A method for extracting high-purity collagen type I

Abstract

The application provides an extraction method of high-purity type I collagen, and belongs to the technical field of biomedical materials. The application realizes efficient and stable extraction of type I collagen by accurately selecting process conditions, significantly improves the purity and extraction efficiency of the target product, and the obtained collagen has complete three-dimensional structure, extremely low impurity residue and water content, greatly improves the biocompatibility and storage stability of the product, the method is simple in process, easy to control in condition, does not need complex large-scale equipment, is suitable for large-scale production, provides a reliable high-quality raw material source for high-end implant medical devices, and expands the application prospect of collagen in the field of biomedicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for extracting high-purity type I collagen. Background Technology

[0002] Collagen is an important structural protein in animal connective tissue. Type I collagen, in particular, has wide applications in tissue engineering, wound repair, and medical implant materials due to its high strength and biocompatibility. Its biological function is highly dependent on its intact triple helix natural structure.

[0003] Currently, the mainstream methods for extracting type I collagen from animal tissues include acid extraction, enzymatic extraction, or a combination of both. These methods face a common dilemma in practical applications: harsh processing conditions aimed at completely removing impurities such as proteins and lipids to improve purity (e.g., using high-concentration acids, alkalis, or prolonged enzymatic hydrolysis) often cause irreversible damage to the triple helix structure of collagen itself, leading to denaturation or degradation and loss of some bioactivity. Conversely, mild extraction conditions adopted to protect the structure often fail to sufficiently remove impurities, resulting in product purity that does not meet the standards of high-end medical materials. Existing technical solutions mostly focus on optimizing single steps, such as changing the type of enzyme or adjusting the precipitation method, but fail to systematically resolve the inherent contradiction between "high purity" and "structural integrity." Therefore, developing an extraction technology that can synergistically achieve high extraction purity and high-level structural preservation is of great significance for promoting the in-depth application of type I collagen in the biomedical field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting high-purity type I collagen, which solves the technical problems of low product purity and limited product quality caused by easy damage to the natural triple helix structure when extracting type I collagen in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for extracting high-purity type I collagen, comprising the following steps: Animal tissue materials rich in type I collagen were subjected to enzymatic hydrolysis. The enzymatically hydrolyzed material is minced to make meat paste; Anhydrous sodium sulfate solution and sodium hydroxide solution were added to the meat paste for alkalization treatment to obtain an alkalized product; An acid solution is added to the alkalization product for neutralization treatment to obtain the neutralized material. The neutralized material is cleaned and then freeze-dried to obtain type I collagen product.

[0007] Preferably, the animal tissue material rich in type I collagen is animal tendon tissue.

[0008] Preferably, the animal tendon tissue is the bovine Achilles tendon, which is taken from a Simmental bull less than 3 years old; The animal tissue material rich in type I collagen is obtained by a preparation method including the following steps: The tendon is cut 2-4 cm above the bifurcation and 6-10 cm below the bifurcation (see diagram for location). Figure 1 As shown), raw tendon blocks are obtained. After cleaning and freezing, the raw tendon blocks are cut into thin slices with a thickness of less than 2 mm to obtain the animal tissue material rich in type I collagen.

[0009] Preferably, the enzymatic hydrolysis treatment specifically includes: After washing the animal tissue material rich in type I collagen, it was placed in purified water, and after adding potassium dihydrogen phosphate to regulate the system, fig protease was added to carry out enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, the sample is washed and then soaked in sodium chloride solution. The amount of purified water used is 6-7 times the volume of the animal tissue material rich in type I collagen; The amount of potassium dihydrogen phosphate added is 2‰-4‰ of the mass of purified water; The amount of fig protease added is 0.7‰-1.4‰ of the mass of purified water; The reaction temperature for the enzymatic hydrolysis treatment is 37.0 ± 1.0 °C; The enzymatic hydrolysis is carried out under stirring at a speed of 1-2 revolutions per minute. The sodium chloride solution has a mass fraction of 0.5%-2%, the soaking temperature is 29-31℃, and the soaking time is 1-1.5 hours.

[0010] Preferably, in the alkalization treatment, the mass fraction of the anhydrous sodium sulfate solution is 5%-15%; The amount of the anhydrous sodium sulfate solution is 3-5 times the volume of the meat paste. In the alkalization treatment, the sodium hydroxide solution has a mass fraction of 1%-3% and is used to adjust the pH value of the mixture to 10-13. The alkalization treatment is carried out at a reaction temperature of 20-26°C for 38-45 hours.

[0011] Preferably, the acid solution used in the neutralization process is a sulfuric acid solution, the pH value of the neutralized material is 4.5-7.0, and the neutralization time is 10-30 minutes.

[0012] Preferably, the cleaning step involves at least six cleaning cycles using water for injection, with the amount of water used in each cleaning cycle being 3-5 times the weight of the animal tissue material rich in type I collagen.

[0013] Preferably, the freeze-drying includes the following steps: The material is pre-frozen at -30℃ for 1-2 hours; then, under a vacuum of 30-40 Pa, it is subjected to segmented sublimation drying at -11℃, -4℃ and 0℃ in sequence; and then subjected to desorption drying at 25℃ and a vacuum of 25 Pa.

[0014] The present invention also provides a high-purity type I collagen, which is obtained by the above extraction method.

[0015] This invention also provides the application of high-purity type I collagen in the preparation of implantable medical materials.

[0016] The beneficial effects of this invention are: This invention achieves efficient and stable extraction of type I collagen through precise selection of process conditions, significantly improving the purity and extraction efficiency of the target product. The obtained collagen has a complete three-dimensional structure, extremely low impurity residue and water content, greatly enhancing the product's biocompatibility and storage stability. The method is simple, the conditions are easy to control, and it does not require complex large-scale equipment, making it suitable for large-scale production. It provides a reliable source of high-quality raw materials for high-end implantable medical devices and expands the application prospects of collagen in the biomedical field. Attached Figure Description

[0017] Figure 1 A diagram showing the site where the Achilles tendon is harvested in a cow. Figure 2 This is a graph showing the viral load decline of pseudorabies virus (PRV, CVCC AV249). Figure 3 The viral load curve for vesicular stomatitis virus (VSV, ATCC VR1238) is shown. Figure 4 The viral decline curve of Reovirus (Reo3, ATCC VR824); Figure 5 The graph shows the viral load decline of porcine parvovirus (PPV, CVCC AV31). Figure 6 Photograph of collagen before freeze-drying; Figure 7 Photos of collagen after freeze-drying; Figure 8 Photographs showing the state of alkalization treatment with anhydrous sodium sulfate solution; Figure 9Photographs showing the state of alkalinization treatment at the same pH level without the protection of anhydrous sodium sulfate solution. Detailed Implementation

[0018] This invention provides a method for extracting high-purity type I collagen. This method aims to efficiently isolate highly pure type I collagen with intact natural triple helix structure from specific animal tissues through a systematic, mild, and controllable process, thereby meeting the requirements of high-end biomedical materials for core protein raw materials.

[0019] Specifically, the method for extracting high-purity type I collagen includes a series of sequential steps. First, animal tissue material rich in type I collagen is enzymatically hydrolyzed. Enzymatic hydrolysis is a key step that utilizes proteases to specifically cleave the non-helical regions of the terminal peptides of collagen, thereby dissolving it from the tissue. In this invention, the enzymatic hydrolysis is preferably carried out under mild pH and temperature conditions to maximize the preservation of the natural structure of collagen. After enzymatic hydrolysis, the hydrolyzed material is mechanically minced to produce a homogeneous meat paste. This step helps to significantly increase the reaction surface area for subsequent chemical treatments, ensuring sufficient and uniform contact between the reagents and the collagen.

[0020] Subsequently, a series of treatments are carried out on the meat paste by adding a chemically formulated solution. First, anhydrous sodium sulfate solution and sodium hydroxide solution are added for alkalization, yielding an alkalized product. Alkalinization serves both sterilization and purification purposes in this method. Next, an acid solution is added to the alkalized product for neutralization, adjusting the pH of the system to near neutral, resulting in a neutralized material. Neutralization helps stabilize the charge state of collagen, preventing aggregation or denaturation. Then, the neutralized material is thoroughly washed to remove various salts, residual reagents, and small molecule impurities introduced or generated in the preceding steps. After washing, freeze-drying is performed, removing moisture from the material through low-temperature vacuum sublimation, ultimately obtaining a dry, porous, network-structured type I collagen product. Freeze-drying is a key drying method for maintaining the loose structure and bioactivity of collagen. This invention provides a high-salt environment by adding anhydrous sodium sulfate solution before the "alkalization" step, allowing the collagen solution to undergo preliminary salting-out precipitation. The high ionic strength also stabilizes the collagen structure, resisting collagen denaturation and acting as a buffer during the intense alkalization reaction, thus maintaining the structural stability of the target protein during the alkalization process. Simultaneously, the saponification effect of the strong alkaline environment removes lipid impurities and other protein contaminants from the solution, improving the purity of the target protein.

[0021] In a preferred embodiment of the present invention, the animal tissue material rich in type I collagen is animal tendon tissue. Animal tendon tissue is a natural source with extremely high enrichment of type I collagen, its fibrous structure is dense, and collagen content usually accounts for the majority of its dry weight. More preferably, the animal tendon tissue is bovine Achilles tendon. Bovine Achilles tendon, especially that of cattle of specific breeds and ages, is often regarded as a high-quality raw material for extracting medical-grade collagen due to its high type I collagen content, coarse fibers, and regular structure. Further preferably, the bovine Achilles tendon is obtained from Simmental cattle under 3 years of age. Simmental cattle are a common dual-purpose breed of beef cattle, and their Achilles tendon tissue is well-developed with moderate collagen cross-linking, making it easy to extract. The selection of age is to ensure the freshness of collagen and low intermolecular cross-linking, which is beneficial to improving the extraction rate and product solubility.

[0022] The animal tissue material rich in type I collagen was obtained through a sophisticated pretreatment process. Specifically, this included: first, precise cutting of the bovine Achilles tendon at specific locations, namely 2-4 cm above the tendon bifurcation and 6-10 cm below the bifurcation (see [reference to cutting locations]). Figure 1 (Illustrative diagram). This area is considered to have the highest purity of type I collagen and relatively low content of impurities such as fat and blood vessels. After cutting, raw tendon blocks are obtained. Subsequently, the raw tendon blocks are washed with pure water or purified water to remove surface dirt and some soluble impurities. After washing, they are subjected to low-temperature freezing to facilitate subsequent machining. After freezing, the raw tendon blocks are cut into slices with a thickness of less than 2 mm, for example, 0.5 mm, 1.0 mm, 1.5 mm, or 2.0 mm, preferably 0.5-1.5 mm, more preferably about 1.0 mm, thereby obtaining the animal tissue material rich in type I collagen with a significantly increased specific surface area. This step facilitates the uniform penetration and action of subsequent enzymatic hydrolysis reagents and solutions.

[0023] In this invention, the enzymatic hydrolysis process is specifically optimized. First, the pretreated animal tissue material rich in type I collagen is further washed to remove debris and residues that may have been generated during the cutting process. After washing, it is placed in purified water. The purified water refers to high-purity water that has undergone deionization, reverse osmosis, and other processes, and whose resistivity is typically greater than 1 MΩ·cm. Then, potassium dihydrogen phosphate is added to the system. Adding potassium dihydrogen phosphate solution as an electrolyte before enzymatic hydrolysis provides high ionic strength, which can break the ionic bonds between the amino and carboxyl groups of insoluble collagen, thus disrupting the tightly cross-linked fiber structure. The high-strength anions also chelate calcium ions in collagen fibers, further breaking down the dense packing of collagen fibers. Simultaneously, a weakly alkaline environment is provided, allowing collagen fibers to absorb water and swell, increasing their volume and softening the overall structure. This provides the prerequisite for fig protease to specifically cleave the non-helical regions of terminal peptides, effectively creating an environment conducive to efficient enzymatic hydrolysis, releasing the target soluble triple-helix collagen, and thus improving the collagen extraction rate. After adjusting the system, fig protease is added for enzymatic hydrolysis. Fig protease is a protease derived from the fig tree that effectively cleaves the terminal peptides of collagen. After the enzymatic hydrolysis is complete, the material is washed to terminate the enzymatic reaction and remove soluble impurities from the hydrolysis products. After washing, the material is soaked in sodium chloride solution. Soaking in sodium chloride solution helps to further stabilize collagen and may precipitate and remove certain impurities.

[0024] The process parameters involved in the enzymatic hydrolysis treatment have been optimized. The amount of purified water used is 6-7 times the volume of the animal tissue material rich in type I collagen, for example, 6, 6.5, or 7 times, preferably 6.5-7 times. The amount of potassium dihydrogen phosphate added is 2‰-4‰ of the mass of the purified water used, for example, 2‰, 3‰, or 4‰, preferably 2.5‰-3.5‰, more preferably about 3‰. The amount of fig protease added is 0.7‰-1.4‰ of the mass of the purified water used, for example, 0.7‰, 0.9‰, 1.1‰, or 1.3%, preferably 0.9‰-1.2‰, more preferably about 0.98‰. The reaction temperature of the enzymatic hydrolysis treatment is 37.0±1.0℃, that is, the reaction temperature is controlled between 36.0℃ and 38.0℃, preferably maintained at 37.0℃, which is the common temperature range for fig protease to exert its optimal activity. The enzymatic hydrolysis is carried out under stirring at a speed of 1-2 revolutions per minute, such as 1 rpm, 1.5 rpm, or 2 rpm, to achieve gentle mixing and mass transfer and avoid excessive mechanical shearing of the collagen fibers. The sodium chloride solution has a mass fraction of 0.5%-2%, such as 0.5%, 1%, or 2%, preferably 0.8%-1.5%. The soaking temperature is 29-31°C, such as 29°C, 30°C, or 31°C, preferably 30±0.5°C. The soaking time is 1-1.5 hours, such as 1 hour, 1.25 hours, or 1.5 hours, preferably 1-1.25 hours.

[0025] The reagents and conditions involved in the alkalization treatment are also specified in detail. The anhydrous sodium sulfate solution has a mass fraction of 5%-15%, for example 5%, 10% or 15%, preferably 8%-12%, more preferably about 10%. The amount of anhydrous sodium sulfate solution added is 3-5 times the volume of the meat paste weight, for example 3 times, 4 times or 5 times the volume, preferably 3.5-4.5 times the volume, more preferably about 4 times the volume. The mass fraction of the sodium hydroxide solution is 1%-3%, for example 1%, 2% or 3%, preferably 1.5%-2.5%. The amount added is sufficient to adjust the pH of the entire reaction mixture to the range of 10-13, for example pH 10, pH 11, pH 12 or pH 13, preferably pH 11-12.5, more preferably around pH 12. The reaction temperature of the alkalization treatment is controlled at 20-26°C, for example 22°C, 24°C or 26°C, preferably 25±0.5°C. The reaction time lasts for 38-45 hours, such as 40 hours, 42 hours, or 45 hours, preferably 41-44 hours, and more preferably about 42 hours. This long-term alkalization treatment at specific temperatures and pH levels has been proven to effectively inactivate any viruses that may be present, while the triple helix structure of collagen is well maintained under the protection of a high-concentration salt solution.

[0026] For the neutralization treatment, the acid solution used is preferably a sulfuric acid solution, such as a dilute sulfuric acid solution. The pH value of the material after the neutralization treatment should be adjusted to the range of 4.5-7.0, for example, pH 4.5, pH 5.5, pH 6.5, or pH 7.0, preferably pH 4.5-6.5, more preferably pH 5.0-6.0, and most preferably about pH 5.5. The neutralization treatment time, i.e., the soaking time of the material at the target pH, is 10-30 minutes, for example, 10 minutes, 20 minutes, or 30 minutes, preferably 15-25 minutes, and more preferably about 15 minutes.

[0027] In the cleaning step, water for injection is used for cleaning. The water for injection is sterile, pyrogen-free, high-purity water that meets pharmacopoeia standards. The cleaning is performed at least 6 times, for example, 6, 7, or 8 times, to ensure thorough removal of impurities. The amount of water for injection used in each cleaning is 3-5 times the volume of the animal tissue material (initial sheet) rich in type I collagen, for example, 3, 4, or 5 times the volume, preferably 3.5-4.5 times the volume.

[0028] The freeze-drying process specifically includes three stages. First, pre-freezing: the washed and drained collagen material is pre-frozen at -30°C for 1-2 hours, for example, 1 hour, 1.5 hours, or 2 hours, to completely freeze the moisture in the material. Then, sublimation drying: under a vacuum of 30-40 Pa, for example, 30 Pa, 35 Pa, or 40 Pa, sublimation drying is performed in stages at -11°C, -4°C, and 0°C. The temperature control for each stage is as follows: -11°C is preferably -10°C to -12°C, -4°C is preferably -3°C to -5°C, and 0°C is preferably -1°C to 1°C. The duration of each stage can be adjusted according to the amount of material to ensure sufficient sublimation of the ice crystals. Finally, desorption drying: the temperature is raised to 25°C (for example, 24-26°C), and desorption drying is performed under a vacuum of 25 Pa (for example, 20-30 Pa) to remove bound water, ultimately obtaining a collagen product with extremely low water content (for example, less than 5%, preferably less than 4%).

[0029] This invention also provides a high-purity type I collagen. The type I collagen is obtained by any of the extraction methods described above. Due to the use of the specific raw materials and precisely controlled processes described above, the obtained type I collagen product has the following characteristics: extremely high purity (e.g., type I collagen content can reach over 98%), effective preservation of the complete triple helix structure, extremely low impurity residues (such as viruses, endotoxins, and other proteins), low water content, and a white or off-white loose porous solid with good rehydration and biocompatibility.

[0030] This invention also provides the application of the high-purity type I collagen in the preparation of implantable medical materials. Implantable medical materials refer to biomaterials implanted into the human body for repairing, replacing, or assisting the function of human tissues or organs. The applications include, but are not limited to, using the high-purity type I collagen as a main component or additive in the preparation of various biodegradable implantable medical devices or materials. For example, it can be processed into different forms such as sponges, films, fibers, hydrogels, and microspheres, and applied to: bone defect repair materials (such as bone-filling sponges), cartilage repair scaffolds, skin tissue engineering scaffolds, nerve conduits, vascular patches, dura mater patches, heart valve matrix, medical sutures, and drug-releasing carriers. In these applications, the high-purity type I collagen can be used alone or in combination with other biocompatible materials, such as hyaluronic acid, chondroitin sulfate, hydroxyapatite, and polylactic-co-glycolic acid copolymer (PLGA), to comprehensively improve the mechanical properties, bioactivity, or degradation rate of the materials. The implantable medical material is preferably a Class III implantable biodegradable medical device. This type of device has the highest requirements for the biosafety, purity, and structural function of the raw materials, and the collagen product provided by this invention can meet these requirements.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 This embodiment provides a method for extracting type I collagen, the steps of which are as follows: The raw materials are sourced as follows: cattle breed: Simmental, age <3 years; Slaughter: Fresh live cattle are slaughtered and harvested on the same day; Transportation: Store in stainless steel drums filled with ice water at 0~4℃, and clean daily; Extracting the beef Achilles tendon: The beef tendon is extracted on the same day it is received. The tendon sheath of the beef tendon, which was originally stored in ice water, is cut open with scissors to remove the tendon. The tendon sheath and fascia are removed from the beef tendon. It is cut off 2-4cm above the fork and cut off 6-10cm below the fork.

[0033] Frozen beef Achilles tendon: After cutting, the beef Achilles tendon containing blood and yellow tissue is removed, cleaned with purified water, packaged in a plastic cup, and stored in a freezer below 18°C.

[0034] Cutting the beef Achilles tendon: The beef tendon frozen for up to 12 months is cut into thin slices with a thickness of 0.75 mm and a size of about 2.6 square centimeters using a slicer. The total weight of the beef Achilles tendon slices is 6000g. After cutting the beef Achilles tendon, the blood flakes and yellow flakes in the slices are removed to improve the purity of the collagen extracted in the end.

[0035] Enzymatic hydrolysis: Wash the weighed beef Achilles tendon slices with purified water until no visible grease is visible on the surface of the water. After washing, add purified water to the beef Achilles tendon slices at a ratio of beef Achilles tendon slice weight (g): purified water volume (ml) = 1:6.5. Place the purified water in a 316L stainless steel reaction vessel and adjust the water temperature to 25℃ through a circulating temperature control system. Add potassium dihydrogen phosphate to the reaction vessel, wherein the mass fraction of potassium dihydrogen phosphate is 3.5‰, and mix thoroughly.

[0036] Thin slices of bovine Achilles tendon were placed in a stainless steel basket, which was then placed in a reaction vessel and hung on a rotator. The rotator was rotated at a speed of 1 rpm to raise the water temperature to 37.0°C. 850 ml of the heated purified water was taken out as a solvent, and fig protease with a concentration of 0.98‰ was added. After adding the enzyme solution, the rotator was continuously rotated for 1 hour, and the solution was stirred once every 15 minutes.

[0037] After the reaction is complete, drain all the liquid from the reaction vessel, place the beef Achilles tendon slices in a new stainless steel container, add purified water for washing, the ratio of purified water to beef Achilles tendon slice weight (g): purified water volume (ml) = 3.5, wash 3 times, after washing add the above-mentioned purified water, let stand for 15 minutes, after standing, discard the water in the container, add the above-mentioned volume of purified water again, wash for 8 minutes, wash until the pH value is 6.5, discard the water in the basin.

[0038] The total amount of purified water was calculated according to the ratio of bovine Achilles tendon slice weight (g): purified water volume (ml) = 1:6.5. 5.6% of the water volume was used to dissolve sodium chloride, which was then added to the reaction vessel. The mass fraction of the sodium chloride was 1.5%. The basket containing the bovine Achilles tendon slices was then hung on the rotator and placed in the reaction vessel. The temperature was adjusted to 37±0.5℃, and then reduced to 30℃ after 1.2 hours. The bovine Achilles tendon slices were stirred once every 20 minutes, and the rotator was continuously rotated during this period.

[0039] After cleaning, drain the liquid, remove the beef Achilles tendon slices and place them in a stainless steel container. Add purified water for cleaning, with a purified water ratio of beef Achilles tendon slice weight (g): purified water volume (ml) = 1:3.5. After cleaning, discard the water in the container and repeat the cleaning process once. Then add purified water in the above ratio and let it stand for 10 minutes, discarding the water in the container. Add purified water again until it covers the beef Achilles tendon slices, and clean for 3 minutes, discarding the water in the container. Repeat this step 6 times. After cleaning, load the beef Achilles tendon slices into a 100-mesh nylon bag and remove the water using a MINI centrifuge at 1900 rpm for 2 minutes. After centrifugation, collect the beef Achilles tendon slices for use on the same day.

[0040] Making meat paste: Place the above-mentioned beef tendon slices in a meat grinder and stir, weigh them, prepare anhydrous sodium sulfate solution with a mass fraction of 15%, weigh purified water according to the ratio of meat paste weight (g): purified water volume (ml) = 1:6.3, and add anhydrous sodium sulfate.

[0041] First, mix anhydrous sodium sulfate solution with the minced meat in a ratio of 1:1.5 (minced meat weight (g): anhydrous sodium sulfate volume (ml)). Place the mixture in a meat grinder and stir for 60 minutes. Store the mixture on the same day after stirring.

[0042] Alkalization: Add the prepared anhydrous sodium sulfate solution to the above-stirred meat paste. The ratio of anhydrous sodium sulfate added is 1:3.5 (weight of meat paste (g): anhydrous sodium sulfate solution (ml)). Then add a 2.5% sodium hydroxide solution to adjust the pH to 12.8. Pour the meat paste into the reaction vessel and mix thoroughly.

[0043] The temperature was controlled at 25±1℃ using a temperature control system for 44 hours. Temperatures were checked at 1 hour, 17 hours, 25 hours, and 42 hours after the first day's reaction to ensure compliance with requirements. The remaining anhydrous sodium sulfate was used as a neutralization solution.

[0044] Neutralization: In the mixture after alkalization, part of the alkalization liquid is discharged. The discharge amount is the weight of the meat paste (g): the volume of the discharged liquid (ml) = (2~3). The remaining anhydrous sodium sulfate solution is divided into two parts in a 6:4 ratio. 50% sulfuric acid solution is added to the anhydrous sodium sulfate solution as a neutralization solution. The temperature is controlled below 20℃. The process involves two neutralization steps. For the first step, a 6:1 mixture of anhydrous sodium sulfate solution and 50% sulfuric acid is added to the alkalization tank. Before adding the neutralizing solution, the meat paste is removed, the solution is mixed thoroughly, and then the meat paste is added back in and mixed again. The meat paste is then squeezed to release the alkali. After neutralization, the meat paste is removed again. For the second step, a 4:1 mixture of anhydrous sodium sulfate solution and 50% sulfuric acid is added to the alkalization tank again. Before adding the neutralizing solution, the meat paste is removed, the solution is mixed thoroughly, and then the meat paste is added back in and mixed again. Finally, an appropriate amount of 5% H₂SO₄ or 5% NaOH is used to adjust the pH to 4.5. The meat paste is soaked for 15 minutes, and the pH is measured to be 4.5. Neutralization is then complete, and the meat paste is removed.

[0045] Washing: The neutralized meat paste is washed with water injection. The water volume is 3.8 times the weight of the beef tendon slices (g): the volume of water injection (ml). The washing is repeated six times. After the first five washings, the pH value is measured to be 4.5. The sixth washing is carried out with the same proportion of water injection. After washing, the material is taken out and some water is squeezed out. The moisture content is between 65% and 70%.

[0046] Freeze-drying: The retrieved material (collagen) is evenly spread in a freeze-drying tray, and the collagen is freeze-dried using vacuum freeze-drying technology. The freeze-drying parameters are shown in Table 1 below: Table 1 Freeze-drying parameters

[0047] Packaging and storage: After freeze-drying, pack the product into a medical-grade all-plastic packaging bag and heat-pack it. Store it at -18°C or below.

[0048] Example 2 This embodiment provides a method for extracting high-purity type I collagen, the steps of which are as follows: Procurement and raw material pretreatment: Simmental cattle aged 2 years were selected, and the fresh Achilles tendons were immediately stored in a container filled with ice water at 2°C after slaughter on the same day, and transported and processed on the same day.

[0049] After receiving the raw material, the tendon sheath is cut open with scissors and the bovine Achilles tendon is removed, along with the tendon sheath and fascia. The tendon is cut 3 cm above the bifurcation and 8 cm below the bifurcation to obtain a specific segment of bovine Achilles tendon tissue.

[0050] The cut beef Achilles tendon was cleaned with purified water to remove visible blood and yellow tissue, then packaged and frozen in a -20°C freezer.

[0051] The frozen beef Achilles tendon was sliced ​​into thin slices with a thickness of 1 mm and a size of about 2 square centimeters using a slicer. The total weight of the slices was 7000 grams, and blood and yellow pieces were removed from the slices.

[0052] Enzymatic hydrolysis: Wash 7000g of beef Achilles tendon slices with purified water until no visible grease is visible on the surface of the water. Place the slices in a reaction vessel and add 49000ml of purified water (the mass-to-volume ratio of beef Achilles tendon slices to purified water is 1:7). Adjust the water temperature to 25℃, add potassium dihydrogen phosphate to make its mass fraction in the system 3‰, and mix thoroughly.

[0053] Place the basket containing the flakes into the reaction vessel and rotate it at 1.5 rpm. Raise the system temperature to 37.0°C, then dissolve the fig protease in 900 mL of heated purified water to a concentration of 0.95‰ and add it to the reaction vessel. Continue rotating the reaction vessel at 37.0°C for 1 hour, stirring every 12 minutes during this time.

[0054] After the reaction is complete, drain the reaction solution. Transfer the bovine Achilles tendon slices to a new container and wash twice with 28,000 ml of purified water (ratio 1:4). Then add the same volume of purified water and let stand for 12 minutes. Discard the water and then add purified water again to wash for 8 minutes until the pH of the washing water is 6.5.

[0055] Take 5.5% of the total purified water (49000 ml), i.e., 2695 ml, dissolve sodium chloride to prepare a 1% sodium chloride solution, and add it to the reaction vessel. Place the beef Achilles tendon slices back into the vessel, maintain the temperature at 37.0°C, and then lower the temperature to 30°C over 1.2 hours, stirring every 30 minutes during this process, while the rotator continues to operate.

[0056] After treatment with sodium chloride solution, drain the liquid. Wash the slices once with 28,000 ml of purified water (1:4), discard the water, and repeat the washing process once more. Then add 28,000 ml of purified water and let stand for 12 minutes, then discard the water. Finally, add purified water to submerge the slices, wash for 3 minutes, discard the water, and repeat this washing step 6 times.

[0057] After cleaning, place the thin film into a 100-mesh nylon bag and centrifuge at 1900 rpm for 2 minutes to remove excess water. Collect the film for later use.

[0058] Making meat paste and alkalization: The processed beef tendon slices are minced using a meat grinder to obtain meat paste.

[0059] Prepare a 10% anhydrous sodium sulfate solution. First, take 1.5 times the weight of the minced meat and mix it with the minced meat in a meat grinder, stirring for 65 minutes.

[0060] Next, add four times its weight in volume of the above-mentioned anhydrous sodium sulfate solution to the meat paste. Then, add a 2% sodium hydroxide solution to adjust the pH of the mixture to 12, and transfer it to a reaction vessel to mix thoroughly.

[0061] The temperature of the reaction system was controlled at 25°C using a temperature control system, and the alkalization reaction lasted for 42 hours.

[0062] Neutralization and Cleansing: After alkalization, drain the alkalization solution, which is equivalent to 2.5 times the weight of the minced meat. Divide the remaining liquid into two parts (in a 6:4 volume ratio) and add sulfuric acid solution to each part to prepare a neutralization solution. The temperature of the neutralization solution should be controlled below 15°C.

[0063] Perform two neutralization processes: First, pour most of the neutralization solution (60%) into the reaction vessel and mix it thoroughly with the meat paste; then add the remaining neutralization solution (40%) and mix. Finally, precisely adjust the pH of the system to 5.5 using a 5% sulfuric acid solution and soak under this condition for 15 minutes. After neutralization, remove the meat paste.

[0064] The neutralized meat paste was washed with water for injection, using 28,000 ml of water for each wash (ratio 1:4), for a total of 6 washes. After the first five washes, the pH value of the aqueous phase was measured to be 4.5. After the sixth wash, the meat paste was removed and some water was squeezed out.

[0065] Freeze-drying and storage: The cleaned collagen meat paste was evenly spread in a freeze-drying tray and then subjected to vacuum freeze-drying. The freeze-drying parameters were the same as in Example 1.

[0066] After freeze-drying, the obtained collagen products are packaged in medical-grade all-plastic bags and thermosealed, and stored at -20°C or below.

[0067] Example 3 This embodiment provides a method for extracting high-purity type I collagen, the steps of which are as follows: Procurement and raw material pretreatment: Simmental cattle aged 1 year were selected, and the fresh Achilles tendons were immediately stored in a container filled with ice water at 0°C after slaughter on the same day, and transported and processed on the same day.

[0068] After receiving the raw material, the tendon sheath is cut open with scissors and the bovine Achilles tendon is removed, along with the tendon sheath and fascia. The tendon is cut 2 cm above the bifurcation and 6 cm below the bifurcation to obtain a specific segment of bovine Achilles tendon tissue.

[0069] The cut bovine Achilles tendon was cleaned with purified water to remove visible blood and yellow tissue, then packaged and frozen in a -18°C freezer.

[0070] The frozen beef Achilles tendon was sliced ​​into thin slices with a thickness of 0.5 mm and a size of about 1 square centimeter using a slicer. The total weight of the slices was 6000 grams, and blood and yellow pieces were removed from the slices.

[0071] Enzymatic hydrolysis: Wash 6000g of beef Achilles tendon slices with purified water until no visible grease is visible on the surface of the water. Place the slices in a reaction vessel and add 36000ml of purified water (the mass-to-volume ratio of beef Achilles tendon slices to purified water is 1:6). Adjust the water temperature to 23℃, add potassium dihydrogen phosphate to make its mass fraction in the system 2‰, and mix thoroughly.

[0072] Place the basket containing the flakes into the reaction vessel and rotate it at 1 rpm. Raise the system temperature to 37.0°C, then dissolve the fig protease in 800 mL of heated purified water to a concentration of 0.90‰ and add it to the reaction vessel. Continue rotating the reaction vessel at 37.0°C for 1 hour, stirring every 10 minutes during this time.

[0073] After the reaction is complete, drain the reaction solution. Transfer the bovine Achilles tendon slices to a new container and wash three times with 18,000 ml of purified water (ratio 1:3). Then add the same volume of purified water and let stand for 10 minutes. Discard the water and then add purified water again to wash for 5 minutes until the pH of the washing water is 5.0.

[0074] Take another 5% (1800 ml) of the total purified water (36000 ml), dissolve sodium chloride to prepare a 0.5% sodium chloride solution, and add it to the reaction vessel. Place the beef Achilles tendon slices back into the vessel, maintain the temperature at 37.0°C, and then lower the temperature to 29°C over 1 hour, stirring every 10 minutes during this process, while the rotator continues to operate.

[0075] After treatment with sodium chloride solution, drain the liquid. Wash the slices once with 18000 ml of purified water (1:3), discard the water, and repeat the washing process once more. Then add 18000 ml of purified water and let stand for 10 minutes, then discard the water. Finally, add purified water to submerge the slices, wash for 3 minutes, discard the water, and repeat this washing step 6 times.

[0076] After cleaning, place the thin film into a 100-mesh nylon bag and centrifuge at 1800 rpm for 2 minutes to remove excess water. Collect the film for later use.

[0077] Making meat paste and alkalization: The processed beef tendon slices are minced using a meat grinder to obtain meat paste.

[0078] Prepare a 5% anhydrous sodium sulfate solution. First, take an equal volume of this solution (one times the weight of the minced meat) and mix it with the minced meat in a meat grinder, stirring for 50 minutes.

[0079] Next, add three times its weight in volume of the above-mentioned anhydrous sodium sulfate solution to the meat paste. Then, add a 1% sodium hydroxide solution to adjust the pH of the mixture to 10, and transfer it to a reaction vessel to mix thoroughly.

[0080] The temperature of the reaction system was controlled at 25°C using a temperature control system, and the alkalization reaction lasted for 40 hours.

[0081] Neutralization and Cleansing: After alkalization, drain an alkalization solution with a volume equivalent to twice the weight of the minced meat. Divide the remaining liquid into two parts (in a 6:4 volume ratio) and add sulfuric acid solution to each part to prepare a neutralization solution. The temperature of the neutralization solution should be controlled below 10°C.

[0082] Perform two neutralization processes: First, pour most of the neutralization solution (60%) into the reaction vessel and mix thoroughly with the meat paste; then add the remaining neutralization solution (40%) and mix. Finally, precisely adjust the pH of the system to 4.5 using a 5% sodium hydroxide solution and soak under this condition for 15 minutes. After neutralization, remove the meat paste.

[0083] The neutralized meat paste was washed with water for injection, using 18,000 ml of water for each wash (ratio 1:3), for a total of 6 washes. After the first five washes, the pH value of the aqueous phase was measured to be 4.0. After the sixth wash, the meat paste was removed and some water was squeezed out.

[0084] Freeze-drying and storage: The cleaned collagen meat paste was evenly spread in a freeze-drying tray and then subjected to vacuum freeze-drying. The freeze-drying parameters were the same as in Example 1.

[0085] After freeze-drying, the obtained collagen products are packaged in medical-grade all-plastic bags and thermosealed, and stored at -18°C or below.

[0086] Experimental Example The detection was performed based on the scheme in Example 1: Compared to methods of sterilizing raw materials using ethanol or n-hexane, this invention can effectively kill viruses that may be present in animal-derived raw materials to a great extent. These viruses are mostly DNA or RNA viruses, including pseudorabies virus (PRV, CVCC AV249), vesicular stomatitis virus (VSV, ATCC VR1238), reovirus (Reo3, ATCCVR824), and porcine parvovirus (PPV, CVCC AV31). In this experimental example, collagen before the alkalization process was infected with these viruses, and the results were simultaneously measured during the alkalization process. The results showed that the alkalization process achieved a reduction limit of 4 logs, as shown in Table 2 below. Table 2 Virus restriction test results

[0087] The virus decline curve during the "alkalization" process is as follows: Figures 2-5 As shown.

[0088] Collagen extraction rate test: Based on the scheme of Example 1, multiple batches of extraction and testing were conducted. The weight of the freeze-dried collagen was divided by the weight of the bovine Achilles tendon, and the amount of material fed in each batch was fixed at 6000g, as shown in Table 3 below: Table 3 Results of Collagen Extraction Rate Detection

[0089] Collagen content testing: The protein content was further determined using the Kjeldahl method, as shown in Table 4 below: Table 4. Results of Collagen Content Detection

[0090] Record the state of collagen before and after freezing, such as... Figures 6-7 As shown, the specific freeze-drying steps used in this invention can ensure that the water in collagen forms crystal particles near the eutectic point, gradually removing the water in collagen, while avoiding high temperature damage to the three-dimensional structure of collagen and ensuring a low water content.

[0091] Moisture content testing: The collagen was baked at 105℃ for 4 hours. After cooling in a desiccator, the water content of the collagen was measured by weighing, as shown in Table 5 below. Table 5. Moisture content test results

[0092] Based on the scheme of Example 1, the following comparative scheme was set up for verification: Control Experiment 1: Example 1 is a treatment group with alkalization under protection containing anhydrous sodium sulfate solution, and the control group is a treatment group with alkalization under the same pH without protection containing anhydrous sodium sulfate solution. Both are from the same batch of collagen production, and appropriate amounts of meat paste samples were taken from each group during the meat paste making process.

[0093] The results are shown in Table 6 below. Figures 8-9 As shown: Table 6 Results of Control Experiment 1

[0094] Figure 8 The beef tendon paste, after being protected with anhydrous sodium sulfate solution according to Example 1 and then alkalized with sodium hydroxide, appears as clear milky white granules. It is uniformly dispersed overall, has relatively low viscosity, and possesses a certain degree of elasticity and mechanical strength.

[0095] Figure 9 To avoid the need for protection with anhydrous sodium sulfate solution, sodium hydroxide solution of the same pH was directly added as an alkalizing solution. As can be clearly seen from the two pictures above, the color of the treated collagen turned yellowish-brown, and the volume increased significantly, indicating severe swelling. The transparency also increased, and the viscosity was visibly higher than that of collagen protected with anhydrous sodium sulfate solution.

[0096] Based on the structure and denaturation characteristics of collagen, strong alkaline solutions contain a large amount of OH-. - The strong alkali will disrupt the hydrogen and ionic bonds between or within collagen molecules, thereby destroying the triple helix structure. Furthermore, the added strong alkali will produce an exothermic reaction, which will also cause collagen molecules to hydrolyze into polypeptide fragments of varying molecular weights, ultimately leading to the loss of macroscopic fiber structure and physical strength. On the other hand, the high concentration of ions in the added excess anhydrous sodium sulfate solution will produce a salting-out effect, thereby protecting the collagen structure from damage caused by the strong alkali solution, shrinking the collagen, and the excess salt ions can be washed away by subsequent processes to release the required collagen.

[0097] Depend on Figure 8 and Figure 9 The comparison and the reaction principle of the reagents show that the addition of anhydrous sodium sulfate solution for protection before alkalization is irreplaceable and necessary.

[0098] Control Experiment 2: Example 1 involved adding potassium dihydrogen phosphate to the enzymatic hydrolysis treatment, while the control group did not add potassium dihydrogen phosphate during enzymatic hydrolysis. All other steps and parameters remained the same.

[0099] The results are shown in Table 7 below: Table 7 Results of Control Experiment 2

[0100] As shown in Table 7, the extraction rate of the group without potassium dihydrogen phosphate was only 7.5%, the purity was only 1.9%, and the product was not of type I collagen structure. This indicates that the addition of potassium dihydrogen phosphate is crucial for ensuring enzymatic hydrolysis efficiency and product purity.

[0101] Control Experiment 3: The scheme of Example 1 was a step-by-step sublimation drying program (sublimation at -11℃, -4℃, and 0℃ in stages), and the control scheme was a conventional one-step sublimation program (sublimation directly from -30℃ to 25℃ after pre-freezing). All other steps and parameters were kept the same.

[0102] The results are shown in Table 8 below: Table 8 Results of Control Experiment 3

[0103] As shown in Table 8, the conventional single-sublimation group exhibited melting or vitrification during sublimation, resulting in a dense product; the step-sublimation group did not show melting, and the product had a loose texture. This indicates that the step-sublimation process plays a crucial role in preventing collagen from melting during freeze-drying and maintaining its porous structure.

[0104] As demonstrated by the above embodiments, this invention provides a stable and efficient extraction method for preparing high-purity type I collagen. This method, through rigorous screening and precise extraction of raw materials, combined with optimized enzymatic hydrolysis, salt-protected alkali inactivation, fine neutralization, and stepwise freeze-drying processes, successfully achieves efficient extraction and purification of type I collagen. The prepared collagen product exhibits extremely high purity, intact structure, significantly lower water content than conventional levels, and consistent quality across batches, fully meeting the stringent requirements for raw materials in high-end biomedical materials and possessing excellent potential for industrial application.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting high-purity type I collagen, characterized in that, Includes the following steps: Animal tissue materials rich in type I collagen were subjected to enzymatic hydrolysis. The enzymatically hydrolyzed material is minced to make meat paste; Anhydrous sodium sulfate solution and sodium hydroxide solution were added to the meat paste for alkalization treatment to obtain an alkalized product; An acid solution is added to the alkalization product for neutralization treatment to obtain the neutralized material. The neutralized material is cleaned and then freeze-dried to obtain type I collagen product.

2. The extraction method according to claim 1, characterized in that, The animal tissue material rich in type I collagen is animal tendon tissue.

3. The extraction method according to claim 2, characterized in that, The animal tendon tissue is the bovine Achilles tendon, which is taken from a Simmental bull less than 3 years old. The animal tissue material rich in type I collagen is obtained by a preparation method including the following steps: The tendon is cut 2-4 cm above the bifurcation and 6-10 cm below the bifurcation to obtain raw tendon blocks. The raw tendon blocks are then cleaned, frozen, and cut into thin slices less than 2 mm thick to obtain the animal tissue material rich in type I collagen.

4. The extraction method according to claim 1, characterized in that, The enzymatic hydrolysis process specifically involves: After washing the animal tissue material rich in type I collagen, it was placed in purified water, and after adding potassium dihydrogen phosphate to regulate the system, fig protease was added to carry out enzymatic hydrolysis. After the enzymatic hydrolysis reaction is completed, the sample is washed and then soaked in sodium chloride solution. The amount of purified water used is 6-7 times the volume of the animal tissue material rich in type I collagen; The amount of potassium dihydrogen phosphate added is 2‰-4‰ of the mass of purified water; The amount of fig protease added is 0.7‰-1.4‰ of the mass of purified water; The reaction temperature for the enzymatic hydrolysis treatment is 37.0 ± 1.0 °C; The enzymatic hydrolysis is carried out under stirring at a speed of 1-2 revolutions per minute. The sodium chloride solution has a mass fraction of 0.5%-2%, the soaking temperature is 29-31℃, and the soaking time is 1-1.5 hours.

5. The extraction method according to claim 1, characterized in that, In the alkalization treatment, the mass fraction of the anhydrous sodium sulfate solution is 5%-15%; The amount of the anhydrous sodium sulfate solution is 3-5 times the volume of the meat paste. In the alkalization treatment, the sodium hydroxide solution has a mass fraction of 1%-3% and is used to adjust the pH value of the mixture to 10-13. The alkalization treatment is carried out at a reaction temperature of 20-26°C for 38-45 hours.

6. The extraction method according to claim 1, characterized in that, The acid solution used in the neutralization process is a sulfuric acid solution, the pH value of the material after neutralization is 4.5-7.0, and the neutralization time is 10-30 minutes.

7. The extraction method according to claim 1, characterized in that, The cleaning step involves at least six cleaning cycles using injectable water, with each cycle using an amount of injectable water equal to 3-5 times the weight of the animal tissue material rich in type I collagen.

8. The extraction method according to claim 1, characterized in that, The freeze-drying process includes the following steps: The material is pre-frozen at -30℃ for 1-2 hours; then, under a vacuum of 30-40 Pa, it is subjected to segmented sublimation drying at -11℃, -4℃ and 0℃ in sequence; and then subjected to desorption drying at 25℃ and a vacuum of 25 Pa.

9. A high-purity type I collagen, characterized in that, The type I collagen is obtained by the extraction method according to any one of claims 1 to 8.

10. The use of the high-purity type I collagen according to claim 9 in the preparation of implantable medical materials.

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