Preparation method of acylated collagen
By using the combination method of the secondary salt detergent of terminal peptide collagen and the pretreatment solution in the collagen extraction and purification process, the problems of complex collagen extraction and purification process and high contamination risk in the prior art are solved, and the acylated collagen with low endotoxin and high purity are achieved, which simplifies the process flow and reduces the cost.
Patent Information
- Application Number
- CN202510205000.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-05-06
AI Technical Summary
The existing collagen extraction and purification process is complex, easy to introduce bacterial contamination, and the acylation process is high, the cycle is long, and the pollution risk is high, making it difficult to meet the needs of industrial production.
The secondary salt detergent of terminal peptide collagen is acylated, and the purity of collagen is sterilized by pretreatment and irradiation of hydrogen peroxide, sodium hydroxide, TritonX-114 and other solutions is reduced to the endotoxin and the purity of collagen is improved.
Low endotoxin and high purity acylated collagen is achieved, the process flow is simplified, the cost and pollution risk is reduced, and the product dissolution speed and transparency are improved.
Smart Images
Figure CN119932143A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing acylated collagen. Background Art
[0002] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% or more in some organisms. Therefore, collagen has good biocompatibility, biodegradability and biological activity, and is widely used in food, medicine, tissue engineering, cosmetics and other fields. Natural collagen has problems such as poor formula compatibility and poor solubility. It is greatly restricted in use. Acylated type I collagen liquid has better water solubility and compatibility, and can better meet the needs of biomedicine and cosmetics.
[0003] The process of extracting collagen with traditional technology is quite complicated: first, the bovine Achilles tendon is pretreated with a series of reagents in sequence, including soaking in peracetic acid, then soaking in tributyl phosphate and Tween, then soaking in sodium hydroxide and Triton X-114, and then soaking in sodium chloride and sodium carbonate. The process is numerous and complicated, which is more likely to introduce the risk of bacterial contamination, which is not conducive to subsequent purification treatment, making the collagen purification process after extraction more complicated. It must also be combined with the process of acylated collagen. In the conventional collagen acylation process, the crude collagen extract is diluted and ultrafiltered and then concentrated. The process cycle is long and the cost is high, and the risk of pollution is high, which is extremely unfavorable for industrial scale-up production.
[0004] How to improve the collagen extraction or purification process and reduce the endotoxin in the acylated collagen solution is a problem that needs to be solved at present. Summary of the invention
[0005] Based on this, one embodiment of the present application provides a method for preparing acylated collagen, wherein the acylated collagen prepared by the method has low endotoxin.
[0006] The technical solution includes:
[0007] A method for preparing acylated collagen, comprising the steps of preparing a salt-precipitated atelopeptide collagen and acylating the salt-precipitated atelopeptide collagen;
[0008] Wherein, the steps of preparing the atelocollagen salting-out product include:
[0009] Step 10: Soak the beef Achilles tendon in hydrogen peroxide solution and wash with water;
[0010] Step 20: After soaking in the Triton X-114 solution, continue soaking in the sodium hydroxide solution, wash with water until neutral, squeeze out the water and then freeze-irradiate for sterilization;
[0011] Step 30: taking the sterilized bovine Achilles tendon powder, soaking it in a citric acid solution, dispersing and crushing it, and performing acid enzymatic extraction with an acetic acid solution and pepsin to prepare an enzymatic solution;
[0012] Step 40: After filtering the enzymatic hydrolysate, centrifuging and collecting the upper clear transparent liquid, and salting it out with saturated saline to prepare a primary salting out product of atelocollagen; and,
[0013] Step 50: The atelocollagen primary salt-precipitated product is added to an acetic acid solution, the upper clear transparent liquid is collected, and salt-precipitated with saturated saline to prepare the atelocollagen secondary salt-precipitated product.
[0014] In one embodiment, in step 10, the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 1%-5%, and the immersion time is 1h-3h.
[0015] In one embodiment, in step 20, one or more of the following conditions are met:
[0016] (1) The mass fraction of Triton X-114 in the Triton X-114 solution is 0.1%-1%, and the immersion time is 0.5h-1.5h;
[0017] (2) the concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L, and the soaking time is 0.5 h-1.5 h; and,
[0018] (3) The irradiation dose used for cryo-irradiation sterilization is 5kgy-15kgy.
[0019] In one embodiment, step 30 satisfies one or more of the following conditions:
[0020] a. The mass fraction of citric acid in the citric acid solution is 4%-6%, and the swelling time is 1h -2h.
[0021] b. the concentration of acetic acid in the acetic acid solution is 0.5 mol / L-1 mol / L; and,
[0022] c. The amount of pepsin used is 1%-5% of the mass of the bovine Achilles tendon powder, the enzymolysis temperature is 18°C-25°C, and the enzymolysis time is 72h-120h.
[0023] In one embodiment, in step 40, filters with 10 mesh, 50 mesh, 100 mesh and 300 mesh are used for step-by-step filtration.
[0024] In one embodiment, the step of acylation of the atelocollagen secondary salting-out product comprises:
[0025] Step 100: adding the atelocollagen secondary salting out product into water to swell at low temperature to form a collagen solution, making the collagen concentration 5 mg / mL-10 mg / mL, adding acetone solution and sodium hydroxide solution containing succinic anhydride, controlling the pH to 8-9, and performing acylation reaction at a temperature of 25°C-30°C; and,
[0026] Step 200: After the reaction, the supernatant is collected, dialyzed with ultrapure water, and concentrated to 2-5 mg / mL by ultrafiltration after dialysis. The filtrate is obtained by membrane filtration to prepare acylated collagen.
[0027] In one embodiment, the step of acylation of the atelocollagen secondary salting out product satisfies one or more of the following conditions:
[0028] (1) The amount of succinic anhydride added is 20%-40% of the mass of collagen;
[0029] (2) the mass fraction of succinic anhydride in the acetone solution is 5%-10%; and,
[0030] (3) The concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L.
[0031] In one embodiment, the filtrate is freeze-dried to prepare acylated collagen sponge and acylated collagen.
[0032] Optionally, the acylated collagen sponge is dissolved in purified water, and a preservative is added to prepare an acylated collagen solution.
[0033] In one embodiment, the preservative is selected from one or more of butylene glycol, pentylene glycol, hexylene glycol, benzyl alcohol and phenoxyethanol; or / and,
[0034] The amount of the preservative added is 1%-10% based on the total mass of the acylated collagen solution.
[0035] In one embodiment, the bovine Achilles tendon is thawed, the surface fascia is removed, the tendon is sliced, and the tendon is frozen and crushed with liquid nitrogen to prepare the bovine Achilles tendon powder.
[0036] Compared with the traditional technology, this application has the following beneficial effects:
[0037] This application uses a process of immersing bovine Achilles tendons broken by liquid nitrogen in hydrogen peroxide, sodium hydroxide, TritonX-114 and other solutions in turn, combined with a radiation sterilization process, which uses less reagents and is simple to operate, and can better remove fat on the Achilles tendon, improve the purity of collagen, and reduce the endotoxin level of the product. At the same time, by using a secondary salting-out process, the viscosity of the dissolved collagen solution is reduced by half compared to the collagen solution of the same concentration that is ultrafiltered after the first salting-out. The benefit of the second salting-out of this application is also conducive to the subsequent acylation reaction, and a product with good transparency and low viscosity is obtained, while the product obtained by acylation of the first salting-out product under the same conditions has a slow dissolution rate and a higher viscosity. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.
[0039] Figure 1 The results of gel electrophoresis test; Note: α1 and α2 are two polypeptide chains of collagen, and β chain is a dimer of α chain. DETAILED DESCRIPTION
[0040] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.
[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0042] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0043] "mPa·s" is milliPascal·second, which is the unit of dynamic viscosity. Dynamic viscosity is a measure of the viscosity of a fluid, which indicates the fluid's ability to resist deformation or flow.
[0044] The present application discovered that the pretreatment process of bovine Achilles tendon has a great influence on the performance of collagen, and the study found that the reasonable selection of reagents and processes in the pretreatment process, through reasonable ratio, controlling the concentration of various reagents will significantly affect the triple helix structure of the obtained collagen. In the pretreatment step, the present application successively soaks the bovine Achilles tendon crushed by liquid nitrogen in hydrogen peroxide, sodium hydroxide, TritonX-114 and other solutions combined with irradiation sterilization process, which can better remove the fat on the Achilles tendon, improve the purity of collagen, and reduce the endotoxin level of the product.
[0045] The present application adds a secondary salting-out process and directly uses the collagen solution after the secondary collagen salting-out product is dissolved for subsequent tests, which not only further reduces the viscosity of the collagen solution re-dissolved by the primary salting-out product, but also facilitates the subsequent acylation reaction to obtain a fast-dissolving, transparent, and low-viscosity product.
[0046] One embodiment of the present application provides a method for preparing acylated collagen, the method comprising the steps of preparing a salting-out product of atelocollagen;
[0047] Wherein, the preparation of the atelocollagen salting-out product comprises steps 10 to 50:
[0048] Step 10: Soak the beef Achilles tendon in hydrogen peroxide solution and wash with water.
[0049] In a specific example, before soaking, the bovine Achilles tendon is thawed, the surface fascia is removed, sliced, and frozen and crushed with liquid nitrogen to prepare bovine Achilles tendon powder.
[0050] In a specific example, the bovine Achilles tendon powder is rinsed with clean water until there is no blood, and then added to a hydrogen peroxide solution and immersed for 1h-3h for disinfection and sterilization.
[0051] In a specific example, the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 1%-5%, which can be 1%, 2%, 3%, 4%, 5%, or a range consisting of any two of the above values.
[0052] Step 20: After soaking in Triton X-114 solution, continue soaking in sodium hydroxide solution, wash with water until neutral, squeeze out the water and freeze-irradiate for sterilization.
[0053] After the bovine Achilles tendon powder is soaked in hydrogen peroxide solution and then combined with Triton X-114 and sodium hydroxide solution, it can better remove endotoxins and achieve protein degreasing.
[0054] In a specific example, the mass fraction of Triton X-114 in the Triton X-114 solution is 0.1%-1%, which can be 0.1%, 0.3%, 0.5%, 0.8%, 1%, or a range consisting of any two of the above values.
[0055] In a specific example, the soaking time in the Triton X-114 solution is 0.5h-1.5h, which can be 0.5h, 1h, 1.5h, or a range consisting of any two of the above values.
[0056] In a specific example, the concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L, which can be 0.5 mol / L, 1.0 mol / L, 1.5 mol / L or a range consisting of any two of the above values.
[0057] In a specific example, the soaking time in the sodium hydroxide solution is 0.5h-1.5h, which can be 0.5h, 1h, 1.5h, or a range consisting of any two of the above values.
[0058] In a specific example, the irradiation dose used for cryo-irradiation sterilization is 5kgy-15kgy, which can be 5kgy, 6kgy, 7kgy, 8kgy, 9kgy, 10kgy, 11kgy, 12kgy, 13kgy, 14kgy, 15kgy, or a range consisting of any two of the above values.
[0059] Step 30: Take the sterilized bovine Achilles tendon powder, swell it with citric acid solution, disperse and crush it, and extract it by acid enzymatic method with acetic acid solution and pepsin to obtain enzymatic solution.
[0060] In a specific example, the mass fraction of citric acid in the citric acid solution is 4%-6%, optionally 4%, 5% or 6%, and the swelling time is 1h-2h.
[0061] In a specific example, the concentration of acetic acid in the acetic acid solution is 0.5 mol / L-1 mol / L, which can be 0.5 mol / L, 0.8 mol / L or 1 mol / L.
[0062] In a specific example, the amount of pepsin used is 1%-5% of the mass of the bovine Achilles tendon powder, which can be 1%, 2%, 3%, 4% or 5%. In a specific example, the enzymolysis temperature is 18°C-25°C, and the enzymolysis time is 72h-120h.
[0063] Step 40: After the enzymatic hydrolysate is filtered step by step, the upper clear liquid is collected after centrifugation, and salted out with saturated saline to obtain a primary salted out product of atelocollagen.
[0064] In a specific example, filters with mesh sizes of 10-300 (eg, 10 mesh, 50 mesh, 100 mesh, and 300 mesh) are used for step-by-step filtration.
[0065] Step 50: The atelocollagen primary salt-precipitate is added to an acetic acid solution to a concentration of 5 mg / mL-10 mg / mL, the upper clear liquid is collected, and salt-precipitated with saturated saline to prepare the atelocollagen secondary salt-precipitate.
[0066] The present application can reduce the endotoxin in the collagen solution after subsequent acylation, increase the acylation rate of collagen, reduce the viscosity of the collagen solution after acylation, and make the acylated collagen freeze-dried product dissolve quickly by improving the collagen extraction or purification process.
[0067] In a specific example, the preparation method further includes the step of acylation of the above-mentioned atelocollagen salting product.
[0068] The raw material used in the acylation process of the present application is the secondary salting out of the atelocollagen. After the acylation process is completed, centrifugation, dialysis, ultrafiltration concentration, filtration sterilization, and freeze-drying are performed to fully remove endotoxins and avoid pyrogenic reactions.
[0069] In a specific example, the acylation of the secondary salting-out product of atelocollagen includes steps 100 to 200:
[0070] Step 100: Add the secondary salting out of the atelocollagen to purified water and swell it at low temperature to form a collagen solution, so that the collagen concentration is 5 mg / mL-10 mg / mL, add acetone solution and sodium hydroxide solution containing succinic anhydride, control the pH to 8-9, and carry out acylation reaction at a temperature of 25°C-30°C.
[0071] In a specific example, the added amount of succinic anhydride is 20%-40% of the mass of collagen, which can be 20%, 25%, 30%, 35%, 40%, or a range consisting of any two of the above values.
[0072] In a specific example, the mass fraction of succinic anhydride in the acetone solution is 5%-10%, which can be 5%, 6%, 7%, 8%, 9%, 10%, or a range consisting of any two of the above values.
[0073] In a specific example, the concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L, which can be 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, or a range consisting of any two of the above values.
[0074] Step 200: After the reaction, the supernatant is collected, dialyzed with water, and concentrated to 2 mg / mL-5 mg / mL by ultrafiltration. The filtrate is obtained by membrane filtration to prepare acylated type I collagen.
[0075] In one specific example, the dialysis molecular weight cut-off is 10W Daltons.
[0076] In a specific example, the ultrafiltration membrane package has a molecular weight cut-off of 100 kDa-300 kDa.
[0077] In one specific example, the filtrate is freeze-dried to obtain an acylated collagen sponge to prepare acylated collagen. In one specific example, the acylated collagen sponge is dissolved in purified water and a preservative is added to prepare an acylated collagen solution.
[0078] The collagen liquid provided in the present application is prepared by freeze-drying low-endotoxic acylated collagen and then storing it in a frozen state. When used, the collagen is mixed with other solvents, thereby improving the safety performance of the product, avoiding the decomposition of collagen, and improving the stability of the product.
[0079] In a specific example, the preservative is selected from one or more of butylene glycol, pentylene glycol, hexylene glycol, benzyl alcohol and phenoxyethanol.
[0080] In a specific example, the mass ratio of the preservative is 1%-10% based on the total mass, and can be optionally 1%, 2%, 5%, 8%, 10%, or a range consisting of any two of the above values.
[0081] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.
[0082] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
[0083] Example 1
[0084] A method for preparing an acylated type I collagen solution comprises the following steps:
[0085] 1. Preparation of atelocollagen type I salt precipitation:
[0086] ① After thawing the bovine Achilles tendon, remove the surface fascia, and then slice it. The sliced bovine Achilles tendon is frozen and crushed with liquid nitrogen to obtain bovine Achilles tendon powder;
[0087] ② Rinse the bovine Achilles tendon powder with clean water until there is no blood, soak it in a 1%wt hydrogen peroxide solution for 2 hours, drain it and wash it three times with ultrapure water;
[0088] ③ After soaking in 0.5%wt Triton X-114 for 0.5h, continue soaking in 1M sodium hydroxide solution for 1h, then wash with ultrapure water until the filtrate is neutral, squeeze out the water and freeze-irradiate for sterilization, with an irradiation dose of 10kgy.
[0089] ④ Take the sterilized bovine Achilles tendon powder and soak it in 5%wt citric acid solution for 60 minutes, then use a high-speed disperser to further break it up, and then use 0.5M acetic acid solution and pepsin for acid enzymatic extraction. The amount of pepsin used is 2% of the mass of the bovine Achilles tendon, the enzymatic hydrolysis temperature is 18°C, and the enzymatic hydrolysis time is 72h to obtain the enzymatic hydrolyzate.
[0090] ⑤ After filtering the enzymatic hydrolyzate through 10 mesh, 50 mesh, 100 mesh and 300 mesh in sequence, centrifuge the filtrate at 12000 rpm and take the upper clear transparent liquid as the crude collagen extract, and salt out with saturated saline to obtain the first salting out product of atelocollagen type I.
[0091] ⑥ Add all the collagen primary salting out products to 0.5M acetic acid solution at a concentration of 5.0 mg / mL. After complete dissolution, take the upper clear liquid as the filtrate by high-speed centrifugation, and salt out with saturated saline to obtain the atelocollagen type I secondary salting out product 1.
[0092] 2. Type I collagen acylation process
[0093] ① Take the secondary salting out product 1 of type I collagen without telopeptide and add purified water to swell at low temperature to form collagen solution 1, the collagen concentration is about 5 mg / mL, add dropwise an acetone solution containing succinic anhydride, the amount of succinic anhydride added is 20% of the mass of collagen, and its concentration in the acetone solution is 5%, and at the same time, add dropwise a 0.5M sodium hydroxide solution, control the pH at 9, and the temperature at 30°C;
[0094] ② After the addition reaction is completed, the reaction is continued for 2 hours, and the supernatant is taken by centrifugation and dialyzed with ultrapure water at 2-8°C for 72 hours. After dialysis to remove salt and solvent, ultrafiltration is concentrated to 2 mg / mL. The ultrafiltration membrane package has a molecular weight cutoff of 300 kDa, and then it is sterilized by filtration through a 0.22 μm filter membrane. The filtrate is lyophilized to obtain acylated type I collagen sponge.
[0095] 3. Preparation of acylated type I collagen solution
[0096] Take 250 mg of acylated type I collagen sponge (water content 20%) and dissolve it completely in 95 g of purified water, add 4 g of pentanediol and 1 g of hexanediol and mix well to obtain acylated type I collagen solution with a concentration of 2 mg / mL.
[0097] Example 2
[0098] A method for preparing an acylated type I collagen solution comprises the following steps:
[0099] 1. Preparation of atelocollagen type I salt precipitation
[0100] ① After thawing the bovine Achilles tendon, remove the surface fascia, and then slice it. The sliced bovine Achilles tendon is frozen and crushed with liquid nitrogen to obtain bovine Achilles tendon powder;
[0101] ② Rinse the beef Achilles tendon powder with clean water until there is no blood, then soak it in 1% hydrogen peroxide for 1 hour, drain and wash it three times with ultrapure water;
[0102] ③ After soaking in 0.5%wt Triton X-114 for 0.5h, continue soaking in 1M sodium hydroxide solution for 1h, then wash with ultrapure water until the filtrate is neutral, squeeze out the water and freeze-irradiate for sterilization, with an irradiation dose of 10kgy.
[0103] ④ Take the sterilized bovine Achilles tendon powder and soak it in 5%wt citric acid solution for 60 minutes, then use a high-speed disperser to further break it up, and then use 0.8M acetic acid solution and pepsin for acid enzymatic extraction. The amount of pepsin used is 4% of the bovine Achilles tendon, the enzymatic hydrolysis temperature is 24°C, and the enzymatic hydrolysis time is 96h to obtain the enzymatic solution.
[0104] ⑤ After filtering the enzymatic hydrolyzate through 10 mesh, 50 mesh, 100 mesh and 300 mesh in sequence, centrifuge the filtrate at 12000 rpm and take the upper clear transparent liquid as the crude collagen extract, and salt out with saturated saline to obtain the first salting out product of atelocollagen type I.
[0105] ⑥ Add all the collagen primary salting out products to 0.5M acetic acid solution at a concentration of 8 mg / mL. After complete dissolution, take the upper clear liquid from the filtrate by high-speed centrifugation as the secondary salting out product 2 of de-telopeptide type I collagen by salting out with saturated saline.
[0106] 2. Type I collagen acylation process
[0107] ① Take the secondary salting out product 2 of type I collagen without telopeptide and add purified water to swell at low temperature to form collagen solution 2, the collagen concentration is about 5 mg / mL, add dropwise an acetone solution containing succinic anhydride, the addition amount of succinic anhydride is 30% of the mass of collagen, and its concentration in the acetone solution is 10%, and at the same time, add dropwise a 1M sodium hydroxide solution, control the pH at 8, and the temperature at 25°C;
[0108] ② After the addition reaction is completed, the reaction is continued for 2 hours, and the supernatant is taken by centrifugation and dialyzed with ultrapure water at 2-8°C for 72 hours. After dialysis to remove salt and solvent, ultrafiltration is concentrated to 2 mg / mL. The ultrafiltration membrane package has a molecular weight cutoff of 300 kDa, and then it is sterilized by filtration through a 0.22 μm filter membrane. The filtrate is lyophilized to obtain acylated type I collagen sponge.
[0109] 3. Preparation of acylated type I collagen solution
[0110] Take 250 mg of acylated type I collagen sponge (water content 20%) and dissolve it completely in 95 g of purified water, add 4 g of pentanediol and 1 g of hexanediol and mix well to obtain acylated type I collagen solution with a concentration of 2 mg / mL.
[0111] Example 3
[0112] A method for preparing an acylated type I collagen solution comprises the following steps:
[0113] 1. Preparation of atelocollagen type I salt precipitation:
[0114] ① After thawing the bovine Achilles tendon, remove the surface fascia, and then slice it. The sliced bovine Achilles tendon is frozen and crushed with liquid nitrogen to obtain bovine Achilles tendon powder;
[0115] ② Rinse the bovine Achilles tendon powder with clean water until there is no blood, soak it in a 3%wt hydrogen peroxide solution for 3 hours, drain it and wash it three times with ultrapure water;
[0116] ③ After soaking in 0.1%wt Triton X-114 for 1.5h, soak in 0.5M sodium hydroxide solution for another 1.5h, then wash with ultrapure water until the filtrate is neutral, squeeze out the water and freeze-irradiate for sterilization, with an irradiation dose of 5kgy.
[0117] ④ Take the sterilized bovine Achilles tendon powder and soak it in a 4%wt citric acid solution for 120 minutes, then use a high-speed disperser to further break it up, and then use 1M acetic acid solution and pepsin for acid enzymatic extraction. The amount of pepsin used is 5% of the mass of the bovine Achilles tendon, the enzymatic hydrolysis temperature is 25°C, and the enzymatic hydrolysis time is 120h to obtain the enzymatic solution.
[0118] ⑤ After filtering the enzymatic hydrolyzate through 10 mesh, 50 mesh, 100 mesh and 300 mesh in sequence, centrifuge the filtrate at 12000 rpm and take the upper clear transparent liquid as the crude collagen extract, and salt out with saturated saline to obtain the first salting out product of atelocollagen type I.
[0119] ⑥ Add all the collagen primary salting out products to 1M acetic acid solution at a concentration of 10 mg / mL. After complete dissolution, take the upper clear liquid from the filtrate by high-speed centrifugation as the secondary salting out product 1 of de-telopeptide type I collagen by salting out with saturated saline.
[0120] 2. Type I collagen acylation process
[0121] The steps are the same as in Example 1.
[0122] 3. Preparation of acylated type I collagen solution
[0123] The steps are the same as in Example 1.
[0124] Example 4
[0125] A method for preparing an acylated type I collagen solution comprises the following steps:
[0126] 1. Preparation of atelocollagen type I salt precipitation:
[0127] ① After thawing the bovine Achilles tendon, remove the surface fascia, and then slice it. The sliced bovine Achilles tendon is frozen and crushed with liquid nitrogen to obtain bovine Achilles tendon powder;
[0128] ② Rinse the bovine Achilles tendon powder with clean water until there is no blood, soak it in a 5%wt hydrogen peroxide solution for 1 hour, drain it and wash it three times with ultrapure water;
[0129] ③ After soaking in 1%wt Triton X-114 for 0.5h, soak in 1.5M sodium hydroxide solution for another 0.5h, then wash with ultrapure water until the filtrate is neutral, squeeze out the water and freeze-irradiate for sterilization, with an irradiation dose of 15kgy.
[0130] ④ Take the sterilized bovine Achilles tendon powder and soak it in a 6%wt citric acid solution for 120 minutes, then use a high-speed disperser to further break it up, and then use 0.5M acetic acid solution and pepsin for acid enzymatic extraction. The amount of pepsin used is 2% of the mass of the bovine Achilles tendon, the enzymatic hydrolysis temperature is 18°C, and the enzymatic hydrolysis time is 72h to obtain the enzymatic hydrolyzate.
[0131] ⑤ After filtering the enzymatic hydrolyzate through 10 mesh, 50 mesh, 100 mesh and 300 mesh in sequence, centrifuge the filtrate at 12000 rpm and take the upper clear transparent liquid as the crude collagen extract, and salt out with saturated saline to obtain the first salting out product of atelocollagen type I.
[0132] ⑥ Add all the collagen primary salting out products to 0.5M acetic acid solution at a concentration of 10 mg / mL. After complete dissolution, take the upper clear liquid from the filtrate by high-speed centrifugation as the secondary salting out product 1 of de-telopeptide type I collagen by salting out with saturated saline.
[0133] 2. Type I collagen acylation process
[0134] The steps are the same as in Example 1.
[0135] 3. Preparation of acylated type I collagen solution
[0136] The steps are the same as in Example 1.
[0137] Example 5
[0138] A method for preparing an acylated type I collagen solution comprises the following steps:
[0139] 1. Preparation of atelocollagen type I salt precipitation:
[0140] The steps are the same as in Example 1.
[0141] 2. Type I collagen acylation process
[0142] ① Take the secondary salting out product 1 of type I collagen without telopeptide and add purified water to swell at low temperature to form collagen solution 1, the collagen concentration is about 5 mg / mL, add dropwise an acetone solution containing succinic anhydride, the amount of succinic anhydride added is 40% of the mass of collagen, and its concentration in the acetone solution is 10%, and at the same time, add dropwise a 1.5M sodium hydroxide solution, control the pH at 8, and the temperature at 25°C;
[0143] ② After the addition reaction is completed, the reaction is continued for 1 hour, and the supernatant is centrifuged and dialyzed with ultrapure water at 2-8°C for 72 hours. After dialysis to remove salts and solvents, ultrafiltration is concentrated to 2 mg / mL. The ultrafiltration membrane package has a molecular weight cutoff of 300 kDa, and then it is filtered through a 0.22 μm filter membrane for sterilization. The filtrate is lyophilized to obtain acylated type I collagen sponge.
[0144] 3. Preparation of acylated type I collagen solution
[0145] Take 250 mg of acylated type I collagen sponge (water content 20%) and dissolve it completely in 95 g of purified water, add 4 g of pentanediol and 1 g of hexanediol and mix well to obtain acylated type I collagen solution with a concentration of 2 mg / mL.
[0146] Comparative Example 1
[0147] Compared with Example 1, the difference is that there is no step ⑥ in the preparation of the salting-out product of de-telopeptide type I collagen, the salting-out product used in step ① in the acylation process of type I collagen is the primary salting-out product of de-telopeptide type I collagen, and the other conditions remain unchanged.
[0148] Comparative Example 2
[0149] Compared with Example 1, the difference is that the process of step ⑥ in the preparation of the salting out of de-telopeptide type I collagen is replaced by adding all the collagen primary salting out to 0.5M acetic acid solution at a concentration of 5mg / mL. After complete dissolution, purified water is gradually added to dilute to 0.5mg / mL, and the tangential flow ultrafiltration membrane package is continuously circulated and ultrafiltered and concentrated until the conductivity no longer changes ≤200μS / cm, and the collagen concentration is 5mg / mL to obtain comparative example collagen ultrafiltrate 2. In addition, the secondary salting out of de-telopeptide type I collagen in step ① during the acylation of type I collagen is replaced with comparative example collagen ultrafiltrate 2 of the same concentration, and the other conditions remain unchanged.
[0150] Comparative Example 3
[0151] A method for preparing an acylated type I collagen solution comprises the following steps:
[0152] 1. Preparation of atelocollagen type I salt precipitation
[0153] ① After thawing the bovine Achilles tendon, remove the surface fascia, and then slice it. The sliced bovine Achilles tendon is frozen and crushed with liquid nitrogen to obtain bovine Achilles tendon powder;
[0154] ② Rinse the beef Achilles tendon powder with clean water until there is no blood, then soak it in 1% hydrogen peroxide, drain and wash it three times with ultrapure water;
[0155] ③ After soaking in 0.5%wt Triton X-114, continue soaking in 1M sodium hydroxide solution, then wash with ultrapure water until the filtrate is neutral, squeeze out the water and freeze-irradiate for sterilization, with an irradiation dose of 10kgy.
[0156] ④ Take the sterilized bovine Achilles tendon powder and swell it with citric acid, then use a high-speed disperser to further break it up, and then use 0.8M acetic acid solution and pepsin for acid enzymatic extraction. The amount of pepsin used is 4% of the bovine Achilles tendon, the enzymatic hydrolysis temperature is 15°C, and the enzymatic hydrolysis time is 120h to obtain the enzymatic hydrolyzate.
[0157] ⑤ After filtering the enzymatic hydrolyzate through 10 mesh, 50 mesh, 100 mesh and 300 mesh in sequence, centrifuge the filtrate at 12000 rpm and take the upper clear transparent liquid as the crude collagen extract, and salt out with saturated saline to obtain the first salting out product of atelocollagen type I.
[0158] ⑥ Add all the collagen primary salting out products to 0.5M acetic acid solution at a concentration of 5-10mg / mL. After complete dissolution, take the upper clear liquid from the filtrate by high-speed centrifugation as the secondary salting out product 3 of the comparative example de-telopeptide type I collagen by salting out with saturated saline.
[0159] 2. Type I collagen acylation process
[0160] ① Take the comparative example of de-telopeptide type I collagen secondary salting out product 3, add purified water to swell at low temperature to form a comparative example of collagen solution 3, the collagen concentration is about 5 mg / mL, add dropwise an acetone solution containing succinic anhydride, the amount of succinic anhydride added is 30% of the amount of collagen, and its concentration in the acetone solution is 10%, and at the same time, add dropwise a 1M sodium hydroxide solution, control the pH at 7, and the temperature at 20°C;
[0161] ② After the addition reaction is completed, the reaction is continued for 2 hours, and the supernatant is taken by centrifugation and dialyzed with ultrapure water at 2-8°C for 72 hours. After dialysis to remove salt and solvent, ultrafiltration is concentrated to 2 mg / mL. The ultrafiltration membrane package has a molecular weight cutoff of 300 kDa, and then it is sterilized by filtration through a 0.22 μm filter membrane. The filtrate is lyophilized to obtain acylated type I collagen sponge.
[0162] 3. Preparation of acylated type I collagen solution
[0163] Take 250 mg of acylated type I collagen sponge (water content 20%) and dissolve it completely in 95 g of purified water, add 4 g of pentanediol and 1 g of hexanediol and mix well to obtain acylated type I collagen solution with a concentration of 2 mg / mL.
[0164] test
[0165] 1. Viscosity test
[0166] Install the Brookfield viscometer on a stable table, rotate the three screws at the bottom to center the bubble in the top level, and ensure that the instrument is in a horizontal state. Connect the power supply and check whether the voltage meets the instrument requirements, which is generally 220V, 50Hz. Select a suitable rotor according to the viscosity range of the sample to be tested. Different rotors have different corresponding viscosity measurement ranges. In general, the smaller the rotor volume (the larger the number), the lower the speed, and the larger the corresponding range; vice versa. Hold the top of the connector and tighten the rotor to the left. Start the motor of the viscometer and rotate the rotor at a constant speed in the sample. After the viscometer reading is stable, record the viscosity value at this time. The test samples are the collagen solutions before and after acylation in the embodiment and the comparative example.
[0167] The viscosity test results are shown in Table 1.
[0168] Table 1
[0169]
[0170] 2. Acylation degree detection
[0171] The acylation effect of acylated collagen is expressed by the acylation rate. Amino acid analysis of type 1 collagen shows that among the side chain amino acids of collagen, only lysine belongs to the ε-amino group, accounting for about 3.13%. Therefore, the detection of the acylation rate is to detect the reduction rate of lysine. The trinitrobenzene sulfonic acid method (TNBS) was used to determine the free amino acid, and the amount of ε-amino group in collagen was detected by the quantitative reaction of trinitrobenzene sulfonic acid with the ε-amino group in collagen. TNBS reacts with the ε-amino group under weak alkaline conditions to form an intermediate complex with a chromophore. The collagen samples before and after acylation were dissolved in 0.1 mol / L acetic acid or purified water to prepare a 4 mg / mL collagen solution, and a certain amount of borax buffer and TNBS solution were added respectively, placed in a 50°C water bath to avoid light for 60 min, and the reaction was terminated with hydrochloric acid, cooled, and the absorbance of the sample was measured at a wavelength of 340 nm. Type I collagen was used as a control sample, and the acylated collagen was measured in parallel 3 times, and the average value was taken. Acylation rate (%) = (absorbance (type I collagen) - absorbance (acylated collagen)) / absorbance (type I collagen). The test results are shown in Table 2.
[0172] Table 2
[0173]
[0174] 3. Determination of dissolution time
[0175] Take a whole bottle of freeze-dried acylated collagen freeze-dried sponge, add a certain amount of purified water and shake until fully dissolved, then record the time required for dissolution at 25°C. Repeat the experiment 4 times and take the average value. The time is shown in Table 3.
[0176] Table 3
[0177]
[0178] 4. Endotoxin detection
[0179] The bacterial endotoxin (photometric method) detection standard was established with reference to the 2020 edition of the Chinese Pharmacopoeia 1143 Bacterial Endotoxin Test Method 2 Photometric Method and YY / T 0618-2017 Test Method for Bacterial Endotoxins in Medical Devices. Test equipment ET microplate reader (Thermo Fisher / Multiskan FC). All samples (acylated type I collagen solution) were diluted to the same multiple to a concentration of less than 0.5 mg / mL for testing. The test results are shown in Table 4.
[0180] Table 4
[0181]
[0182] 5. Acylated type I collagen gel electrophoresis test
[0183] Polyacrylamide gel analysis: Dilute bovine serum albumin with ultrapure water to a gradient concentration, then take 20 µL of each concentration of bovine serum albumin solution and the sample to be tested (acylated type I collagen solution) and load them. Connect the electrodes and start electrophoresis at a voltage of 110 V. When the blue color of bromophenol blue reaches the metal wire, stop the electrophoresis. Remove the film, add an appropriate amount of Coomassie brilliant blue staining solution, let it stand overnight for use, then decolorize the sample with Coomassie brilliant blue decolorizing solution and take photos for analysis. See the results of gel electrophoresis test example 1. Figure 1 .
[0184] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0185] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.
Claims
1. A method for preparing acylated collagen, characterized in that: The method comprises the steps of preparing a salting-out product of atelocollagen and acylating the salting-out product of atelocollagen; Wherein, the steps of preparing the atelocollagen salting-out product include: Step 10: Soak the beef Achilles tendon in hydrogen peroxide solution and wash with water; Step 20: After soaking in the Triton X-114 solution, continue soaking in the sodium hydroxide solution, wash with water until neutral, squeeze out the water and then freeze-irradiate for sterilization; Step 30: taking the sterilized bovine Achilles tendon powder, soaking it in a citric acid solution, dispersing and crushing it, and performing acid enzymatic extraction with an acetic acid solution and pepsin to prepare an enzymatic solution; Step 40: After filtering the enzymatic hydrolysate, centrifuging and collecting the upper clear liquid, and salting it out with saturated saline to prepare a primary salting out product of atelocollagen; and, Step 50: The atelocollagen primary salt-precipitated product is added to an acetic acid solution, the upper clear transparent liquid is collected, and salt-precipitated with saturated saline to prepare the atelocollagen secondary salt-precipitated product.
2. The preparation method according to claim 1, characterized in that: In step 10, the mass fraction of hydrogen peroxide in the hydrogen peroxide solution is 1%-5%, and the soaking time is 1h-3h.
3. The preparation method according to claim 1 or 2, characterized in that: In step 20, one or more of the following conditions are met: (1) The mass fraction of Triton X-114 in the Triton X-114 solution is 0.1%-1%, and the immersion time is 0.5h-1.5h; (2) the concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L, and the soaking time is 0.5 h-1.5 h; and, (3) The irradiation dose used for cryo-irradiation sterilization is 5kgy-15kgy.
4. The preparation method according to claim 1 or 2, characterized in that: Step 30: satisfy one or more of the following conditions: a. The mass fraction of citric acid in the citric acid solution is 4%-6%, and the swelling time is 1h -2h; b. the concentration of acetic acid in the acetic acid solution is 0.5 mol / L-1 mol / L; and, c. The amount of pepsin used is 1%-5% of the mass of the bovine Achilles tendon powder, the enzymolysis temperature is 18°C-25°C, and the enzymolysis time is 72h-120h.
5. The preparation method according to claim 1 or 2, characterized in that: In step 40, filter screens of 10 mesh, 50 mesh, 100 mesh and 300 mesh are used for step-by-step filtration.
6. The preparation method according to claim 1 or 2, characterized in that: The step of acylation of the atelocollagen secondary salting-out product comprises: Step 100: adding the atelocollagen secondary salting out product into water to swell at low temperature to form a collagen solution, making the collagen concentration 5 mg / mL-10 mg / mL, adding acetone solution and sodium hydroxide solution containing succinic anhydride, controlling the pH to 8-9, and performing acylation reaction at a temperature of 25°C-30°C; and, Step 200: After the reaction, the supernatant is collected, dialyzed with ultrapure water, and concentrated to 2 mg / mL-5 mg / mL by ultrafiltration. The filtrate is filtered through a filter membrane to prepare acylated collagen.
7. The preparation method according to claim 6, characterized in that: The step of acylation of the atelocollagen secondary salting-out product satisfies one or more of the following conditions: (1) The amount of succinic anhydride added is 20%-40% of the mass of collagen; (2) the mass fraction of succinic anhydride in the acetone solution is 5%-10%; and, (3) The concentration of sodium hydroxide in the sodium hydroxide solution is 0.5 mol / L-1.5 mol / L.
8. The preparation method according to claim 6, characterized in that: The filtrate is freeze-dried to prepare an acylated collagen sponge and an acylated collagen; Optionally, the acylated collagen sponge is dissolved in purified water, and a preservative is added to prepare an acylated collagen solution.
9. The preparation method according to claim 8, characterized in that: The preservative is selected from one or more of butylene glycol, pentylene glycol, hexylene glycol, benzyl alcohol and phenoxyethanol; or / and, The amount of the preservative added is 1%-10% based on the total mass of the acylated collagen solution.
10. The preparation method according to claim 1 or 2, characterized in that: After the bovine Achilles tendon is thawed, the surface fascia is removed, the tendon is sliced, and the tendon is frozen and broken with liquid nitrogen to prepare the bovine Achilles tendon powder.