A method for rapid detection of active collagen content

Abstract

The present invention belongs to the technical field of natural active collagen, and specifically relates to a method for rapidly detecting the content of active collagen. The present invention prepares a series of standard samples with different triple helical structure contents by mixing a collagen reference substance with a complete triple helical structure and a denatured collagen that has lost its triple helical structure after denaturation treatment in different proportions. The active collagen content is characterized and detected using self-assembly properties. PBS buffer is added to the collagen solution, and in vitro self-assembly is carried out under appropriate conditions (collagen concentration, pH value, ionic strength, temperature, etc.). The turbidity change of the collagen solution is measured in real time at a set wavelength to obtain a quantitative standard curve for calculating the active collagen content in the test sample. The method for detecting the active collagen content of the present invention is faster and simpler. Compared with the traditional protease enzymatic hydrolysis method, it not only greatly shortens the detection time but also improves the detection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of natural active collagen, and particularly relates to a method for rapidly detecting the content of active collagen. Background Art

[0002] Collagen is an important structural protein in animals, widely present in skin, bones, muscles, and other connective tissues. Collagen's triple helical structure is key to its function and stability, and possesses strong biological activity. However, during extraction and processing, collagen often suffers varying degrees of damage, resulting in partial or complete loss of the triple helical structure, thereby affecting its biological efficacy and application value. Therefore, testing the integrity of collagen's triple helical structure, especially the content of active collagen, is of great significance for collagen quality control and evaluation.

[0003] Patent CN108659117A proposes a detection method based on the enzymatic hydrolysis properties of trypsin. This method utilizes the fact that collagen with an intact triple helical structure cannot be hydrolyzed by trypsin, while collagen with damaged structure can be hydrolyzed. The method quantitatively detects the integrity of the triple helical structure by measuring the change in hydroxyproline content in the solution before and after trypsin hydrolysis. However, this method has certain limitations, such as the need for expensive enzymatic hydrolysis reagents and lengthy experimental procedures.

[0004] Patent CN109884153A proposes a method for detecting the integrity of collagen's triple helix structure based on SDS-PAGE electrophoresis. This method analyzes the degradation products of collagen under the action of proteases and uses electrophoresis to visualize the decomposed peptides. While this method can accurately determine the integrity of collagen's triple helix structure, its practical application is limited by the expensive equipment, complex operation, and time-consuming detection process.

[0005] The prior art discloses a denatured collagen evaluation method (Chen Liyuan, Liang Xing, Peng Qihui, et al. Quantitative analysis method of denatured collagen based on pepsin resistance [J]. Journal of Biology, 2023, 40(01): 9-13.). This method selects pepsin for enzymatic hydrolysis of denatured collagen based on the structural characteristics, thermosensitive characteristics, and amino acid sequence of collagen. Then, based on the density of denatured collagen and non-denatured collagen, centrifugal precipitation is selected to analyze the two. Finally, collagen characteristic amino acids (hydroxyproline) are used as markers for quantitative detection. The quantitative detection results of the denatured collagen content in the sample are obtained by calculation. This method is complex to operate, has a long detection cycle, and has problems such as low detection efficiency and high labor costs.

[0006] Therefore, the existing technology generally has disadvantages such as expensive equipment, cumbersome operation, and time-consuming detection. There is an urgent need to develop a new method for detecting active collagen content that is low-cost, simple to operate, and fast to meet the needs of practical applications. Summary of the Invention

[0007] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a faster and simpler method for detecting the active collagen content. Compared with the traditional protease hydrolysis method, it not only greatly shortens the detection time but also improves the detection efficiency.

[0008] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a method for detecting the content of active collagen, comprising the following steps:

[0010] S1. Mixing an active collagen solution with denatured collagen in different proportions to prepare a gradient standard sample solution;

[0011] S2. Inducing the standard sample solution to self-assemble, measuring the turbidity change of the collagen solution in real time at a set wavelength to obtain a quantitative standard curve;

[0012] S3. Self-assemble the test sample under the same induction conditions as in step S2, measure the turbidity change value, and then calculate the content of active collagen in the test sample according to the quantitative standard curve;

[0013] In step S2, the specific method of inducing the standard sample solution to self-assemble is: adding a buffered saline solution to the standard sample solution to dilute it to a concentration of 0.5-1.0 mg / mL, and adjusting the pH value to 7.0-7.4;

[0014] The buffered saline solution is PBS buffer solution, and the concentration of the PBS buffer solution is 0.005-0.015M.

[0015] The present invention uses the self-assembly kinetics (turbidimetric method) as an indicator for evaluating collagen functionality, thereby enabling quantitative detection of active collagen components. By mixing active collagen with an intact triple helix structure with denatured collagen in varying proportions to form standard samples with varying triple helix content, the active collagen content is quantitatively determined by turbidity changes, providing accurate collagen functionality testing. The core of the turbidimetric method is based on the turbidity changes produced when collagen self-assembles under appropriate conditions, which are directly correlated with the active collagen content. By constructing a standard curve, the active collagen content can be effectively quantified, demonstrating its high practicality. Furthermore, this method avoids some of the complex steps and potential errors associated with traditional methods, enabling more efficient collagen quality control and functional analysis. This method has broad application prospects and holds significant significance for the quantitative detection of active collagen in pharmaceutical development, cosmetics, and the food industry.

[0016] Moreover, experimental investigations have found that, during the induction of self-assembly, the concentration of collagen directly affects the efficiency of its self-assembly process. An appropriate concentration can ensure that the collagen molecules have a sufficient concentration in the solution to form a triple helical structure. As the concentration increases, the self-assembly rate gradually accelerates. The pH value also has an important influence on the structural stability and self-assembly process of collagen; an appropriate pH value helps to maintain the natural triple helical structure of collagen and promote its self-assembly, while a pH value that is too high or too low will affect the charge state of collagen, thereby affecting the self-assembly process. In addition, during the collagen self-assembly process, changes in the ionic strength in the solution will affect the charge shielding effect between molecules, thereby affecting the attraction and repulsion between molecules, and thus affecting the aggregation or self-assembly degree of collagen. The present invention selects a specific concentration of PBS buffer to induce the self-assembly of collagen. Its appropriate ionic strength helps to maintain the stability of the triple helical structure of collagen, promotes the orderly arrangement and self-assembly of collagen, and the PBS buffer can maintain the pH of the solution in a relatively constant range, thereby effectively preventing the drastic changes in the collagen conformation caused by pH fluctuations during the self-assembly process, thereby improving the accuracy of the test results.

[0017] Therefore, these parameters synergistically determine the self-assembly effect of collagen. If these parameters exceed the above preferred ranges, the collagen will lose its original structure and self-assembly ability, thus affecting the turbidity measurement and the accurate calculation of active collagen content. Therefore, strict control of these conditions in the experiment is crucial to ensure the reliability and consistency of the results.

[0018] Preferably, in step S2, the temperature of the self-assembly is controlled to be 36-38°C.

[0019] Experimental research found that during the induced self-assembly process, temperature also has a certain impact on the structural stability and self-assembly process of collagen; too high a temperature will cause thermal denaturation or excessive aggregation of collagen, resulting in structural damage or self-assembly failure, and too low a temperature will make the self-assembly reaction too slow, or even lack sufficient power to promote intermolecular interactions, thereby affecting the sensitivity and accuracy of the detection.

[0020] More preferably, the specific method of inducing the standard sample solution to self-assemble is: adding a buffered saline solution to the standard sample solution to dilute it to a concentration of 1.0 mg / mL, and adjusting the pH value to 7.2.

[0021] More preferably, the concentration of the PBS buffer is 0.01M.

[0022] More preferably, the temperature of the self-assembly is controlled to be 37°C.

[0023] Through experimental research, it was found that precisely controlling the important parameters involved in the detection method under the above-mentioned optimal conditions can maximize the accuracy and repeatability of the detection results.

[0024] Preferably, in step S1, before the active collagen solution is mixed with the denatured collagen solution, the collagen standard with a complete triple helical structure is diluted to a concentration of 2-4 mg / mL using pure water or an acid solution.

[0025] Preferably, the acid solution includes an acetic acid solution or a hydrochloric acid solution, the mass concentration of the acetic acid is 0.05-0.15%, and the concentration of the hydrochloric acid is 0.005-0.015 mol / L.

[0026] Preferably, in step S1, the denaturation treatment includes heat treatment or chemical denaturation treatment.

[0027] Preferably, the specific conditions of the heat treatment are: heating at 60-80° C. for 40-60 min, followed by ice bathing for 8-12 min.

[0028] Preferably, in step S2, the set wavelength is 310-313 nm.

[0029] Preferably, in step S2, before measuring the turbidity change of the collagen solution, the collagen solution needs to be degassed, and the specific conditions of the degassing treatment are: degassing at 2000-4000 rpm for 3-7 minutes.

[0030] Preferably, in step S2, the specific method for measuring the turbidity change of the collagen solution in real time at a set wavelength is: continuously detecting the curve of the change of the solution turbidity over time, ending the measurement after the curve shows an obvious platform feature, and taking the absorbance value when the platform feature is shown as the measured value, and the turbidity value is the difference between the sample absorbance measurement value and the blank tube absorbance value.

[0031] Preferably, the test sample includes a collagen product, and the dosage form of the collagen product includes a solution, a sponge and a powder.

[0032] The detection method of the present invention is suitable for detecting the content of active collagen in different high-level structures. The method has wide applicability and can be used to detect the content of active collagen in various dosage forms of collagen products such as collagen solutions, powders, and sponges.

[0033] In a second aspect, the present invention provides the use of the method for detecting the active collagen content in detecting the active collagen content in a collagen product.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention provides a faster and simpler method for determining the content of active collagen. Compared with the traditional protease hydrolysis method, it not only greatly shortens the detection time but also improves the detection efficiency;

[0036] (2) The present invention uses the specificity of the collagen triple helix structure to characterize the self-assembly performance, providing an accurate and reliable basis for collagen research and application. This innovative technical approach not only expands the technical means in the collagen field, but also provides technical support for the development and quality control of related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the active collagen content-turbidity quantitative curve of the collagen solution standard sample in Example 1;

[0038] Figure 2 This is the active collagen content-turbidity quantitative curve of the collagen powder standard sample in Example 2. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0041] Example 1

[0042] This embodiment provides a method for detecting the active collagen content in a collagen solution, comprising the following steps:

[0043] S1. Preparation of gradient standard sample solution

[0044] 1. Active collagen solution: Dilute the active collagen solution standard with a complete triple helix structure with purified water to 4 mg / mL, mix well and set aside;

[0045] 2. Denatured collagen solution: Transfer 5 mL of the diluted active collagen solution standard to a centrifuge tube with a lid, tightly cap the tube, heat at 60°C for 1 hour, and immediately place on ice for 10 minutes to obtain the denatured collagen solution.

[0046] 3. Add active collagen solution and denatured collagen solution according to the ratio in Table 1 to prepare 6 groups of standard sample solutions with different active collagen contents.

[0047] Table 1

[0048]

[0049] S2. Self-assembly

[0050] 1. Solution preparation:

[0051] 0.01M PBS buffer: Weigh 0.27g of potassium dihydrogen phosphate, 1.14g of disodium hydrogen phosphate, 0.20g of potassium chloride, and 8.00g of sodium chloride, add 800mL of water, adjust the pH to 7.4 with 0.1mol / L hydrochloric acid or sodium hydroxide, and add water to 1000mL;

[0052] 2. In a cold water bath (6°C), add 0.01M PBS buffer to the standard sample solution obtained in step S1 to make a 1 mg / mL working solution, adjust the pH to 7.2, transfer to a refrigerated centrifuge, and degas at 4°C, 3000 rpm for 5 minutes. Then remove the solution; simultaneously set up a blank tube with purified water as the sample;

[0053] 3. Take 200 μL of the working solution prepared above and add it to a 96-well plate. Set the detection wavelength to 313 nm and control the temperature at 37°C. Use a microplate reader to record the change in turbidity over time (△A = A 测定管 -A 空白管 ), the measurement was terminated after reaching the plateau phase (40 min);

[0054] 4. Preparation of standard curve

[0055] (1) According to 2 and 3 in the above step S2, 6 groups of standard samples with different active collagen contents (M00, M20, M40, M60, M80, M100) were self-assembled, and the change of turbidity over time was measured. The results are shown in Table 2;

[0056] Table 2 Changes in turbidity of collagen solution standard samples over time

[0057]

[0058] (2) With the active collagen content (%) as the horizontal axis, the turbidity change value (△A=A 测定管 -A 空白管 ) is the vertical coordinate, and the linear regression equation is fitted: y=0.0071x+0.0084, R 2 =0.9974.

[0059] S3. Determination of active collagen content

[0060] 1. Dilute the sample with purified water to a total protein concentration of 4 mg / mL according to the total protein concentration of the sample, and mix well to obtain the sample solution;

[0061] 2. According to step S2, the test solution was self-assembled and the change of turbidity over time was measured. The results are shown in Table 3;

[0062] Table 3 Changes in turbidity of collagen solution over time

[0063]

[0064] 3. The active collagen content of the collagen solution, obtained using the standard curve equation, was 101.38%. "Active collagen content" as used herein refers to the ratio of the percentage of self-assembling collagen in the sample to the percentage of self-assembling collagen in the standard sample. Those skilled in the art will appreciate that an active collagen content exceeding 100% is due to potential statistical error in the calculated result after correction using the standard curve.

[0065] In order to verify the test results obtained by the technical solution of the present invention, this example uses the results of the standard protease enzymatic method disclosed in the prior art (Chen Liyuan, Liang Xing, Peng Qihui, et al. Quantitative analysis method of denatured collagen based on pepsin resistance [J]. Journal of Biology, 2023, 40(01): 9-13.) for comparative study. The specific method is as follows: the collagen solution is diluted with 0.1 mol / L acetic acid solution to a collagen concentration of 1 mg / mL, digested by pepsin digestion, and the digestion solution is recovered by NaCl precipitation. The content of non-denatured collagen contained in the collagen solution is quantitatively analyzed by detecting the hydroxyproline content of the collagen solution and the recovered precipitate. The final results are shown in Table 4.

[0066] Table 4 Comparison of active collagen content in collagen solution obtained by the present invention and prior art methods

[0067]

[0068] The content of active collagen in the collagen solution measured by the detection method of the present invention is 101.38%, which is relatively close to the results obtained by the prior art method, indicating that the method of the present invention is close to the existing industry standard results and has a certain degree of accuracy. In addition, the ratio of the active collagen content obtained by the detection method of the present invention to the non-denatured collagen content measured by the detection method disclosed in the existing industry shows that the recovery rate of active collagen in this example is 106.46%, which is in line with the recovery rate range (80%-120%) generally required for biological product detection methods, and the recovery rate is close to 100%, further demonstrating the reliability and accuracy of the detection method of the present invention.

[0069] Example 2

[0070] This embodiment provides a method for detecting the active collagen content in collagen powder, comprising the following steps:

[0071] S1. Preparation of gradient standard sample solution

[0072] 1. Active collagen solution: Accurately weigh an appropriate amount of active collagen powder standard with a complete triple helix structure, add purified water to dissolve it to make a 4 mg / mL collagen solution, and mix well for later use;

[0073] 2. Denatured collagen solution: Take 5 mL of the dissolved active collagen powder standard solution into a centrifuge tube with a lid, tightly cover the tube, heat at 80°C for 40 minutes, and immediately place on ice for 10 minutes to obtain the denatured collagen solution;

[0074] 3. Add active collagen solution and denatured collagen solution according to the ratio in Table 5 to prepare 6 groups of standard sample solutions with different active collagen contents.

[0075] Table 5

[0076]

[0077] S2. Self-assembly

[0078] 1. Solution preparation: Same as in Example 1;

[0079] 2. In a cold water bath (6°C), add 0.01M PBS buffer to the standard sample solution obtained in step S1 to make a 1 mg / mL working solution and adjust the pH to 7.2. Transfer to a refrigerated centrifuge and degas at 4°C, 3000 rpm for 5 minutes before removing from the centrifuge. Simultaneously set up a blank tube containing purified water as the sample.

[0080] 3. Take 200 μL of the working solution prepared above and add it to a 96-well plate. Set the detection wavelength to 313 nm and control the temperature at 37°C. Use a microplate reader to record the change in turbidity over time (△A = A 测定管 -A 空白管 ), the measurement was ended after reaching the plateau (50 min).

[0081] 4. Preparation of standard curve

[0082] (1) According to 2 and 3 in the above step S2, 6 groups of standard samples with different active collagen contents (M00, M20, M40, M60, M80, M100) were self-assembled, and the change of turbidity over time was measured. The results are shown in Table 6;

[0083] Table 6 Changes in turbidity of collagen powder standard samples over time

[0084]

[0085] (2) With the active collagen content as the horizontal axis and the turbidity change value as the vertical axis, the linear regression equation was fitted: y = 0.0023x + 0.0333, R 2 =0.9912.

[0086] S3. Determination of active collagen content

[0087] 1. Dilute the sample with purified water to a total protein concentration of 4 mg / mL according to the total protein concentration of the sample, and mix well to obtain the sample solution;

[0088] 2. According to step S2, the test solution was self-assembled and the change of turbidity over time was measured. The results are shown in Table 7;

[0089] Table 7 Changes in collagen powder turbidity over time

[0090]

[0091] 3. According to the standard curve equation, the active collagen content in the collagen powder is 61.39%.

[0092] In order to verify the test results obtained by the technical solution of the present invention, this example uses the results of the standard protease enzymatic method disclosed in the prior art (Chen Liyuan, Liang Xing, Peng Qihui, et al. Quantitative analysis method of denatured collagen based on pepsin resistance [J]. Journal of Biology, 2023, 40(01): 9-13.) for comparative study. The specific method is as follows: collagen powder is dissolved in 0.1 mol / L acetic acid solution to a collagen concentration of 1 mg / mL, which is used as a test solution. The test solution is digested by pepsin digestion, and then the digestion solution is recovered by NaCl precipitation. The content of non-denatured collagen contained in the collagen powder is quantitatively analyzed by detecting the hydroxyproline content of the test solution and the recovered precipitate. The final results are shown in Table 8.

[0093] Table 8 Comparison of active collagen content in collagen powder obtained by the present invention and prior art methods

[0094]

[0095] The results in Table 8 show that when measuring the active collagen content in collagen powder, the deviation between the active collagen content measured by the detection method of the present invention and the result measured by the prior art method is also relatively small, and the measured recovery rates are between 80% and 120%, which is in line with the recovery rate range generally required for biological product detection methods, confirming that the detection method of the present invention has good accuracy.

[0096] Comparative Example 1

[0097] This comparative example provides a method for detecting the active collagen content in a collagen solution. The method differs from Example 1 only in that, in step S2, the concentration of the PBS buffer is 0.1 M during the self-assembly of the standard sample solution; the collagen solution is prepared into a 2 mg / mL working solution and the pH is adjusted to 8; and the test sample is self-assembled using the same conditions. The remaining detection process is the same as in Example 1.

[0098] Table 9

[0099]

[0100] The results in Table 9 show that when the PBS buffer concentration, collagen concentration and pH value involved in the self-assembly process of the detection method slightly deviate from the range specified in the present invention, the measured active collagen content deviates greatly from the result measured by the method disclosed in the prior art, and the recovery rate exceeds the recovery rate range generally required for the biological product detection method, that is, the detection accuracy and reliability are significantly reduced.

[0101] Comparative Example 2

[0102] This comparative example provides a method for detecting the active collagen content in a collagen solution. The method differs from Example 1 only in that, in step S2, the concentration of the PBS buffer is 0.001 M during the self-assembly of the standard sample solution; the collagen solution is prepared into a 0.1 mg / mL working solution and the pH is adjusted to 6; and the test sample is self-assembled using the same conditions. The remaining detection process is the same as in Example 1.

[0103] Table 10

[0104]

[0105] The results in Table 10 also show that the PBS buffer concentration, collagen concentration and pH parameters involved in the self-assembly process have an important influence on the accuracy of detecting the active collagen content in the collagen solution.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting active collagen content, characterized in that: The following steps are involved: S1. Mixing an active collagen solution and a denatured collagen solution in different proportions to prepare a gradient standard sample solution; S2. Inducing the standard sample solution to self-assemble, measuring the turbidity change of the collagen solution in real time at a set wavelength to obtain a quantitative standard curve; S3. Self-assemble the test sample under the same induction conditions as in step S2, measure the turbidity change value, and then calculate the content of active collagen in the test sample according to the quantitative standard curve; In step S2, the specific method of inducing the standard sample solution to self-assemble is: adding a buffered saline solution to the standard sample solution to dilute it to a concentration of 0.5-1.0 mg / mL, and adjusting the pH value to 7.0-7.4; The buffered saline solution is PBS buffer, and the concentration of the PBS buffer is 0.005-0.015M; In step S2, the temperature of the self-assembly is controlled to be 36-38°C.

2. The method for detecting active collagen content according to claim 1, wherein: In step S1, before the active collagen solution is mixed with the denatured collagen solution, the collagen standard with a complete triple helical structure is diluted to a concentration of 2-4 mg / mL using pure water or an acid solution.

3. The method for detecting active collagen content according to claim 1, wherein: In step S1, the denaturation treatment includes heat treatment or chemical denaturation treatment.

4. The method for detecting active collagen content according to claim 3, wherein: The specific conditions of the heat treatment are: heating at 60-80° C. for 40-60 minutes, followed by ice bathing for 8-12 minutes.

5. The method for detecting active collagen content according to claim 1, wherein: In step S2, the set wavelength is 310-313 nm.

6. The method for detecting active collagen content according to claim 1, wherein: In step S2, before measuring the turbidity change of the collagen solution, the collagen solution needs to be degassed. The specific conditions of the degassing treatment are: degassing at 2000-4000 rpm for 3-7 minutes.

7. The method for detecting active collagen content according to claim 1, wherein: The test product includes a collagen product, and the dosage form of the collagen product includes a solution, a sponge and a powder.

8. Use of the method for detecting the active collagen content according to any one of claims 1 to 7 in detecting the active collagen content in collagen products.

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