A preparation method of medical grade type III collagen and type III collagen

Abstract

The invention belongs to the field of biomedical materials, and specifically relates to a method for preparing medical-grade type III collagen, comprising the following steps: animal placenta tissue pretreatment, decellularization, defatting, impurity removal, enzyme cutting, salting-out purification, and desalting concentration; the salting-out method comprises: salting-out an animal-derived collagen mixture containing type III collagen under alkaline conditions, and high-purity type III collagen can be obtained by salting-out in one step, with a purity of more than 95%; the prepared animal placenta type III collagen has a good triple helix structure and a characteristic fibrous reticular structure; the endotoxin content is lower than 0.5Eu / mL, reaching the medical standard; and the method can be applied to active skin care products, skin repair dressings, implants, artificial skins, medical devices, health foods and other fields.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a preparation method of medical-grade type III collagen and the type III collagen. Background Art

[0002] Collagen is the most abundant and widely distributed protein in animals and is the main component of connective tissue. Type III collagen is an interstitial collagen composed of three α1(III) chains. It is one of the main fibrin components of tissues such as skin and blood vessels. Type III collagen usually coexists with type I collagen. As a companion collagen, type III collagen forms fibrous tissue with type I collagen in the form of an elastic thin fiber layer in mammals. Type III collagen plays an important role in human wound healing and tissue repair, and has a good application effect in skin repair dressings. Type III collagen presents a unique triple helix structure, which is the key factor for type III collagen to have good biological functions. Therefore, the key to extracting type III collagen from animal tissues is to maintain the structural integrity of type III collagen during the extraction process while ensuring its purity to prevent it from denaturing. At the same time, as a medical material, type III collagen has strict requirements for purity and safety, and strict industry standards are also set for the content of components such as endotoxins contained in collagen.

[0003] The placenta is a special tissue of mammalian viviparous animals that supplies nutrients to the fetus and allows the embryo to grow during pregnancy. It is excreted from the body during childbirth. The placenta has been used as medicine in my country for thousands of years. Mammalian placenta is rich in collagen components, among which type I, type III and type IV collagen account for the highest content. Chinese patent CN1030528C uses pepsin to extract a mixture of different types of collagen from human placenta, among which the content of type III collagen is low. Chinese patent CN101717804B extracts and prepares type III collagen from newborn calf skin. This method uses high concentrations of guanidine hydrochloride and heating to denature collagen, losing the triple helix structure necessary for the biological function of collagen. How to efficiently prepare high-purity type III collagen with a complete triple helix structure needs to be studied urgently.

[0004] Salting out is an important method for protein purification. At present, a multi-step salting out purification process including redissolution, salting out precipitation, and redissolution desalting steps has been established to purify a single type of collagen from a collagen mixture. However, the process steps are cumbersome and the cycle is long, and salting out is carried out under acidic or neutral conditions. In the preparation method of high-purity collagen sponge disclosed in invention patent CN103772734A, salting out is carried out under a pH value of 7.0; in a soluble non-denatured type II collagen and a preparation method thereof disclosed in invention patent CN111748030A, salting out is carried out under a pH value of 2.0-7.0; in the preparation method of sika deer skin collagen and collagen peptide disclosed in invention patent CN113061178A, salting out is carried out under a pH of 7.0; in a preparation method of type I and type V collagen disclosed in invention patent CN103882082B, salting out is carried out under a pH of 7.5.

[0005] During the research process, the inventor unexpectedly discovered that under alkaline conditions, by salting out an animal collagen mixture containing type III collagen, high-purity type III animal collagen can be obtained in one step, and the prepared type III animal collagen has a purity of more than 95%; it maintains a good triple helix structure, is non-denatured type III collagen, and has an endotoxin content of less than 0.5Eu / mL, meeting medical standards; it can be used to prepare skin care products, skin repair dressings, implants, medical devices, health foods, etc. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide a method for extracting and preparing type III collagen from animal tissues. The preparation method comprises: salting out an animal collagen mixture containing type III collagen under alkaline conditions to obtain high-purity type III collagen.

[0007] Preferably, the salting-out conditions are: pH: 8.5-10.0, and salt concentration: 0.1-1.5M.

[0008] Preferably, the animal tissue is animal placenta.

[0009] In a second aspect, the present invention also provides a method for preparing type III collagen, comprising the following steps: animal placental tissue pretreatment, decellularization, defatting, impurity removal, enzymatic digestion, salting out purification, and desalting and concentration. The salting out method is: salting out an animal collagen mixture containing type III collagen under alkaline conditions, and the salting out conditions are: pH: 8.5-10.0, and salt concentration: 0.1-1.5M.

[0010] Preferably, the decellularization method is: treating animal placental tissue with an acid solution, and / or a salt solution, and / or a buffer solution, and / or a surfactant, and / or a peroxide solution; the defatting method is: treating animal placental tissue with n-butanol, and / or a chloroform-methanol solution, and / or a chloroform-methanol-water solution, and / or ether, and / or n-hexane, and / or acetone, and / or an ethanol-n-hexane solution, and / or an ethanol solution, and / or a surfactant; the impurity removal method is: treating the animal placenta with a sodium hydroxide solution, and / or a potassium hydroxide solution.

[0011] Preferably, the preparation method comprises the following steps:

[0012] (1) Animal placenta pretreatment: The animal placenta is cut into small pieces and washed in purified water to remove impurities and extra-tissue blood residues;

[0013] (2) Decellularization: Decellularization of the pretreated animal placenta is performed in a container using a NaCl solution, a surfactant solution, and a peroxide solution in sequence by a constant speed stirring method;

[0014] (3) Degreasing: treating the decellularized animal placenta fragments in a freshly prepared 5%-30% v / v n-butanol solution by constant speed stirring for 3-5 times, each time for 6-18 hours;

[0015] (4) impurity removal: removing impurities from the defatted animal placenta fragments in a 0.01-2.0 M sodium hydroxide solution for 0.5-72 h by constant speed stirring;

[0016] (5) Enzymatic digestion: extracting collagen from the animal placenta after impurity removal by enzymatic digestion to obtain an animal collagen mixture;

[0017] (6) Salting out: adjusting the pH of the animal collagen mixture to alkaline conditions, and performing salting out on the animal collagen mixture to obtain type III collagen precipitate;

[0018] (7) Desalting and concentrating: Desalting and concentrating the obtained type III collagen solution by using one or any combination of ultrafiltration, dialysis and gel chromatography.

[0019] Preferably, in step (2), the sample is immersed in a 5-30% m / v NaCl solution overnight; after being rinsed with purified water until no residue is left, the sample is immersed in a 0.1-2% v / v surfactant solution for 0.5-6 h; after being rinsed with purified water again, the sample is immersed in a 3-10% v / v peroxide solution for 30-60 min.

[0020] Preferably, the surfactant includes any one of SDS / TritonX-100, Na2EDTA, chlorhexidine, benzalkonium chloride / dodecyldimethylbenzyl ammonium chloride, and benzalkonium bromide; the peroxide includes any one of hydrogen peroxide and peracetic acid.

[0021] In a third aspect, the present invention provides a method for preparing medical-grade type III collagen, the method comprising: subjecting the type III collagen prepared by the method to radiation sterilization at a temperature not higher than 25°C.

[0022] In a fourth aspect, the present invention provides a medical grade type III collagen prepared according to the method described.

[0023] In a fifth aspect, the present invention provides a medical grade type III collagen, wherein the medical grade type III collagen comprises more than 95% type III collagen, and the endotoxin content of the medical grade type III collagen is less than 0.5Eu / mL.

[0024] In a sixth aspect, the present invention provides the use of the medical-grade type III collagen in the preparation of active skin care products, skin repair dressings, implants, artificial skin, medical devices, and health foods.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention provides a method for preparing medical-grade type III collagen, wherein an animal collagen mixture containing type III collagen is subjected to salting out under alkaline conditions, and high-purity type III collagen can be prepared by only one step of salting out;

[0027] (2) The preparation method described in the present invention has mild conditions, is green and safe, has simple steps, is easy to operate, has a short cycle, and is low in cost, and is suitable for large-scale preparation.

[0028] (3) The type III collagen prepared by the present invention has a purity of more than 95%, maintains a good triple helical structure and a characteristic fiber morphology, and is non-denatured type III collagen.

[0029] (4) The type III collagen prepared by the present invention has an endotoxin content of less than 0.5Eu / mL, which meets the medical standards;

[0030] (5) The type III collagen prepared by the present invention can be applied to the fields of active skin care products, skin repair dressings, implants, artificial skin, medical devices, health foods, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SDS-PAGE results of type III collagen prepared in the examples of the present invention;

[0032] Note: Batch 1 represents the type III collagen prepared in Example 1; Batch 2 represents the type III collagen prepared in Example 2; Batch 3 represents the type III collagen prepared in Example 3;

[0033] Figure 2 SDS-PAGE results of type III collagen prepared in the comparative example of the present invention;

[0034] Note: Batch 1 represents the collagen prepared in Comparative Example 1; Batch 2 represents the collagen prepared in Comparative Example 2;

[0035] Figure 3 Circular dichroism spectrum of type III collagen prepared by the present invention;

[0036] Figure 4 Thermal curve of type III collagen prepared by the present invention;

[0037] Figure 5 SEM image of type III collagen prepared by the present invention;

[0038] Figure 6 TEM image of type III collagen prepared by the present invention;

[0039] Figure 7 Mass spectrum of type III collagen prepared by the present invention;

[0040] Figure 8 The non-denaturing test result of the type III collagen prepared by the present invention. DETAILED DESCRIPTION

[0041] The present invention is described in detail below through specific embodiments. Any technical solutions that can be conceived by any technician in the field based on the present invention and combined with the common knowledge in the field belong to the protection scope of the present invention.

[0042] Example 1 Preparation of Type III Collagen

[0043] Placental tissue pretreatment: Take yak placental tissue and cut it into pieces, soak and wash it with purified water to remove residual impurities on the surface, and process it into smaller tissue fragments for later use;

[0044] Decellularization: soak the animal placenta tissue obtained after pretreatment in a 5% NaCl solution with constant speed stirring overnight; after the NaCl is completely removed with purified water, the animal placenta tissue is then treated in a 0.1% chlorhexidine acetate (chlorhexidine) solution with constant speed stirring for 6 hours, and the animal placenta tissue is precipitated; after the chlorhexidine acetate is completely removed with purified water, the animal placenta tissue is then soaked in 3% hydrogen peroxide with constant speed stirring for 60 minutes, the hydrogen peroxide is completely removed with purified water, and the animal placenta tissue is precipitated;

[0045] Degreasing: Soak the animal placenta tissue precipitate after decellularization in a newly prepared 5% v / v n-butanol solution, stir at a constant speed for 3 times, each time for 6 hours. After the last degreasing, remove the n-butanol completely with purified water, and take the animal placenta tissue precipitate;

[0046] Impurity removal: soak the defatted animal placenta tissue precipitate in a 0.01M sodium hydroxide solution, stir at a constant speed for 72 hours, then use purified water to completely remove the sodium hydroxide, and take the animal placenta tissue precipitate;

[0047] Enzymatic digestion: Place the animal placenta tissue precipitate after impurity removal in a 0.5M acetic acid solution containing pepsin and stir at a constant speed for 48 hours to obtain an animal collagen mixture;

[0048] Salting out: adjusting the pH of the animal collagen mixture to 8.5, slowly adding sodium chloride solid to a concentration of 0.1 M under stirring conditions to salt out the obtained animal collagen mixture;

[0049] Desalting: The precipitate obtained by salting out is redissolved in acetic acid solution to a uniform solution, and then dialyzed alternately with acetic acid solution and purified water to obtain an aqueous solution of type III collagen.

[0050] The operating temperature of all experiments was not higher than 25°C.

[0051] Example 2 Preparation of Type III Collagen

[0052] Placental tissue pretreatment: Take yak placental tissue and cut it into pieces, soak and wash it with purified water to remove residual impurities on the surface, and process it into smaller tissue fragments for later use;

[0053] Decellularization: soak the animal placenta tissue obtained after pretreatment in a 20% NaCl solution with constant speed stirring overnight; after completely removing NaCl with purified water, soak the animal placenta tissue in a 0.1% chlorhexidine acetate (chlorhexidine) solution with constant speed stirring for 1 hour, and take the animal placenta tissue precipitation; after completely removing chlorhexidine acetate with purified water, soak the animal placenta tissue in 5% hydrogen peroxide with constant speed stirring for 60 minutes, and completely remove hydrogen peroxide with purified water, and take the animal placenta tissue precipitation;

[0054] Degreasing: Soak the animal placenta tissue precipitate after decellularization in a newly prepared 30% v / v n-butanol solution, stir at a constant speed for 3 times, each time for 12 hours. After the last degreasing, use purified water to completely remove the n-butanol and take the animal placenta tissue precipitate;

[0055] Impurity removal: soak the defatted animal placenta tissue precipitate in a 0.5M sodium hydroxide solution, stir at a constant speed for 1 hour, then use purified water to completely remove the sodium hydroxide, and take the animal placenta tissue precipitate;

[0056] Enzymatic digestion: Place the animal placenta tissue precipitate after impurity removal in a 0.5M acetic acid solution containing pepsin and stir at a constant speed for 48 hours to obtain an animal collagen mixture;

[0057] Salting out: adjusting the pH of the animal collagen mixture to 10.0, slowly adding sodium chloride solid to a concentration of 1.5 M under stirring conditions to salt out the obtained animal collagen mixture;

[0058] Desalting: The precipitate obtained by salting out is redissolved in acetic acid solution to a uniform solution, and then dialyzed alternately with acetic acid solution and purified water to obtain an aqueous solution of type III collagen.

[0059] The operating temperature of all experiments was not higher than 25°C.

[0060] Example 3 Preparation of Type III Collagen

[0061] Placental tissue pretreatment: Take yak placental tissue and cut it into pieces, soak and wash it with purified water to remove residual impurities on the surface, and process it into smaller tissue fragments for later use;

[0062] Decellularization: soak the animal placenta tissue obtained after pretreatment in a 20% NaCl solution with constant speed stirring overnight; after completely removing NaCl with purified water, soak the animal placenta tissue in a 0.1% chlorhexidine acetate (chlorhexidine) solution with constant speed stirring for 1 hour, and take the animal placenta tissue precipitation; after completely removing chlorhexidine acetate with purified water, soak the animal placenta tissue in 5% hydrogen peroxide with constant speed stirring for 60 minutes, and completely remove hydrogen peroxide with purified water, and take the animal placenta tissue precipitation;

[0063] Degreasing: Soak the animal placenta tissue precipitate after decellularization in a newly prepared 10% v / v n-butanol solution, stir at a constant speed for 3 times, each time for 6 hours. After the last degreasing, remove the n-butanol completely with purified water, and take the animal placenta tissue precipitate;

[0064] Impurity removal: soak the defatted animal placenta tissue precipitate in a 0.5M sodium hydroxide solution, stir at a constant speed for 1 hour, then use purified water to completely remove the sodium hydroxide, and take the animal placenta tissue precipitate;

[0065] Enzymatic digestion: Place the animal placenta tissue precipitate after impurity removal in a 0.5M acetic acid solution containing pepsin and stir at a constant speed for 48 hours to obtain an animal collagen mixture;

[0066] Salting out: adjusting the pH of the animal collagen mixture to 10.0, slowly adding sodium chloride solid to a concentration of 1.0 M under stirring to salt out the obtained animal collagen mixture;

[0067] Desalting: The precipitate obtained by salting out is redissolved in acetic acid solution to a uniform solution, and then dialyzed alternately with acetic acid solution and purified water to obtain an aqueous solution of type III collagen.

[0068] The operating temperature of all experiments was not higher than 25°C.

[0069] Comparative Example 1

[0070] Placental tissue pretreatment: Take yak placental tissue and cut it into pieces, soak and wash it with purified water to remove residual impurities on the surface, and process it into smaller tissue fragments for later use;

[0071] Decellularization: soak the animal placenta tissue obtained after pretreatment in a 20% NaCl solution with constant speed stirring overnight; after completely removing NaCl with purified water, soak the animal placenta tissue in a 0.1% chlorhexidine acetate (chlorhexidine) solution with constant speed stirring for 1 hour, and take the animal placenta tissue precipitation; after completely removing chlorhexidine acetate with purified water, soak the animal placenta tissue in 5% hydrogen peroxide with constant speed stirring for 60 minutes, and completely remove hydrogen peroxide with purified water, and take the animal placenta tissue precipitation;

[0072] Degreasing: Soak the animal placenta tissue precipitate after decellularization in a newly prepared 10% v / v n-butanol solution, stir at a constant speed for 3 times, each time for 6 hours. After the last degreasing, remove the n-butanol completely with purified water, and take the animal placenta tissue precipitate;

[0073] Impurity removal: soak the defatted animal placenta tissue precipitate in a 0.5M sodium hydroxide solution, stir at a constant speed for 1 hour, then use purified water to completely remove the sodium hydroxide, and take the animal placenta tissue precipitate;

[0074] Enzymatic digestion: Place the animal placenta tissue precipitate after impurity removal in a 0.5M acetic acid solution containing pepsin and stir at a constant speed for 48 hours to obtain an animal collagen mixture;

[0075] Salting out: adjusting the pH of the animal collagen mixture to 7.0, slowly adding sodium chloride solid to a concentration of 1.5 M under stirring conditions to salt out the obtained animal collagen mixture;

[0076] Desalting: The precipitate obtained by salting out is redissolved in acetic acid solution to a uniform solution, and then dialyzed alternately with acetic acid solution and purified water to obtain a collagen aqueous solution.

[0077] The operating temperature of all experiments was not higher than 25°C.

[0078] Comparative Example 2

[0079] Placental tissue pretreatment: Take yak placental tissue and cut it into pieces, soak and wash it with purified water to remove residual impurities on the surface, and process it into smaller tissue fragments for later use;

[0080] Decellularization: soak the animal placenta tissue obtained after pretreatment in a 20% NaCl solution with constant speed stirring overnight; after completely removing NaCl with purified water, soak the animal placenta tissue in a 0.1% chlorhexidine acetate (chlorhexidine) solution with constant speed stirring for 1 hour, and take the animal placenta tissue precipitation; after completely removing chlorhexidine acetate with purified water, soak the animal placenta tissue in 5% hydrogen peroxide with constant speed stirring for 60 minutes, and completely remove hydrogen peroxide with purified water, and take the animal placenta tissue precipitation;

[0081] Degreasing: Soak the animal placenta tissue precipitate after decellularization in a newly prepared 10% v / v n-butanol solution, stir at a constant speed for 3 times, each time for 6 hours. After the last degreasing, remove the n-butanol completely with purified water, and take the animal placenta tissue precipitate;

[0082] Impurity removal: soak the defatted animal placenta tissue precipitate in a 0.5M sodium hydroxide solution, stir at a constant speed for 1 hour, then use purified water to completely remove the sodium hydroxide, and take the animal placenta tissue precipitate;

[0083] Enzymatic digestion: Place the animal placenta tissue precipitate after impurity removal in a 0.5M acetic acid solution containing pepsin and stir at a constant speed for 48 hours to obtain an animal collagen mixture;

[0084] Salting out: adjusting the pH of the animal collagen mixture to 10.0, slowly adding sodium chloride solid to a concentration of 2.0 M under stirring conditions to salt out the obtained animal collagen mixture;

[0085] Desalting: The precipitate obtained by salting out is redissolved in acetic acid solution to a uniform solution, and then dialyzed alternately with acetic acid solution and purified water to obtain a collagen aqueous solution.

[0086] The operating temperature of all experiments was not higher than 25°C.

[0087] Example 4: Quality evaluation of type III collagen

[0088] 1. SDS-PAGE gel electrophoresis experiment

[0089] The type III collagen prepared under the conditions of the example and the comparative example was subjected to SDS-PAGE polyacrylamide gel electrophoresis experiment.

[0090] Figure 1This is the electrophoresis diagram of type III collagen prepared in Example 1-3. It can be seen from the figure that the molecular weight of the prepared products is distributed around 100 KDa, and there is only one band, which is the α1 (III) band, which is consistent with the typical characteristics of type III collagen. There are no other bands at the low molecular weight of the entire lane, indicating that type III collagen has not been degraded during the extraction process.

[0091] Figure 2 This is the electrophoresis diagram of the collagen prepared in Comparative Examples 1-2. It can be seen from the figure that the collagen prepared by the methods of Comparative Examples 1 and 2 is a collagen mixture, with three bands of α1(Ⅲ), α1(Ⅰ) and α2(Ⅰ) distributed from top to bottom near 100KDa, indicating that the collagen prepared in Comparative Examples 1 and 2 is a mixture of type I and III collagen.

[0092] 2. Circular dichroism

[0093] Circular dichroism is a common method for characterizing protein secondary structure and is also a common method for determining the characteristic triple helical structure of collagen. The circular dichroism spectrum of collagen is measured by a circular dichroism spectrometer equipped with a temperature controller. A type III collagen stock solution with a concentration of 1 mg / mL is prepared, and the sample is equilibrated at 4°C for more than 24 hours before the measurement.

[0094] The circular dichroism results of type III collagen prepared in Example 3 are as follows: Figure 3 As shown, type III collagen has a positive peak at 225 nm, indicating that it has the characteristic triple helix structure of collagen.

[0095] 3. Thermal curve

[0096] Thermal stability is a method to characterize the stability and characteristic triple helix structure of collagen. The thermal curve of type III collagen is characterized by detecting the change value of the characteristic CD peak at the positive peak of the circular dichroism spectrum with the temperature gradient. For animal collagen, the specific measurement conditions are: temperature range: 4℃-20℃, heating rate: 1℃ / min, temperature range: 20℃-50℃, heating rate: 0.3℃ / min, temperature range: 50℃-60℃, heating rate: 1℃ / min, equilibrium time is 2min.

[0097] Figure 4 The thermal transition curve of type III collagen prepared in Example 3 is shown, and its thermal transition temperature is 40° C., which is consistent with the characteristics of animal collagen.

[0098] 4. SEM measurement

[0099] The morphology of type III collagen was characterized using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) at an operating voltage of 5.0 kV.

[0100] The SEM results of type III collagen prepared in Example 3 are as follows: Figure 5 As shown, it is shown that the type III collagen obtained by the present invention has a fiber network structure characteristic of natural collagen.

[0101] 5.TEM measurement

[0102] TEM measurements were performed using a Talos F200S transmission electron microscope (FEI, America). The copper mesh used for transmission electron microscopy was laid flat on filter paper, 15 μL of collagen sample solution was dripped on the copper mesh, and 15 μL of 2% uranyl acetate solution was dripped on it after 1 minute to color the sample. After waiting for 1 minute, the water on the copper mesh was dried, dried in a 30°C oven, and then placed at room temperature for at least 6 hours before testing, and TEM images were recorded at 200 kV.

[0103] The TEM results of type III collagen prepared in Example 3 are as follows Figure 6 As shown, it is shown that the type III collagen obtained by the present invention has periodic light and dark stripes characteristic of natural collagen.

[0104] 6. Endotoxin content determination

[0105] Referring to the endotoxin detection process of type I collagen, the endotoxin content of type III collagen prepared in the embodiment of the present invention was measured. The specific detection method is: using ToxinSensor TM The chromogenic LAL endotoxin detection kit was used, and the endotoxin content of type III collagen was determined according to the method described in the kit manual. The determination method is sensitive and reliable. The pharmaceutical industry standard stipulates that the endotoxin content in type III collagen should be less than 0.5Eu / mL. The results of the endotoxin content in the type III collagen prepared in the embodiments of the present invention are shown in Table 1. The endotoxin content of the collagen prepared in Example 1, Example 2, and Example 3 is less than 0.5EU / mL, which meets the requirements of the industry standards.

[0106] Table 1

[0107]

[0108] 7. Purity determination

[0109] Referring to the method for detecting the purity of type I collagen (DB44 / T 2080-2017), the present invention uses SDS-PAGE to detect the purity of type III collagen. Type III collagen is enzymatically hydrolyzed by specific collagenase, and then the content of the impurity protein in the sample that is not digested by the collagenase is detected; at the same time, the staining sensitivity of the experimental method is detected to determine the detection limit of the impurity protein. The SDS-PAGE results are processed by ImageJ software to determine the purity of type III collagen.

[0110] The specific detection method is: characterize type III collagen, type III collagen treated with collagenase, and collagenase by SDS-PAGE; scan the decolorized film in an image analysis system, calculate the density of bands with a molecular weight greater than 100 kDa, the result of type III collagen sample is A, the result of type III collagen sample treated with collagenase is B, and the result of collagenase sample is C, and each density is expressed in %. Calculation of impurity protein content: a) When BC=0, the purity of collagen in the sample (%): (10000-BSA limit net value) / 10000×100%; b) When BC≠0, the purity of collagen in the sample (%)=A-(BC). Under the experimental conditions, the staining limit of Coomassie Brilliant Blue for BSA can reach 100 ng, and impurity proteins with a content of more than 0.5% can be determined.

[0111] The results of collagen purity determination are shown in Table 2. The purity of the collagen prepared in Example 1, Example 2, and Example 3 is greater than 95%. The collagen obtained in Comparative Example 1 and Comparative Example 2 is a collagen mixture, and the purity cannot reach the 95% purity standard of type III collagen.

[0112] Table 2

[0113]

[0114] 8. Non-denaturing detection

[0115] Refer to the polypeptide probe FAM-(GPO)8 described in Patent 2020100021713 to detect whether the type II collagen prepared in the examples and comparative examples is denatured. Detection principle: The polypeptide probe FAM-(GPO)8 can specifically bind to denatured collagen, but does not bind to undenatured collagen with a complete triple helix structure. The more severe the degree of collagen denaturation, the stronger the fluorescence intensity detected.

[0116] Specific detection method: add the sample solution prepared in Example 3 to a 96-well ELISA plate with high binding capacity, place it for adsorption overnight at 4°C, and then dry the sample solution; wash the ELISA plate three times with PBS buffer solution, 3 min / time; add 1% bovine serum albumin solution for blocking, block at 4°C for 1h, and then wash according to the above method; then add 20μM fluorescent peptide probe FAM-(GPO)8, incubate at 4°C for 4h, so that it specifically binds to denatured collagen; wash the plate according to the above method, 5 min / time. Use Infinite M200 multifunctional ELISA reader to detect fluorescence intensity, and repeat three times for each measurement.

[0117] The test results of type III collagen described in Example 3 are as follows: Figure 7 As shown, the fluorescence intensity of the collagen prepared in Example 3 without heat treatment is very weak, while the fluorescence intensity of the type III collagen treated by heating at 100°C for 20 minutes increases significantly, indicating that the type III collagen obtained in Example 3 has a complete triple helix structure and has not been denatured, while the heat-treated type III collagen has undergone severe denaturation.

[0118] In summary, the present invention provides a method for preparing medical-grade type III collagen. Under alkaline conditions, an animal collagen mixture containing type III collagen is salted out. High-purity type III collagen can be prepared by only one step of salting out. The preparation method described in the present invention has mild conditions, is green and safe, has simple steps, is easy to operate, has a short cycle, and is low in cost, and is suitable for large-scale preparation. The type III collagen prepared by the present invention has a purity of more than 95%, maintains a good triple helix structure and a characteristic fiber morphology, and is non-denatured type III collagen. The type III collagen prepared by the present invention has an endotoxin content of less than 0.5Eu / mL, which meets medical standards; it can be used in active skin care products, skin repair dressings, implants, artificial skin, medical devices, health foods and other fields.

Claims

1. A method for preparing type III collagen, characterized in that: The method comprises the following steps: pretreatment of animal placental tissue, decellularization, defatting, impurity removal, enzyme digestion, salting-out purification, and desalting and concentration. The salting-out method comprises the following steps: salting-out an animal collagen mixture containing type III collagen under alkaline conditions. The salting-out conditions are as follows: pH: 8.5-10.0, and salt concentration: 0.1-1.5M.

2. The preparation method according to claim 1, characterized in that The decellularization method is: treating animal placental tissue with an acid solution, and / or a salt solution, and / or a buffer solution, and / or a surfactant, and / or a peroxide solution; the defatting method is: treating animal placental tissue with n-butanol, and / or a chloroform-methanol solution, and / or a chloroform-methanol-water solution, and / or ether, and / or n-hexane, and / or acetone, and / or an ethanol-n-hexane solution, and / or an ethanol solution, and / or a surfactant; the impurity removal method is: treating animal placenta with a sodium hydroxide solution, and / or a potassium hydroxide solution.

3. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Animal placenta pretreatment: mince the animal placenta into small pieces and wash in purified water to remove impurities and extra-tissue blood residues; (2) Decellularization: The pretreated animal placenta is decellularized in a container using a NaCl solution, a surfactant solution, and a peroxide solution in sequence by a constant speed stirring method; (3) Degreasing: Treat the decellularized animal placenta fragments in a freshly prepared 5%-30% v / v n-butanol solution by constant speed stirring for 3-5 times, each time for 6-18 hours; (4) Impurity removal: The defatted animal placenta fragments are treated to remove impurities by constant speed stirring in a 0.01-2.0 M sodium hydroxide solution for 0.5-72 hours; (5) Enzymatic digestion: extracting collagen from the animal placenta after impurity removal by enzymatic digestion to obtain an animal collagen mixture; (6) Salting out: The pH of the animal collagen mixture is adjusted to alkaline conditions, and the animal collagen mixture is subjected to salting out to obtain type III collagen precipitate; (7) Desalting and concentrating: Desalting and concentrating the obtained type III collagen solution by using one or any combination of ultrafiltration, dialysis and gel chromatography.

4. The preparation method according to claim 3, characterized in that: In step (2), the substrate is soaked overnight in a 5%-30% m / v NaCl solution; rinsed with purified water until no residue is left, and then soaked for 0.5-6 hours in a 0.1%-2% v / v surfactant solution; rinsed again with purified water, and then soaked for 30-60 minutes in a 3%-10% v / v peroxide solution solution.

5. The preparation method according to claim 4, characterized in that: The surfactant includes any one of SDS / TritonX-100, Na2EDTA, chlorhexidine, benzalkonium chloride / dodecyldimethylbenzyl ammonium chloride, and benzalkonium bromide; the peroxide includes any one of hydrogen peroxide and peracetic acid.

6. A method for preparing medical grade type III collagen, characterized in that: The method comprises: subjecting the type III collagen prepared by any one of the methods of claims 1 to 5 to radiation sterilization at a temperature not higher than 25° C. to obtain the product.

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