Method for extracting non-denatured collagen and method for identifying collagen

Abstract

The invention belongs to the technical field of collagen, and particularly relates to a method for extracting non-denatured collagen and a method for identifying collagen. The method for extracting the non-denatured collagen comprises the following steps of: a) adding collagen into an incubation solution for incubation and centrifugation to obtain an insoluble substance; b) carrying out enzymolysis, neutralization and salting out on the insoluble substance in sequence to obtain the non-denatured collagen; wherein the incubation solution is selected from Tris-HCl solution, Bis-Tris solution, imidazole buffer solution or trypsin culture solution. Experimental results show that the collagen is incubated by using the incubation solution, so that the denatured collagen can be removed, the non-denatured collagen can be extracted, and the activity of the collagen products can be improved.

Description

technical field

[0001] The invention belongs to the technical field of collagen, and in particular relates to a non-denatured collagen extraction method and a collagen identification method. Background technique

[0002] Protein is the material basis of life, an organic macromolecule, the basic organic matter that constitutes a cell, and the main bearer of life activities. There is no life without protein, and protein is a substance closely related to life and various forms of life activities. Every cell and all important components in the body have protein participation. There are many types of proteins in the human body, with different properties and functions, but they are all composed of more than 20 amino acids in different proportions, and they are continuously processed in the body. Metabolism and renewal. For example, the carrier protein is very important to maintain the normal life activities of the human body, and can carry various substances in the body; more th...

Examples

Embodiment 1

[0067] In this embodiment, five collagens are identified, and the five collagens are denatured collagen, No. 1 collagen, No. 2 collagen, No. 3 collagen and No. 4 collagen. The identification of each collagen includes the following step:

[0068] 1) Weigh 2g of collagen sample in a 250mL heat-resistant glass bottle, add 100-200mL of 1M guanidine hydrochloride solution to the collagen sample, stir overnight in a refrigerator at 4°C, centrifuge at 3000rpm for 2min, remove the supernatant, and obtain the first precipitate Repeat the above steps (add guanidine hydrochloride solution to remove the supernatant) once, add 10-50mL extraction buffer to the first precipitate, stir in a refrigerator at 4°C for 2h, centrifuge at 3000rpm for 2min, remove the supernatant, and obtain the second precipitate Precipitate.

[0069] 2) After adding 50-200mL of trypsin culture solution to the second precipitate, incubate overnight in a water bath at 37°C while stirring continuously; take it out fr...

Embodiment 2

[0074] This embodiment analyzes the result of embodiment 1, please refer to Figure 1 to Figure 8 , figure 1 with figure 2 They are the electrophoresis results of denatured collagen and No. 1 collagen in Example 1 of the present invention, respectively, image 3 It is the electrophoresis result figure of No. 2 collagen and No. 3 collagen in Example 1 of the present invention, Figure 4 It is the electrophoresis result graph of No. 4 collagen in Example 1 of the present invention. Figure 5 to Figure 8 The diagrams are the results of circular dichroism chromatography detection of No. 1 collagen, No. 2 collagen, No. 3 collagen and No. 4 collagen in Example 1 of the present invention in sequence.

[0075] figure 1 It shows that no electrophoresis band is seen in electrophoresis after the treatment of denatured collagen in Example 1, indicating that the present invention adopts trypsin culture solution to incubate and then centrifuge collagen to remove denatured collagen and ...

Embodiment 3

[0079] In this embodiment, the collagen before and after purification using guanidine hydrochloride is analyzed by electrophoresis, and the results are as follows: Figure 9 As shown, the results show that there are many bands of collagen before purification, most of which are small molecular weight peptides. There are protein components whose complete structure is destroyed in the collagen before purification. Miscellaneous proteins are detected, and there are also 140KD The left and right bands indicate that there are collagen and miscellaneous proteins in the collagen before purification; the purified collagen has a single band, which conforms to the typical molecular weight distribution characteristics of type II collagen, and the protein structure is complete and not destroyed, only detected Collagen, the results showed that the sample was purified to remove impurities.

technical field

[0001] The invention belongs to the technical field of collagen, and in particular relates to a non-denatured collagen extraction method and a collagen identification method. Background technique

[0002] Protein is the material basis of life, an organic macromolecule, the basic organic matter that constitutes a cell, and the main bearer of life activities. There is no life without protein, and protein is a substance closely related to life and various forms of life activities. Every cell and all important components in the body have protein participation. There are many types of proteins in the human body, with different properties and functions, but they are all composed of more than 20 amino acids in different proportions, and they are continuously processed in the body. Metabolism and renewal. For example, the carrier protein is very important to maintain the normal life activities of the human body, and can carry various substances in the body; more th...

Examples

Embodiment 1

[0067] In this embodiment, five collagens are identified, and the five collagens are denatured collagen, No. 1 collagen, No. 2 collagen, No. 3 collagen and No. 4 collagen. The identification of each collagen includes the following step:

[0068] 1) Weigh 2g of collagen sample in a 250mL heat-resistant glass bottle, add 100-200mL of 1M guanidine hydrochloride solution to the collagen sample, stir overnight in a refrigerator at 4°C, centrifuge at 3000rpm for 2min, remove the supernatant, and obtain the first precipitate Repeat the above steps (add guanidine hydrochloride solution to remove the supernatant) once, add 10-50mL extraction buffer to the first precipitate, stir in a refrigerator at 4°C for 2h, centrifuge at 3000rpm for 2min, remove the supernatant, and obtain the second precipitate Precipitate.

[0069] 2) After adding 50-200mL of trypsin culture solution to the second precipitate, incubate overnight in a water bath at 37°C while stirring continuously; take it out fr...

Embodiment 2

[0074] This embodiment analyzes the result of embodiment 1, please refer to Figure 1 to Figure 8 , figure 1 with figure 2 They are the electrophoresis results of denatured collagen and No. 1 collagen in Example 1 of the present invention, respectively, image 3 It is the electrophoresis result figure of No. 2 collagen and No. 3 collagen in Example 1 of the present invention, Figure 4 It is the electrophoresis result graph of No. 4 collagen in Example 1 of the present invention. Figure 5 to Figure 8 The diagrams are the results of circular dichroism chromatography detection of No. 1 collagen, No. 2 collagen, No. 3 collagen and No. 4 collagen in Example 1 of the present invention in sequence.

[0075] figure 1 It shows that no electrophoresis band is seen in electrophoresis after the treatment of denatured collagen in Example 1, indicating that the present invention adopts trypsin culture solution to incubate and then centrifuge collagen to remove denatured collagen and ...

Embodiment 3

[0079] In this embodiment, the collagen before and after purification using guanidine hydrochloride is analyzed by electrophoresis, and the results are as follows: Figure 9 As shown, the results show that there are many bands of collagen before purification, most of which are small molecular weight peptides. There are protein components whose complete structure is destroyed in the collagen before purification. Miscellaneous proteins are detected, and there are also 140KD The left and right bands indicate that there are collagen and miscellaneous proteins in the collagen before purification; the purified collagen has a single band, which conforms to the typical molecular weight distribution characteristics of type II collagen, and the protein structure is complete and not destroyed, only detected Collagen, the results showed that the sample was purified to remove impurities.

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