A natural active collagen rapid identification kit, identification method and application thereof

Abstract

The present application belongs to the technical field of biological detection, and particularly relates to a natural active collagen rapid identification kit, an identification method and application thereof. The rapid identification method and the kit are used for specific staining of active collagen with an intact triple helix structure by using a specific staining agent, and a visual dye-collagen complex precipitate is generated. By observing the color, state and amount of the precipitate, the active collagen with the intact triple helix structure can be rapidly qualitatively detected. Compared with the existing detection method, the rapid identification method simplifies the process, shortens the original 1-hour detection to 15 minutes, replaces picric acid with acid and alcohol double reagents, and makes the identification more intuitive, clear and accurate. The naked eye identification does not need to rely on instruments and equipment, and is more conducive to on-site rapid identification.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a natural active collagen rapid identification kit, an identification method and application thereof. BACKGROUND

[0002] Active collagen is a right-handed superhelical fibrous protein formed by three peptide chains with complete triple helix structure, and is an important protein in animal connective tissue. The connective tissue has certain structure and mechanical properties such as tensile strength, tensile force and elasticity due to a high content of collagen, so as to support and protect the body. Due to many excellent properties of active collagen, the biological macromolecular compound has a very wide range of uses, and is widely used in the fields of medicine, chemical industry, cosmetics, food and the like.

[0003] At present, the detection methods for soluble active collagen mainly include hydroxyproline spectrophotometry, biuret method, SDS-PAGE, electrophoresis method, circular dichroism method, high performance liquid chromatography method, mass spectrometry method and immunological detection method. However, the detection methods in the existing standards are difficult to effectively distinguish natural collagen, recombinant collagen and hydrolyzed collagen products on the one hand, and most of the existing detection methods have defects such as long time consumption, complicated operation, dependence on large equipment, high cost and the like, and are difficult to be used for real-time online qualitative and quantitative detection of active collagen and related products.

[0004] The detection method closest to the technical scheme of the present application in the prior art is the Sirius Red colorimetric method. The method is derived from the picric acid Sirius Red F3BA histological specific staining method reported by Sweat in 1964. After being improved by Brentani and others, the method has been used as a classic qualitative detection method for collagen until now. The commercialized kit for collagen tissue staining on the market mainly adopts the method. In 1985, Martino et al. found that Sirius Red can be specifically combined with collagen in acetic acid to generate a red precipitate. After centrifugation, washing and alkali redissolution, the content of collagen is determined by measuring the absorbance at a specific wavelength of 540 nm. The quantitative detection method and application thereof in the process of fish collagen extraction involved in US Patent Publication No. US2003 / 0004315A1 and Chinese Patent Publication No. CN103776778A respectively follow the method, only with changes in the types of acid solvents and centrifugal rates. The Sircol Soluble Collagen Assay detection kit produced by Biocolor has the operation process (see Figure 1), the difference is that the kit still uses picric acid as a solvent, and is mainly used for the detection of collagen extraction. The commercial kit has been noted in the beginning of the instruction that the Sircol detection method is designed for research work, and should be operated in a laboratory with conditions.

[0005] However, the current commonly used Sirius red colorimetric method mainly has the following defects: the operation process needs centrifugation operation above 10000 rpm and detection by ultraviolet spectrophotometer, and the time consumption is long, which is not conducive to real-time online rapid detection of active collagen and related products; the existing commercial detection kit mainly uses picric acid as a solvent, and picric acid is a controlled drug, which is not conducive to wide promotion; the existing method does not describe the state and characteristics of the precipitate, and the identification basis is not clear. SUMMARY

[0006] In order to solve the above technical problems, the inventors have developed a new type of natural active collagen protein rapid identification kit. The detection method described in the present application does not need centrifugation operation, does not need picric acid, and on the basis of the original acid reagent, a group of alcohol reagents are added, and the rapid identification of natural active collagen protein is realized through the naked eye recognition of the state and characteristics of the precipitate.

[0007] Specifically, the present application is realized through the following technical solutions:

[0008] In a first aspect, the present application provides a natural active collagen protein rapid identification kit, which comprises a detection liquid A and a detection liquid B, the detection liquid A is an acid solution of 20-100 μM Sirius red dye, the detection liquid B is an alcohol solution of 20-100 μM Sirius red dye, and the natural active collagen protein is a collagen protein with complete triple helix structure.

[0009] Preferably, the detection liquid A and the detection liquid B are both 50-100 μM.

[0010] As an optional mode, in the above-mentioned kit, the detection liquid A and the detection liquid B are separately placed in the kit, the kit makes the natural active collagen protein generate a visible dye-collagen protein complex precipitate, and by observing the color, state and amount of the precipitate, the active collagen protein with complete triple helix structure can be rapidly qualitatively detected.

[0011] As an optional mode, in the above-mentioned kit, the acid solution is one or more of 0.1-0.6 M acetic acid, hydrochloric acid, citric acid or buffer thereof, and the pH of the detection liquid A is controlled to be 1.50-5.00.

[0012] As an optional mode, in the above-mentioned kit, the alcohol solution is one or more of ethanol, acetone, n-propanol, isopropanol, trichloroacetic acid or formamide organic solvent with a volume percentage concentration of 30-90%.

[0013] In a second aspect, the present application provides a rapid identification method of natural active collagen, which uses the kit of the first aspect described above, and the identification method comprises the following steps:

[0014] (1) respectively take out the detection solution A and the detection solution B;

[0015] (2) take the sample to be tested and drop it into the detection solution A;

[0016] (3) take the sample to be tested and drop it into the detection solution B;

[0017] (4) respectively mix the sample to be tested added in step (2) and step (3) with the detection solution A or the detection solution B uniformly;

[0018] (5) after standing for 2-10 minutes, observe the results.

[0019] Preferably, in the identification method, V 待测样品 :V 检测液 = 1:5-1:10.

[0020] Further preferably, the volume of the detection solution A and the detection solution B is the same; the volume of the sample to be tested used in step (2) and step (3) is the same.

[0021] As an optional mode, in the above-mentioned identification method, the identification method further comprises the following step of reading the observed results:

[0022] Positive: if red precipitate appears in both the detection solution A and the detection solution B, the result is positive, indicating that the sample to be tested contains active collagen;

[0023] Negative: if only the detection solution B precipitates, or neither the detection solution A nor the detection solution B precipitates or the precipitate is not red flocculation, the result is negative, indicating that the sample to be tested does not contain active collagen or the concentration of active collagen is too low, below 0.10 mg / mL;

[0024] Weak positive: if the initial result is negative, and after continuing to add the same volume of the sample to be tested in the detection solution A and the detection solution B in step (2) or step (3) respectively, the result turns to positive, the result is weak positive, indicating that the concentration of active collagen in the sample is relatively low, about 0.10 mg / mL.

[0025] As an optional mode, in the above-mentioned identification method, the detection range of the identification method is 0.15-5 mg / mL, and when the active collagen content in the sample to be detected is lower than the detection limit 0.10 mg / mL, there is no visible precipitate in the detection liquid A and the detection liquid B.

[0026] As an optional mode, in the above-mentioned identification method, the identification method can be completed within 2-15 minutes, and the identification result can be judged by naked eye identification, which is suitable for on-site rapid detection.

[0027] As an optional mode, in the above-mentioned identification method, the sample to be detected should be a solution sample with a protein concentration of 0.5-2 mg / mL, if the sample to be detected is a solid or powder collagen, the sample to be detected should be dissolved and diluted to a protein concentration of 0.5-2 mg / mL by using 0.5M acetic acid, and then detected.

[0028] In a third aspect, the present application provides the use of the kit of the first aspect or the identification method of the second aspect in the rapid identification of natural active collagen.

[0029] As an optional mode, in the above-mentioned use, the detection object of the kit, the identification method or the use is soluble active collagen in cosmetics, medical and cosmetic products, pharmaceutical products or collagen raw materials.

[0030] The rapid identification method of the present application uses specific staining agent to specifically stain the active collagen with complete triple helix structure, and generates visible dye-collagen complex precipitate, and qualitatively detects the active collagen with complete triple helix structure.

[0031] The rapid identification kit of the present application contains two detection liquids A and B. During detection, the dye in the detection liquid A specifically binds with the active collagen with complete triple helix structure in the sample to generate a visible flocculent red precipitate (see Figure 2 ). When the active collagen content is low or high, the precipitate in the detection liquid A is fine. In order to facilitate observation, the kit introduces an enhancing agent-detection liquid B, if the detection liquid B also generates a flocculent red precipitate, it can be determined that the sample contains active collagen with complete triple helix structure. Therefore, the detection principle of the active collagen rapid identification method of the present application is completely different from the core principle of the existing Sirius red colorimetric method.

[0032] The detection range of the method of the present application is 0.15-5 mg / mL (100 μL). When the active collagen content in the sample is lower than the detection limit 0.10 mg / mL (100 μL), there is no visible precipitate in the detection liquid A and the detection liquid B tube.

[0033] The present application has the following advantages over the prior art:

[0034] The rapid identification method and kit of the present application can specifically stain the active collagen with complete triple helix structure with specific staining agent, and generate visible dye-collagen complex precipitate, so as to rapidly qualitatively detect the active collagen with complete triple helix structure. Compared with the existing detection method, the rapid identification method of the present application simplifies the process without centrifugation, so that the original 1-hour detection is shortened to 2-15 minutes; the acid and alcohol double reagent replaces picric acid, so that the identification is more intuitive, clear and accurate; naked eye recognition avoids the dependence on detection instruments such as microscope and ultraviolet spectrophotometer in the original technology, and is more conducive to on-site rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 : Sircol Soluble Collagen Assay detection kit operation process schematic diagram.

[0036] Figure 2 : Effect diagram of precipitate generated after adding active collagen of different concentrations into detection liquid A and detection liquid B.

[0037] Figure 3 : Operation flowchart of the rapid identification method using the rapid identification kit of the present application.

[0038] Figure 4 : Detection result schematic diagram of the rapid identification method using the rapid identification kit of the present application.

[0039] Figure 5 : Specific detection result of the rapid identification method using the rapid identification kit of the present application.

[0040] Figure 6 : Electrophoresis experimental results of various types of collagen.

[0041] Figure 7 : Electrophoresis diagram of bovine skin type I collagen (self-extraction) before and after trypsinolysis. DETAILED DESCRIPTION

[0042] The present application will be further described below with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and do not limit the scope of the present application.

[0043] If a specific technology or condition is not specified in the examples, it is performed according to the technology or condition described in the literature in the art, or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0045] Example:

[0046] 1. Representative examples of the rapid identification kit of the present application

[0047] Example 1

[0048] The rapid identification kit of the present application contains two detection solutions A and B.

[0049] Detection solution A contains: 50 μM Sirius Red dye, 0.5 M acetic acid.

[0050] Detection solution B contains: 50 μM Sirius Red dye, 0.5 M acetic acid, 80% (by volume) ethanol.

[0051] During detection, take the test solution in a ratio of V 待测样品 :V 检测液 = 1:10 into detection solution A, take the test solution in a ratio of V 待测样品 :V 检测液 = 1:10 into detection solution B, mix well several times, and then observe the results after 2-10 minutes of standing.

[0052] Example 2

[0053] The rapid identification kit of the present application contains two detection solutions A and B.

[0054] Detection solution A contains: 50 μM Sirius Red dye, 0.5 M acetic acid-acetate buffer, pH 2.0-5.0.

[0055] Detection solution B contains: 50 μM Sirius Red dye, 0.5 M acetic acid-acetate buffer, pH 2.0-5.0, 80% (by volume) ethanol.

[0056] During detection, take the test solution in a ratio of V 待测样品 :V 检测液 = 1:10 into detection solution A, take the test solution in a ratio of V 待测样品 :V 检测液 = 1:10 into detection solution B, mix well several times, and then observe the results after 2-10 minutes of standing.

[0057] Example 3

[0058] The rapid identification kit of the present application contains two detection solutions A and B.

[0059] Detection solution A contains: 50 μM Sirius Red dye, 0.5 M acetic acid.

[0060] Detection liquid B comprises: 50 μM safranine dye, 0.5 M acetic acid, 80% (volume ratio) ethanol.

[0061] During detection, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:10 to detection liquid A, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:10 to detection liquid B, mix several times, and then observe the results after 2-10 minutes.

[0062] Example 4

[0063] The rapid identification kit of the present application comprises two detection liquids A and B.

[0064] Detection liquid A comprises: 50 μM safranine dye, 0.5 M acetic acid.

[0065] Detection liquid B comprises: 50 μM safranine dye, 0.5 M acetic acid, 80% (volume ratio) n-propanol or isopropanol.

[0066] During detection, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:10 to detection liquid A, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:10 to detection liquid B, mix several times, and then observe the results after 2-10 minutes.

[0067] Example 5

[0068] The rapid identification kit of the present application comprises two detection liquids A and B.

[0069] Detection liquid A comprises: 50 μM safranine dye, 0.5 M acetic acid.

[0070] Detection liquid B comprises: 50 μM safranine dye, 0.5 M acetic acid, 80% (volume ratio) ethanol.

[0071] During detection, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:5 to detection liquid A, take the test liquid in a ratio of V 待测样品 :V 检测液 =1:5 to detection liquid B, mix several times, and then observe the results after 2-10 minutes.

[0072] 2. Exemplary identification procedure of the rapid identification method of the present application

[0073] The exemplary operation steps are as follows (as shown in Figure 3 ):

[0074] (1) Take out the detection liquid A tube and the detection liquid B tube and the double balloon pipette.

[0075] (2) Open the detection liquid A and B tube covers, pinch the uppermost balloon of the double balloon pipette, and suck the sample to be measured (note that the upper end of the solution should reach the first balloon connection, i.e., about 100 μL, to be full, and then squeeze and drop into the detection liquid A tube.

[0076] (3) Repeat operation (2) and squeeze and drop into the detection liquid B tube.

[0077] (4) Tighten the A and B tube covers and mix several times by turning back and forth.

[0078] (5) After standing for 2 minutes, observe the results.

[0079] (6) When there is doubt about the interpretation of the results, the quality control product (white cover tube) in the kit can be used to repeat operations (1)-(5).

[0080] 3. Interpretation of the detection results of the rapid identification method of the present application

[0081] The detection results of the rapid identification method using the rapid identification kit of the present application are shown in the schematic diagram as shown in Figure 4 .

[0082] Positive: Both detection liquid A and B tubes have the red precipitate as shown in the following figure (+, +), which is a positive result.

[0083] Negative: Only the detection liquid B tube has a precipitate (-, +), and both tubes do not have a red precipitate (-, -), which is a negative result.

[0084] Weak positive: The initial result is negative (-, +), and after adding 1 drop of the sample to the A and B tubes, the result becomes positive (+, +), which is a weak positive result.

[0085] Invalid: The quality control product is added to the detection liquid A and B tubes, and no precipitate is generated, which is considered as invalid detection results.

[0086] Positive result indicates that the sample to be measured contains active collagen (with complete triple helix structure).

[0087] Negative result indicates that the sample to be measured does not contain active collagen or the concentration of active collagen is too low (less than 0.10 mg / mL).

[0088] Weak positive result indicates that the sample to be measured contains active collagen with a relatively low concentration (about 0.10 mg / mL).

[0089] Invalid result indicates that the detection liquid or the quality control product has been invalid, or the operating environment temperature is too high (> 45℃).

[0090] The first part of the embodiments of the present application is described in embodiments 1-5 of the first part The rapid identification kits were all successful rapidly qualitatively detecting active collagen with complete triple helix structure.

[0091] 4. Specificity of the rapid identification method of the present application

[0092] As an example, the inventors selected embodiment 1 (in essence, the same detection results were obtained for other embodiments 2-5) to perform specificity experiments on different types of collagen (1 mg / mL). The experimental results are shown in Table 1. Figure 5 As shown in Table 1, bovine type I collagen control, bovine tendon type I collagen, bovine skin type I collagen (self-extracted), rat tail type I collagen, fish swim bladder type I collagen, chicken type II collagen, and human placenta type IV collagen all showed positive results, while other small molecule collagens, recombinant collagens, and collagen hydrolysate gelatin all showed negative results. Combined with the results of SDS-PAGE electrophoresis experiments Figure 6 ), it is shown that the identification method can effectively distinguish active collagen with complete triple helix structure, recombinant collagen, and small molecule collagen, and has good specificity.

[0093] To further demonstrate the above results, the inventors continued to use enzyme degradation experiments to verify the above results. Trypsin can effectively degrade polypeptides, but cannot degrade collagen with complete triple helix structure, and is a commonly used tool enzyme for identifying whether collagen has complete triple helix structure. The following Figure 7 The electrophoresis results of bovine skin type I collagen (self-extracted) show that bovine skin type I collagen (self-extracted) can effectively resist trypsin degradation and show three characteristic polypeptides of type I collagen. It is proved that bovine skin type I collagen (self-extracted) has complete triple helix structure. Bovine type I collagen control, bovine tendon type I collagen, rat tail type I collagen, fish swim bladder type I collagen, chicken type II collagen, and human placenta type IV collagen all show similar electrophoresis results to bovine skin type I collagen. However, after trypsin degradation, small molecule collagens, recombinant collagens, and collagen hydrolysate gelatin all do not show characteristic bands, proving that these molecules do not have complete triple helix structure. The electrophoresis results are consistent with Figure 6 the identification results, proving that the identification method can effectively distinguish collagen with complete triple helix structure, recombinant collagen, and small molecule collagen.

[0094] 5. Summary of the rapid identification method of the present application

[0095] In comparison with the prior art, the active collagen rapid identification method has the following differences and improvements: 1) the current commonly used method does not describe the precipitate after staining in detail, and needs to use the operation after centrifugal reconstitution or microscope observation. The method of the application describes the precipitate after staining in detail, and proposes to directly observe the precipitate state by naked eye for qualitative identification, which greatly simplifies the process and shortens the identification time; 2) the rapid identification method of the application does not need picric acid, but uses commonly used acid and alcohol reagents, which is more convenient for naked eye observation. Picric acid is a controlled drug, and its color (such as Figure 1 ) is very deep, which is not convenient for naked eye observation of the precipitate state; 3) on the basis of the original acid reagent, the rapid identification method of the application adds a group of alcohol reagents, which can quickly identify natural active collagen by naked eye recognition of the precipitate state and characteristics in the two groups of reagents. The identification method is more intuitive and clear. The advantage of adding a group of alcohol reagents is that: in the prior art, only one group of acid reagents is often used, and when the content of active collagen in the sample to be tested is very low or higher than 2 mg / mL, the precipitate is very fine, which is not conducive to observation (see Figure 2 ). After the rapid identification kit of the application introduces a group of alcohol reagents, the fine precipitate can be converted into coarse precipitate which is more easily recognized by naked eye, which widens the detection range.

[0096] Obviously, those skilled in the art can make various modifications and variations to the application without departing from the spirit and scope of the application. Therefore, if these modifications and variations of the application belong to the scope of the claims of the application and their equivalents, the application also intends to include these modifications and variations.

Claims

1. A rapid identification kit for natural active collagen, characterized by: The kit comprises detection liquid A and detection liquid B, the detection liquid A is an acid solution of 50-100 μM Sirius Red dye, the detection liquid B is an alcohol solution of 50-100 μM Sirius Red dye, the natural active collagen is collagen with intact triple helix structure, the detection liquid A and the detection liquid B are separately placed in the kit, the acid solution is one or more of 0.5 M acetic acid, hydrochloric acid, citric acid or buffer thereof, the pH of the detection liquid A is controlled to be 1.50-5.00, and the alcohol solution is 0.5 M acetic acid and 80% ethanol in volume percentage.

2. The kit of claim 1, wherein: The kit enables the precipitation of dye-collagen complex which visualizes the natural active collagen, and the active collagen with intact triple helix structure can be quickly qualitatively detected by observing the color, state and amount of the precipitation, and the time of the quick qualitative detection is 2-15 minutes.

3. A method for rapid identification of natural active collagen, characterized by: The identification method uses the kit of claim 1 or claim 2, and comprises the following steps: (1) separately taking out the detection liquid A and the detection liquid B; (2) taking the sample to be tested and dropping it into the detection liquid A; (3) taking the sample to be tested and dropping it into the detection liquid B; (4) mixing the sample to be tested added in step (2) and step (3) with the detection liquid A or the detection liquid B respectively; (5) after standing for 2-10 minutes, observing the results, In the identification method, V 待测样品 : V 检测液 = 1:5-1:10, the volume of the detection liquid A and the detection liquid B is the same, the volume of the sample to be tested used in step (2) and step (3) is the same, The identification method comprises the following steps of reading and observing the results: Positive: if red precipitate appears in both the detection liquid A and the detection liquid B, the result is positive, indicating that the sample to be tested contains active collagen; Negative: if precipitate appears only in the detection liquid B, or red precipitate does not appear in both the detection liquid A and the detection liquid B, the result is negative, indicating that the sample to be tested does not contain active collagen or the concentration of active collagen is too low, less than 0.10 mg / mL; Weak positive: if the initial result is negative, and after continuing to add the same volume of sample to be tested in step (2) or step (3) into the detection liquid A and the detection liquid B respectively, the result turns to positive, the result is weak positive, indicating that the concentration of active collagen in the sample is relatively low, about 0.10 mg / mL.

4. The method of claim 3, wherein: The detection range of the identification method is 0.15-5 mg / mL, and when the content of active collagen in the sample to be tested is less than the detection limit 0.10 mg / mL, no precipitate can be observed with naked eyes in both the detection liquid A and the detection liquid B.

5. The method of claim 4, wherein: The identification method can be completed within 2-15 minutes, and the identification result can be interpreted only by naked eye recognition, which is suitable for on-site rapid detection.

6. Use of the kit of claim 1 or claim 2 or the identification method of any one of claims 3 to 5 in the rapid identification of natural active collagen.

7. Use according to claim 6, characterized in that: The detection object of the kit, the identification method or the use is soluble active collagen in cosmetics, medical and cosmetic products, pharmaceutical products or collagen raw materials.

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