Concentrated collagen and its concentration process and application

Abstract

The present invention discloses a concentrated collagen protein and its concentration process and application. The collagen protein concentration process comprises the following steps: providing a concentration reagent, the concentration reagent contains salt ions, and the concentration of the salt ions is 3-30 g / L; reacting the collagen protein stock solution with the concentration reagent to obtain the reacted collagen; centrifuging the reacted collagen to obtain the concentrated collagen. The collagen protein obtained by the concentration process of the present invention has good concentration effect, high concentration multiple, and has the effect of removing impurity proteins.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, in particular to concentrated collagen and a concentration process and application thereof. Background Art

[0002] Collagen is the most abundant protein in connective tissue and one of the main components of the extracellular matrix. As we age, we lose collagen, which manifests itself in wrinkles and loss of firmness and elasticity of the skin. Injectable collagen is a common medical aesthetic project. Collagen injected into the body can provide support and filling effects and is relatively safe. In addition, collagen is also widely used in the repair and treatment of various tissues.

[0003] Generally speaking, collagen needs to meet certain concentration requirements to achieve better results. In the existing technology, some methods first use animal-based materials to extract collagen, and then filter it through a hollow fiber tangential flow filter to achieve a concentration effect to increase the collagen concentration. However, the concentration effect is still not good enough, and the concentration multiple is difficult to meet the requirements. Summary of the invention

[0004] The present invention provides concentrated collagen and a concentration process and application thereof. The concentration process can achieve a higher concentration multiple and has the effect of removing impurity proteins.

[0005] In a first aspect, the present invention provides a collagen concentration process, comprising the following steps:

[0006] Providing a concentrated reagent, wherein the concentrated reagent contains salt ions, and the concentration of the salt ions is 3 to 30 g / L;

[0007] Allowing the collagen stock solution to react with the concentrated reagent to obtain the reacted collagen;

[0008] The collagen after the action is centrifuged to obtain concentrated collagen.

[0009] Optionally, the concentration of the salt ions is 3 to 5.5 g / L.

[0010] Optionally, the salt ions include sodium ions and / or potassium ions.

[0011] Optionally, the concentrated reagent includes one or more of NaCl, KCl, Na2HPO4 and KH2PO4.

[0012] Optionally, the mass ratio of NaCl, KCl, Na2HPO4 and KH2PO4 in the concentrated reagent is (180-220):(4-6):(32-40):(4-7).

[0013] Optionally, the mass ratio of NaCl, KCl, Na2HPO4 and KH2PO4 in the concentrated reagent is 200:5:36:6.

[0014] Optionally, the pH value of the concentrated reagent is 7.3-7.6.

[0015] Optionally, the volume ratio of the collagen stock solution to the concentrated reagent is 1:2-10.

[0016] Optionally, when the collagen stock solution is reacted with the concentrated reagent, the temperature of the concentrated reagent is 3-6°C.

[0017] Optionally, the collagen stock solution and the concentrated reagent are allowed to react with each other in a manner of: mixing the collagen stock solution with the concentrated reagent.

[0018] Optionally, the collagen stock solution is allowed to react with the concentrated reagent in the following manner: the concentrated reagent is used as a dialysis external fluid, the collagen stock solution is dialyzed, and the reacted collagen and the dialyzed concentrated reagent are obtained.

[0019] Optionally, the dialysis time is 60 to 90 hours, and the dialysis external fluid is changed every 8 to 16 hours.

[0020] Optionally, the collagen stock solution is obtained by using animal tissue as raw material through defatting, removing impurities and proteins, enzymolysis, salting out and dialysis.

[0021] Optionally, the centrifugal speed is 8000-12000 r / min, and the centrifugal time is 8-20 min.

[0022] Optionally, the temperature during centrifugation is 3-6°C.

[0023] In a second aspect, the present invention provides concentrated collagen obtained by the above-mentioned concentration process.

[0024] In a third aspect, the present invention provides a use of the above-mentioned concentrated collagen in the preparation of wound repair products, medical cosmetic products, bone repair products, products for treating stress urinary incontinence or cosmetics.

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

[0026] 1. In the collagen concentration process of the present invention, the collagen stock solution is reacted with a concentration reagent, wherein the concentration reagent contains salt ions, which can precipitate collagen, and then centrifugation is performed to obtain concentrated collagen. The collagen obtained by the concentration method has a high concentration multiple, and the process has the effect of removing impurity proteins. The present invention limits the concentration of salt ions to 3-30 g / L. The inventor has found through multiple experimental studies that if the salt ion concentration is too low, collagen can basically not be collected. If the salt ion concentration is too high, it will result in a low concentration multiple and a poor concentration effect.

[0027] 2. Furthermore, the present invention limits the concentration of salt ions to 3 to 5.5 g / L. The salt ion concentration is relatively low, much lower than the salt ion concentration used in the conventional salting-out process. This can not only ensure the precipitation of collagen, but also ensure that the concentrated collagen can maintain an osmotic pressure that is not much different from the cellular osmotic pressure in the human body after being injected into the human body, thereby reducing safety risks. The concentration ratio of collagen is higher, reaching up to 11 times of concentration, and the concentration effect is better.

[0028] 3. Furthermore, the inventors of the present application have found in their research that a concentrated reagent prepared with NaCl, KCl, Na2HPO4 and KH2PO4 has a better collagen concentration effect than a concentrated reagent prepared with only neutral salts such as NaCl and / or KCl.

[0029] 4. Furthermore, the present invention has two modes of action of the collagen stock solution and the concentrated reagent. One is direct mixing, which has a simple process and a short process time. The other is to use the concentrated reagent as the dialysis external fluid to dialyze the collagen stock solution. This mode of action has a higher concentration ratio and a better concentration effect.

[0030] 5. The concentrated collagen obtained by the present invention has good concentration effect, high concentration multiple, and almost no impurity protein, and is particularly suitable for wound repair products, medical beauty products, bone repair products, products for treating stress urinary incontinence or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The electrophoresis diagram of the collagen stock solution, concentrated collagen, and standard product of Example 1 obtained by the electrophoresis test method;

[0032] Figure 2 The electrophoresis diagrams are obtained by testing the collagen stock solution of Example 2, the concentrated collagen of Examples 2 to 8 and Comparative Examples 4 to 5, and the standard product using the electrophoresis testing method. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0034] In order to obtain concentrated collagen with good concentration effect, high concentration multiple and less impurity protein, the first aspect of the present invention provides a collagen concentration process, comprising the following steps:

[0035] Providing a concentrated reagent, wherein the concentrated reagent contains salt ions, and the concentration of the salt ions is 3 to 30 g / L;

[0036] Allowing the collagen stock solution to react with the concentrated reagent to obtain the reacted collagen;

[0037] The collagen after the action is centrifuged to obtain concentrated collagen.

[0038] In the present invention, the inventor creatively discovered that in the process of collagen concentration, the concentration reagent contains salt ions. When the collagen stock solution reacts with the concentration reagent, these salt ions can precipitate collagen, and then after centrifugation, concentrated collagen can be obtained. The collagen obtained by this concentration method has a high concentration multiple, which can reach up to 11 times, and the process is helpful to remove impurities.

[0039] The salt ion concentration is extremely important for the concentration process of the present invention. The present invention is to obtain collagen on the basis of salting out and dialysis, and then obtain concentrated collagen through a concentration process. In some embodiments, the concentration of the salt ion is 3 to 30 g / L. Exemplarily, the concentration of the salt ion is 3 g / L, 5 g / L, 9.83 g / L, 15 g / L, 19.66 g / L, 25 g / L and 30 g / L, or any value between any two of them. The inventor has found through repeated experimental studies that if the salt ion concentration is too low, collagen can hardly be collected. If the salt ion concentration is too high, when the collagen stock solution acts on the concentration reagent, it will become a transparent precipitate. At this time, the collagen may have been denatured, which is not conducive to the concentration of collagen, and will cause the concentration ratio to be greatly reduced, and the concentration effect cannot be achieved. Under normal circumstances, when the collagen stock solution acts on the concentration reagent, a white precipitate is presented. Further, the concentration of the salt ion is 3 to 5.5 g / L; optionally, the concentration of the salt ion is 3.25 to 5.23 g / L, and illustratively, the concentration of the salt ion is 3.25 g / L, 3.54 g / L, 3.77 g / L, 4.71 g / L and 5.23 g / L, or any value between any two of them. The salt ion concentration is low, much lower than the salt ion concentration used in the conventional salting-out process (generally 4 mol / L). The inventors of this application considered in their research that in the process of collagen concentration, not only a higher concentration ratio needs to be achieved, but also the concentration reagent used needs to consider the influence of osmotic pressure, because the concentrated collagen obtained may be used as an injection filler to be injected into the human body, and its osmotic pressure should be not much different from the cellular osmotic pressure in the human body. If the concentration of the concentrated reagent salt ions remaining in the concentrated collagen is too high, it may cause dehydration of cells in the human body, and there are certain safety risks. The concentrated collagen obtained under this salt ion concentration has a higher concentration ratio, better concentration effect, and higher safety.

[0040] In some embodiments, the salt ions include sodium ions and / or potassium ions; optionally, the concentrated reagent includes one or more of NaCl, KCl, Na2HPO4 and KH2PO4; in some embodiments, the mass ratio of NaCl, KCl, Na2HPO4 and KH2PO4 in the concentrated reagent is (180-220):(4-6):(32-40):(4-7). The inventors of the present application found in their research that the concentrated reagent prepared with NaCl, KCl, Na2HPO4 and KH2PO4 has a better collagen concentration effect than the concentrated reagent prepared with only neutral salts such as NaCl and / or KCl. Optionally, the pH value of the concentrated reagent is 7.3-7.6, for example, 7.3, 7.4, 7.5 or 7.6.

[0041] The collagen stock solution of the present invention is obtained by defatting, removing impurities and proteins, enzymolysis, salting out and dialysis using animal tissue as raw material. The animal tissue can be animal skin tissue, tendon tissue or organ tissue, etc. For example, the animal can be pig, cattle, sheep, fish, etc. In some embodiments of the present application, the animal tissue is pig skin.

[0042] In some embodiments, the volume ratio of the collagen stock solution to the concentrated reagent is 1:2 to 10; illustratively, the volume ratio of the collagen stock solution to the concentrated reagent is any one of 1:2, 1:3, 1:5, 1:6, 1:8 and 1:10 or a value between any two of them. The volume ratio of the collagen stock solution to the concentrated reagent will also have an important impact on the concentration effect. If the amount of the concentrated reagent is large, the concentration ratio will be reduced.

[0043] The inventors of the present application have found that the collagen stock solution reacts with the concentrated reagent at low temperature and centrifuges at low temperature to avoid collagen denaturation. In some embodiments, the temperature of the concentrated reagent when the collagen stock solution reacts with the concentrated reagent is 3 to 6°C; exemplarily, the temperature of the concentrated reagent when the collagen stock solution reacts with the concentrated reagent is any one of 3°C, 4°C, 5°C and 6°C or a value between any two of them. In some embodiments, the temperature during centrifugation is 3 to 6°C; exemplarily, the temperature during centrifugation is any one of 3°C, 4°C, 5°C and 6°C or a value between any two of them.

[0044] There are two modes of action of the collagen stock solution and the concentrated reagent in the present invention. One is: mixing the collagen stock solution with the concentrated reagent. This mode of action is simple to operate and shortens the process time. For example, the concentration can be completed in only 5 to 8 hours.

[0045] The second is: using the concentrated reagent as the dialysis external solution, dialyzing the collagen stock solution to obtain the acted collagen and the dialyzed concentrated reagent, which has a higher concentration ratio and a better concentration effect. In some embodiments, the dialysis time is 60 to 90 hours, and the dialysis external solution is changed every 8 to 16 hours.

[0046] After the collagen stock solution reacts with the concentration reagent, it is centrifuged, and the resulting precipitate is the concentrated collagen. In some embodiments, the centrifugal rate is 8000-12000 r / min, and the centrifugal time is 8-20 min; illustratively, the centrifugal rate is any one of 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min and 12000 r / min, or a value between any two of them, and the centrifugal time is any one of 8 min, 10 min, 15 min, 18 min and 20 min, or a value between any two of them.

[0047] In a second aspect, the present invention provides a concentrated collagen obtained by the above-mentioned concentration process. The concentrated collagen of the present invention has good concentration effect, high concentration multiple, and is substantially free of impurity proteins.

[0048] In a third aspect, the present invention provides a use of the above-mentioned concentrated collagen in wound repair products, medical cosmetic products, bone repair products, products for treating stress urinary incontinence or cosmetics.

[0049] The above is a detailed description of the present invention, and the following are embodiments of the present invention.

[0050] Example 1

[0051] The collagen concentration process of this embodiment includes the following steps:

[0052] (1) preparing a concentrated reagent, the preparation steps of the concentrated reagent comprising: adding 8 g NaCl, 200 mg KCl, 1.44 g Na2HPO4 and 240 mg KH2PO4 (mass ratio is 200:5:36:6) into 800 mL of water, adding purified water to make the volume to 1 L, and the pH of the concentrated reagent is 7.4;

[0053] (2) using pig skin to obtain collagen stock solution after defatting, removing impurities and proteins, enzymolysis, salting out and dialysis, wherein the salting out is performed using 4 mol / L sodium chloride; putting 50 mL of the collagen stock solution into a dialysis bag, dialyzing in a concentrated reagent at 4° C. for 3 days, replacing the dialysis external solution twice a day, to obtain the acted collagen; wherein the volume ratio of the collagen stock solution to the concentrated reagent is 1:6;

[0054] (3) The collagen after the reaction is centrifuged to obtain concentrated collagen; wherein the centrifugal speed is 10000 r / min, the centrifugal time is 10 min, and the centrifugal temperature is 4°C.

[0055] Example 2

[0056] Compared with Example 1, the only difference is that the concentrated reagent preparation process in step (1) is to add 8 g NaCl and 200 mg KCl to 1 L of water.

[0057] Example 3

[0058] Compared with Example 1, the only difference is that the concentrated reagent process in step (1) is to add 9 g of NaCl to 1 L of water.

[0059] Example 4

[0060] Compared with Example 1, the only difference is that the concentrated reagent preparation process in step (1) is to add 9g KCl to 1L of water; and the volume ratio of the collagen stock solution to the concentrated reagent in step (2) is 1:3.

[0061] Example 5

[0062] Compared with Example 3, the only difference is that the step (2) of Example 3 "Put 50 mL of collagen stock solution into a dialysis bag, dialyze in a concentrated reagent at 4°C for 3 days, replace the dialysis external fluid twice a day to obtain the acted collagen" is replaced by: Mix the collagen stock solution with the concentrated reagent at 4°C and stir for 6 hours to obtain the acted collagen.

[0063] Example 6

[0064] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 50 g of NaCl to 1 L of water.

[0065] Example 7

[0066] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 10 g KCl to 1 L of water;

[0067] In step (2), the volume ratio of the collagen stock solution to the concentrated reagent is 1:2;

[0068] The centrifugal treatment rate in step (3) is 8000r / min and the centrifugal time is 20min.

[0069] Example 8

[0070] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 10 g KCl to 1 L of water;

[0071] In step (2), the volume ratio of the collagen stock solution to the concentrated reagent is 1:10;

[0072] The centrifugal treatment rate in step (3) is 12000r / min and the centrifugal time is 8min.

[0073] Example 9

[0074] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 7.7 g of NaCl to 1 L of water, and the salt ion concentration is 3 g / L.

[0075] Example 10

[0076] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 25 g NaCl to 1 L of water, and the salt ion concentration is 9.83 g / L.

[0077] Embodiment 11

[0078] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 76.3 g of NaCl to 1 L of water, and the salt ion concentration is 30 g / L.

[0079] Example 12

[0080] (1) preparing a concentrated reagent, the preparation steps of the concentrated reagent comprising: adding 7.2 g NaCl, 160 mg KCl, 1.28 g Na2HPO4 and 280 mg KH2PO4 (mass ratio is 180:4:32:7) into 800 mL of water, and adding purified water to make the volume to 1 L;

[0081] (2) using pig skin to obtain collagen stock solution after defatting, removing impurities and proteins, enzymolysis, salting out and dialysis, wherein the salting out is performed using 4 mol / L sodium chloride; putting 50 mL of the collagen stock solution into a dialysis bag, dialyzing in a concentrated reagent at 4° C. for 3 days, replacing the dialysis external solution twice a day, to obtain the acted collagen; wherein the volume ratio of the collagen stock solution to the concentrated reagent is 1:6;

[0082] (3) The collagen after the reaction is centrifuged to obtain concentrated collagen; wherein the centrifugal speed is 10000 r / min, the centrifugal time is 10 min, and the centrifugal temperature is 4°C.

[0083] Embodiment 13

[0084] (1) preparing a concentrated reagent, the preparation steps of the concentrated reagent comprising: adding 8.8 g NaCl, 240 mg KCl, 1.60 g Na2HPO4 and 160 mg KH2PO4 (mass ratio is 220:6:40:4) into 800 mL of water, and adding purified water to make the volume to 1 L;

[0085] (2) using pig skin to obtain collagen stock solution after defatting, removing impurities and proteins, enzymolysis, salting out and dialysis, wherein the salting out is performed using 4 mol / L sodium chloride; putting 50 mL of the collagen stock solution into a dialysis bag, dialyzing in a concentrated reagent at 4° C. for 3 days, replacing the dialysis external solution twice a day, to obtain the acted collagen; wherein the volume ratio of the collagen stock solution to the concentrated reagent is 1:6;

[0086] (3) The collagen after the reaction is centrifuged to obtain concentrated collagen; wherein the centrifugal speed is 10000 r / min, the centrifugal time is 10 min, and the centrifugal temperature is 4°C.

[0087] Comparative Example 1

[0088] The source of collagen stock solution is the same as that in Example 1. After the collagen stock solution is mixed with water at a volume ratio of 1:5, it is filtered using a tangential flow filter with a membrane pore size of 30 kd. The concentration of the collagen stock solution changes from 6.16 to 9.85, and the concentration ratio is 1.6 times.

[0089] Comparative Example 2

[0090] Compared with Example 1, the only difference is that in step (1) during the preparation of the concentrated reagent, 1.44 g Na2HPO4 and 240 mg KH2PO4 are added to 800 mL of water, and then the volume is fixed to 1 L.

[0091] The concentration of the collagen stock solution is 6.16 mg / ml. The inventors of the present application found during the research and testing process that after the collagen was mixed with the concentrated reagent, the collagen was dissolved and almost no collagen was collected by centrifugation.

[0092] Comparative Example 3

[0093] Same as Example 5, except that, in step (1), the concentrated reagent preparation process is to add 5 g of NaCl to 1 L of water;

[0094] Among them, the concentration of the collagen stock solution is 7.15 mg / ml. During the research and testing process, the inventors of the present application found that after the collagen stock solution was mixed with the concentrated reagent, there was basically no collagen precipitation, and basically no collagen was collected after centrifugation.

[0095] Comparative Example 4

[0096] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 100 g of NaCl into 1 L of water.

[0097] Comparative Example 5

[0098] Compared with Example 5, the only difference is that the concentrated reagent preparation process in step (1) is to add 130 g of NaCl to 1 L of water.

[0099] Comparative Example 6

[0100] The collagen concentration process of this comparative example comprises the following steps:

[0101] (1) Provide a concentrated reagent, which is a NaOH solution with a concentration of 1 mol / L.

[0102] (2) using pig skin to obtain collagen stock solution after defatting, removing impurities and proteins, enzymolysis, salting out and dialysis, wherein the salting out is performed using 4 mol / L sodium chloride; mixing 20 mL of the collagen stock solution with 1 mol / L NaOH solution and adjusting the pH of the system to 8 to obtain the collagen after the action;

[0103] (3) The collagen after the reaction is centrifuged to obtain concentrated collagen; wherein the centrifugal speed is 10000 r / min, the centrifugal time is 10 min, and the centrifugal temperature is 4°C.

[0104] The concentration of the collagen stock solution is 6.16 mg / ml. The inventors of the present application found during the research and testing process that the collagen was dissolved in step (2) with almost no precipitation, and almost no collagen came out after centrifugation.

[0105] Comparative Example 7

[0106] Compared with Comparative Example 3, the only difference is that in step (2), 20 mL of collagen stock solution is mixed with 1 mol / L NaOH solution and the pH of the system is set to 9.

[0107] The concentration of the collagen stock solution is 6.16 mg / ml. The inventors of the present application found during the research and testing process that the collagen was dissolved in step (2) with almost no precipitation, and almost no collagen came out after centrifugation.

[0108] Test Example 1

[0109] The total protein content of the collagen stock solutions of Examples 1 to 8 and Comparative Examples 1 to 7 was tested by the Kjeldahl method, and the total protein content of the concentrated collagen protein of Examples 1 to 8, Comparative Example 1 and Comparative Examples 4 to 5 was tested, and the results are recorded in Table 1.

[0110] Table 1

[0111]

[0112] As can be seen from Table 1, under the salt ion concentration limit of the concentrated reagent, in the embodiments of the present invention, embodiments 1 to 4 use dialysis to react the collagen stock solution with the concentrated reagent, and embodiments 5 to 8 use direct mixing to react the collagen stock solution with the concentrated reagent. The concentrated collagen obtained has a good concentration effect, and the collagen concentration ratio obtained by the dialysis mode is higher, which can reach 5.4 times or more, and the concentration effect is better. From the comparison between embodiment 1 and embodiment 2 to 3, it can be seen that the concentrated reagent prepared by using NaCl, KCl, Na2HPO4 and KH2PO4 has a higher concentration effect ratio of collagen than the concentrated reagent prepared by using only neutral salts such as NaCl and / or KCl; further, when the mass ratio of NaCl, KCl, Na2HPO4 and KH2PO4 is 200:5:36:6 (that is, the salt ion concentration is 3.77g / L), the concentration effect is optimal, and the concentration ratio can be as high as 11 times. From the comparison between Example 7 and Example 8, it can be seen that the volume ratio of collagen stock solution to the concentration reagent will also have an important influence on the concentration effect. A larger amount of the concentration reagent will result in a lower concentration ratio.

[0113] Comparative Example 1 is a process for concentrating by a traditional filter method. It can be seen that the collagen concentration ratio obtained is only 1.6 times, which is significantly less than the embodiments of the present invention. From the comparison of Example 1 and Comparative Example 2, it can be seen that not all phosphate solutions can achieve the technical effect described in the present invention. The concentrated reagent prepared by the phosphate solution defined by the present invention can obtain the collagen with a high concentration ratio described in the present invention. From the comparison of Examples 5 to 6 and Comparative Examples 2 to 5, it can be seen that when the salt ion concentration is too low (such as Comparative Examples 2 to 3), collagen is basically not collected. As the salt ion concentration increases (such as Examples 5 to 6), the concentrated protein can be gradually collected, and the concentration ratio also increases accordingly, but when the salt ion concentration is too high (such as Comparative Examples 4 to 5), the concentration ratio will be significantly reduced, and the concentration effect will deteriorate. It can be seen from Comparative Examples 6 to 7 that if an alkaline reagent is used as the concentration reagent and the pH value of the mixed system of the collagen stock solution and the concentration reagent is too high, the collagen after the reaction will be dissolved and basically no precipitation will occur, and basically no collagen will come out after centrifugation.

[0114] Test Example 2

[0115] (1) SDS-PAGE electrophoresis was performed on the collagen stock solution, concentrated collagen, and standard product of Example 1 according to the fifth method of SDS-polyacrylamide gel electrophoresis in 0541 of the 2020 edition of the Chinese Pharmacopoeia. The results are as follows: Figure 1 shown.

[0116] (2) According to the fifth method of SDS-polyacrylamide gel electrophoresis in 0541 of the 2020 edition of the Chinese Pharmacopoeia, the collagen stock solution of Example 2, the concentrated collagen of Examples 2 to 8 and Comparative Examples 4 to 5, and the standard product were subjected to SDS-PAGE electrophoresis test. The results are as follows: Figure 2 shown.

[0117] Figure 1 The electrophoresis diagram obtained by the electrophoresis test method in Example 1, from left to right are Marker, bovine type I collagen standard provided by the China National Institute for Food and Drug Control, collagen stock solution (2 strips), and concentrated collagen (2 strips) obtained in Example 1. Figure 1 It can be seen that the concentrated collagen obtained in Example 1 retains the original active groups, the concentrated collagen characteristic bands α1 and α2 are both wider, and the bands corresponding to 50KD and 40KD are significantly lighter, indicating that the concentration process in Example 1 is helpful in removing impurities.

[0118] Depend on Figure 2 From the electrophoresis diagram, it can be seen that the concentrated collagen obtained in Examples 2 to 8 retains the original active groups, just like the bovine type I collagen standard. The characteristic bands α1 and α2 of the concentrated collagen are both broadened, and the band corresponding to 40KD is significantly weakened, indicating that the concentration process of Examples 2 to 8 is helpful in removing impurity proteins.

[0119] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A collagen concentration process, characterized in that: The following steps are involved: A concentrated reagent is provided, wherein the concentrated reagent contains salt ions, wherein the cation concentration of the salt ions is 3 to 30 g / L; the salt ions in the concentrated reagent are NaCl, KCl, Na2HPO4 and KH2PO4, and the mass ratio thereof is (180 to 220):(4 to 6):(32 to 40):(4 to 7); The collagen stock solution is reacted with the concentrated reagent to obtain the reacted collagen, wherein the collagen stock solution is obtained by defatting, removing impurities and proteins, enzymolysis, salting out and dialysis using animal tissue as raw material; the collagen stock solution is reacted with the concentrated reagent in the following manner: the collagen stock solution is mixed with the concentrated reagent or the concentrated reagent is used as a dialysis external fluid to dialyze the collagen stock solution; the volume ratio of the collagen stock solution to the concentrated reagent is 1:(2-10); The collagen after the action is centrifuged to obtain concentrated collagen.

2. The collagen concentration process according to claim 1, characterized in that: The cation concentration in the salt ions is 3 to 5.5 g / L.

3. A concentrated collagen obtained according to the collagen concentration process according to claim 1 or 2.

4. The concentrated collagen according to claim 3, characterized in that The concentrated collagen is used to prepare any one of wound repair products, medical cosmetic products and bone repair products.

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