple helix ratio of purchased collagen samples was determined using the methods described in Examples 4 and 5, respectively, as follows:

[0141] 1. Sample preparation

[0142] Sample 1: Dissolve the purchased eel swim bladder collagen (100% triple helix) in distilled water, adjust the pH to 6 and 8 respectively, and bring the volume to 1 mg / mL;

[0143] Sample 2: Dissolve the purchased eel swim bladder collagen (100% triple helix) in distilled water, adjust the pH to 6 and 8 respectively, and bring the volume to 1 mg / mL. Heat at 100℃ for 10 min, cool and set aside.

[0144] Sample 3: Sample 1 and Sample 2 were mixed in a certain proportion to prepare a collagen sample with a concentration of 1 mg / mL, pH of 6 and 8 respectively, and a triple helix ratio of 35%.

[0145] Sample 4: Sample 1 and Sample 2 were mixed in a certain proportion to prepare a collagen sample with a concentration of 1 mg / mL, pH of 6 and 8 respectively, and a triple helix ratio of 45%.

[0146] 2. Standard Curve Plotting

[0147] Sample 1 and Sample 2 were mixed in different proportions to prepare collagen standards with different triple-helix ratios (10%, 20%, 40%, 60%, and 80%, respectively), and the total protein concentration after mixing was 1 mg / mL for each standard. The collagen standards with different triple-helix ratios and a concentration of 1 mg / mL were stained with Sirius red (staining, washing, dye release, and detection steps were the same as in Example 1), and the absorbance was measured. A triple-helix ratio standard curve was plotted based on the triple-helix ratio and absorbance of the collagen standards with different triple-helix ratios (to ensure parallelism and accuracy, the standard curve was re-plotted for each experiment). Figure 6 and Figure 7 As shown.

[0148] 3. Determination of the sample to be tested

[0149] The sample to be tested was stained with Sirius red (the staining, washing, dye release, and detection steps were the same as in Example 1), and the absorbance was measured. Substituting the absorbance of the sample into the standard curve yielded the triple helix ratio of the sample. The detection results are shown in Table 1 (where pH 6 represents the detection method of Example 4, and pH 8 represents the detection method of Example 5). The results show that the triple helix ratio detection result and the actual ratio are on the same order of magnitude with an error of <5%, indicating that the triple helix ratio detection method of the present invention has high accuracy and can meet the detection purpose.

[0150] Table 1. Results of Trihexyrometer Ratio Tests on Samples

[0151]

[0152] The above-mentioned samples were tested at least three times, and the results showed that the consistency of the test results was good.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the proportion of collagen triple helixes, characterized by, The method includes: mixing triple-helix collagen standard and collagen monomer standard in different proportions to obtain collagen standard with different triple-helix ratios; detecting the absorbance of the collagen standard with different triple-helix ratios using Sirius red staining method; and constructing a standard curve based on the triple-helix ratio and absorbance value. The preparation method of the collagen monomer standard includes: treating the triple helix collagen standard under pH 6-8 and 65-100℃ conditions; The collagen standards with different triple helix ratios all had the same concentration.

2. The method for detecting the proportion of collagen triple helix according to claim 1, characterized in that, The processing time is 10-30 minutes.

3. The method for detecting the triple helix ratio of collagen according to claim 1, characterized in that, The triple-helix collagen standard is a 100% triple-helix collagen standard. And / or, in the collagen standards with different triple helix ratios, the triple helix ratio is 0-100%.

4. The method for detecting the triple helix ratio of collagen according to any one of claims 1 to 3, characterized in that, The method further includes: detecting the absorbance of the sample to be tested using the Sirius red staining method, and calculating the triple helix ratio of the sample to be tested based on the standard curve.

5. The method for detecting the triple helix ratio of collagen according to any one of claims 1 to 3, characterized in that, The staining and washing solutions used in the Sirius red staining method contain surfactants.

6. The method for detecting the triple helix ratio of collagen according to claim 5, characterized in that, The surfactant is Triton X-100; the volume percentage of Triton X-100 in the dyeing solution and washing solution is 0.08-0.15%.

7. The method for detecting the triple helix ratio of collagen according to claim 5, characterized in that, The staining solution used in the Sirius red staining method comprises the following components: 0.8-1.2 g / L Sirius red, 12-13 g / L picric acid, and 0.08-0.15% Triton X-100; And / or, the washing solution used in the Sirius red staining method comprises the following components: 0.4-0.6M acetic acid, 140-160mM sodium chloride, and 0.08-0.15% Triton X-100.

8. The method for detecting the triple helix ratio of collagen according to any one of claims 1 to 3, characterized in that, The Sirius red staining method includes: mixing the standard or the sample to be tested with the staining solution for staining, collecting the precipitate and washing the precipitate with a washing solution, dissolving the washed precipitate in a reconstitution solution, and detecting the absorbance value at a wavelength of 540-560 nm.

9. The method for detecting the triple helix ratio of collagen according to claim 6 or 7, characterized in that, The Sirius red staining method includes: mixing the standard or the sample to be tested with the staining solution for staining, collecting the precipitate and washing the precipitate with a washing solution, dissolving the washed precipitate in a reconstitution solution, and detecting the absorbance value at a wavelength of 540-560 nm.

10. A method for producing recombinant collagen, characterized in that, The method includes: culturing host cells capable of expressing recombinant collagen, and detecting the triple helix ratio of recombinant collagen in the culture medium using the method for detecting the triple helix ratio of collagen according to any one of claims 1 to 9.

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