A method for preparing non-denatured type ii collagen for increasing bone density and improving bone elasticity

Abstract

The application provides a preparation method of non-denatured type II collagen for increasing bone density and improving bone elasticity, and belongs to the technical field of biotechnology. The application adopts EDTA-citric acid buffer containing beta-mercaptoethanol to decalcify cartilage, adopts supercritical CO2 extraction technology to defat, adopts alkaline protease and collagenase II to perform matrix directional enzymolysis, and performs gradient digestion; then three-phase centrifugal separation is adopted to separate non-denatured type II collagen, and biomimetic H column chromatography purification is adopted, so that non-denatured type II collagen with high extraction rate, high purity and high structural integrity can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity. Background Technology

[0002] In today's society, bone health issues are increasingly becoming a focus of public attention. As people age, especially as they enter middle and old age, calcium loss from bones intensifies, bone density decreases, and bone elasticity weakens, leading to a series of conditions such as osteoporosis and osteoarthritis, which seriously affect their quality of life.

[0003] For a long time, traditional methods of maintaining bone health have mainly focused on calcium and vitamin D supplementation. Calcium, as an important inorganic component of bones, provides basic structural support and rigidity; vitamin D helps the intestines absorb calcium and promotes its deposition in bones. However, relying solely on these two elements has gradually revealed its limitations when facing complex bone health issues. Bones are not merely simple accumulations of calcium salts, but a complex structure in which organic and inorganic components are tightly bound together. Among the organic components of bones, collagen plays a crucial role, accounting for over 90% of bone organic matter, and its importance cannot be ignored.

[0004] The collagen family comprises many types, among which type II collagen plays a unique role in bone health. It is primarily found in articular cartilage and some specialized connective tissues, and is the main protein that makes up articular cartilage. With its unique triple helix structure, type II collagen weaves a dense network, providing articular cartilage with strong elasticity and resilience. Like the steel frame in a building, it supports the overall structure of the cartilage, ensuring that joints can move flexibly and freely during daily activities, while effectively cushioning external impacts and protecting joints from damage.

[0005] Undenatured type II collagen is extracted while maintaining the integrity of its natural triple helix structure. Compared to denatured collagen, undenatured type II collagen better preserves its biological activity and functional properties. Intake of undenatured type II collagen can promote chondrocyte metabolism, enhance the ability of chondrocytes to synthesize cartilage matrix, making cartilage more resilient and elastic. It can also regulate the dynamic balance between osteoclasts and osteoblasts, promote bone formation, inhibit bone resorption, comprehensively improve bone density, and strengthen the overall strength of bones.

[0006] However, the extraction of non-denatured type II collagen still faces certain challenges, mainly due to the following reasons:

[0007] (1) The structure is easily damaged: Non-denatured type II collagen needs to maintain the complete triple helix structure. The extraction process requires extremely strict conditions such as temperature, pH value, and enzyme use. If not careful, its structure will be damaged and it will lose its biological activity.

[0008] (2) Low extraction rate: Traditional extraction methods such as salting out and washing have the problem of low protein extraction rate; while some new methods such as ultrasound-assisted extraction and enzymatic extraction have certain advantages, they also face problems such as unstable extraction rate in practical applications.

[0009] (3) Low purity: In addition to non-denatured type II collagen, animal cartilage also contains other proteins, fats and other impurities. During the extraction process, these impurities are difficult to completely remove, which affects the purity of the product. Summary of the Invention

[0010] To address the problems of existing extraction methods for non-denatured type II collagen, such as easy structural damage, low extraction rate, and low purity, this invention provides a method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity. The method involves decalcifying cartilage using an EDTA-citric acid buffer containing β-mercaptoethanol, defatting using supercritical CO2 extraction, matrix-directed enzymatic hydrolysis using alkaline protease and collagenase II, gradient digestion, separation of non-denatured type II collagen using three-phase centrifugation, and purification using biomimetic H-column chromatography. This method yields non-denatured type II collagen with high extraction rate, high purity, and high structural integrity. The specific technical solution is as follows:

[0011] A method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity includes the following steps:

[0012] S1, Decalcification: Prepare cartilage, soak it in EDTA-citric acid buffer, remove it, rinse it, and obtain decalcified cartilage;

[0013] S2, Degreasing: Decalcified cartilage is degreased by supercritical CO2 extraction and removed to obtain degreased cartilage;

[0014] S3, Matrix-directed enzymatic hydrolysis: The defatted cartilage was added to Tris-HCl buffer containing alkaline protease and collagenase II and digested in a gradient manner. Specifically, the cartilage was first shaken, then allowed to stand, and then proteinase K inhibitor was added and shaken again to obtain an enzymatic hydrolysate containing undenatured type II collagen.

[0015] S4, Three-phase centrifugal separation:

[0016] S4.1 Initial centrifugation to remove residue: Centrifuge to separate the residue and obtain supernatant A;

[0017] S4.2 Secondary centrifugation to precipitate impurity proteins: Add PEG to the supernatant A, centrifuge to separate the precipitate, and obtain supernatant B;

[0018] S4.3 Filtration: Filter the supernatant B and take the filtrate to obtain a collagen solution;

[0019] S5, Biomimetic fiber column chromatography purification: The collagen solution is loaded into the HA column and eluted with NaCl aqueous solution using a linear gradient elution method. The eluents of different concentrations of NaCl aqueous solution are collected and combined to obtain the purified solution.

[0020] S6, Dynamic pH Stabilization Treatment: Adjust the pH of the purification solution; add glycerol; mix in laminar flow mode using a microfluidic device to obtain the target protein solution;

[0021] S7, dried, yields undenatured type II collagen.

[0022] Furthermore, the above preparation method includes:

[0023] S1, Decalcification: Prepare cartilage by immersing it in pre-cooled EDTA-citric acid buffer solution, removing the cartilage, rinsing it, and obtaining decalcified cartilage;

[0024] S2, Degreasing: The decalcified cartilage is degreased using supercritical CO2 extraction; after degreasing, the cartilage is removed to obtain degreased cartilage;

[0025] S3, Matrix-directed enzymatic hydrolysis: The defatted cartilage was added to Tris-HCl buffer containing alkaline protease and collagenase II for gradient digestion. Specifically, the cartilage was first shaken, then allowed to stand, and then proteinase K inhibitor was added and shaken again to obtain an enzymatic hydrolysate containing non-denatured type II collagen.

[0026] S4, Three-phase centrifugal separation:

[0027] S4.1 Initial centrifugation to remove residue: Centrifuge at 3000g~3500g to separate the residue and obtain supernatant A;

[0028] S4.2 Secondary centrifugation to precipitate impurity proteins: Add PEG8000 to the supernatant A, centrifuge at 8000g~10000g to separate the precipitate and obtain supernatant B;

[0029] S4.3 Filtration: Filter the supernatant B, take the filtrate, and obtain a collagen solution;

[0030] S5, Biomimetic Fiber Column Chromatography Purification: The collagen solution was loaded into an HA column, and a linear gradient elution was performed sequentially with 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, and 0.35 mol / L NaCl aqueous solutions. The eluents of 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L NaCl aqueous solutions were collected and combined to obtain the purified solution.

[0031] S6, Dynamic pH Stabilization Treatment: Adjust the pH of the purification solution; add glycerol; mix in laminar flow mode using a microfluidic device to obtain the target protein solution;

[0032] S7, dried, yields undenatured type II collagen.

[0033] The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity includes the following steps:

[0034] S1, Decalcification: Prepare clean cartilage. The cartilage is soaked in EDTA-citric acid buffer solution pre-cooled to 3℃-4℃. The EDTA-citric acid buffer solution is changed every 6h-8h. The soaking time is 48h-56h. The cartilage is then removed and rinsed with purified water to obtain decalcified cartilage.

[0035] S2, Degreasing: The decalcified cartilage is degreased using supercritical CO2 extraction technology; after degreasing, the cartilage is removed to obtain degreased cartilage;

[0036] S3, Matrix-directed enzymatic hydrolysis: Prepare a Tris-HCl buffer containing alkaline protease and collagenase II; the defatted cartilage : Tris-HCl buffer = 1 : (10-15) by mass ratio. Add the defatted cartilage to the Tris-HCl buffer and perform gradient digestion at 37℃-40℃: first, shake at 50 rpm-60 rpm for 2-2.5 h, then let stand for 12-14 h, then add 0.0004 g / mL-0.0006 g / mL proteinase K inhibitor and continue shaking at 50 rpm-60 rpm for 2-4 h to obtain an enzymatic hydrolysate containing non-denatured type II collagen.

[0037] S4, Three-phase centrifugal separation:

[0038] S4.1 Initial centrifugation to remove residue: Centrifuge at 3000g-3500g for 20-25 minutes to separate the residue and obtain supernatant A;

[0039] S4.2 Secondary centrifugation to precipitate impurity proteins: Add PEG8000 to the supernatant and centrifuge at 8000g~10000g for 30min~40min to separate the precipitate and obtain supernatant B;

[0040] S4.3 Filtration: Filter the supernatant B through a 0.22μm ceramic membrane, collect the filtrate, and obtain a collagen solution;

[0041] S5, Biomimetic Fiber Column Chromatography Purification: The filtered collagen solution was loaded into an HA column, and then linear gradient elution was performed sequentially with NaCl aqueous solutions of concentrations of 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, and 0.35 mol / L. Each gradient elution consisted of 1 to 1.5 HA column volumes. The eluents of 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L NaCl aqueous solutions were collected and combined to obtain the purified solution.

[0042] S6, Dynamic pH Stabilization Treatment: The pH of the purified solution was immediately adjusted to 5.5-6.0 with 0.1mol / L-0.12mol / L acetate buffer; glycerol was added as a cryoprotectant; then the solution was slowly mixed in laminar flow mode using a microfluidic device to avoid local pH abrupt changes, and the target protein solution was obtained.

[0043] S7, vacuum freeze-drying, to obtain a dried, non-denatured type II collagen product.

[0044] In step S1 of the above preparation method, the cartilage is chicken breast cartilage or shark fin cartilage. The cartilage is cut into fragments ranging from 1 mm to 5 mm in size. The concentration of the EDTA-citric acid buffer is 0.5 mol / L to 0.6 mol / L, and it contains 1 mmol / L to 1.5 mmol / L β-mercaptoethanol, with a pH of 6.7 to 6.9. The soaking process is performed with stirring at a speed of 100 rpm to 150 rpm.

[0045] In step S2 of the above preparation method, the degreasing method includes: placing the decalcified cartilage into the extraction vessel of a supercritical CO2 extraction device, setting the extraction temperature to 34℃~36℃, the pressure to 10MPa~12MPa, and the extraction time to 30min~40min.

[0046] In step S3 of the above preparation method, the Tris-HCl buffer contains 5 mmol / L–6 mmol / L CaCl2, 2 mmol / L–3 mmol / L ZnCl2, 0.001 g / mL–0.003 g / mL trehalose, 0.001 g / mL–0.002 g / mL alkaline protease, and 0.0005 g / mL–0.0008 g / mL collagenase II. The pH of the Tris-HCl buffer is 7.3–7.5.

[0047] In the above preparation method, S4, the three-phase centrifugal separation includes:

[0048] S4.1 Initial centrifugation to remove residue: Centrifuge the enzymatic hydrolysate at 3000g to 3500g for 20 to 25 minutes at 4℃ to 6℃ to separate and obtain supernatant A containing collagen and impurity proteins.

[0049] S4.2 Secondary centrifugation to precipitate impurity proteins: Add 0.1 g / mL to 0.15 g / mL PEG8000 to the supernatant A, and then centrifuge at 8000 g to 10000 g for 30 min to 40 min to selectively precipitate impurity proteins. PEG8000 can change the physical properties of the solution, causing impurity proteins to precipitate, while collagen remains in the supernatant, resulting in supernatant B.

[0050] S4.3 Filtration: The supernatant B is filtered through a 0.22μm ceramic membrane to remove residual microparticle impurities, resulting in a relatively pure collagen solution.

[0051] In step S4.3 of the above preparation method, the ceramic membrane is pre-cooled to 2°C to 4°C.

[0052] In step S5 of the above preparation method, the HA column is a hydroxyapatite column containing 2wt%–4wt% hyaluronic acid to simulate the extracellular matrix (ECM) environment of chondrocytes. The HA column is equilibrated with a equilibration buffer before use. The equilibration buffer is a 0.05 mol / L–0.06 mol / L Tris-HCl buffer containing 0.12 mol / L–0.15 mol / L NaCl and 1.5 mmol / L–2 mmol / L CaCl2, with a pH of 7.0–7.2. The elution flow rate is controlled at 1 mL / min–1.5 mL / min. The temperature is maintained at 4℃–5℃ throughout the entire biomimetic fiber column chromatography purification process.

[0053] In step S6 of the above preparation method, the pH value of the 0.1 mol / L to 0.12 mol / L acetate buffer is 4.8 to 4.9. The 0.1 mol / L to 0.12 mol / L acetate buffer is pre-cooled to 4°C to 6°C. The amount of glycerol added is 3% to 3.5% of the purification liquid volume. The channel diameter of the microfluidic device is 200 μm.

[0054] In step S7 of the above preparation method, the vacuum freeze-drying method includes: pre-freezing the target protein solution to -80°C, and after complete freezing, sublimating it at a vacuum of 0.01 mBar and a temperature of -45°C for 48 h to 50 h to remove the water and obtain a dried, non-denatured type II collagen product.

[0055] When using the dried, non-denatured type II collagen product of the present invention, it is rehydrated and reconstituted with PBS containing 5 mmol / L CaCl2 at pH 7.2 to restore it to its usable state.

[0056] The present invention provides a method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity, with the following beneficial effects:

[0057] The EDTA-citric acid complex system is gentler in decalcification and can effectively reduce damage to the structure of non-denatured type II collagen, while β-mercaptoethanol can inhibit the breaking of disulfide bonds and maintain the original structure of collagen.

[0058] Second, CO2 in its supercritical state possesses characteristics of both gas and liquid, exhibiting excellent solubility and diffusivity. It can effectively dissolve the fatty components in cartilage, achieving degreasing. The degreasing rate is high, and no residue remains, ensuring the purity of the subsequently extracted collagen.

[0059] Third, CaCl2 and ZnCl2 can synergistically stabilize the conformation of collagen, while trehalose protects hydrogen bonds and maintains the stability of collagen through water molecule replacement. Low-temperature enzymatic hydrolysis reduces the risk of thermal denaturation.

[0060] Fourth, low-temperature enzymatic hydrolysis can effectively reduce the risk of thermal denaturation and ensure the structural integrity of non-denatured type II collagen.

[0061] 5. Add proteinase K inhibitor to control the enzymatic reaction process and avoid excessive enzymatic degradation that could damage the collagen structure.

[0062] VI. Alkaline protease can assist collagenase II in more comprehensive decomposition of cartilage matrix.

[0063] 7. Trehalose and glycerol provide dual low-temperature protection to reduce ice crystal damage.

[0064] In summary, this invention uses EDTA-citric acid buffer containing β-mercaptoethanol to decalcify cartilage, employs supercritical CO2 extraction for defatting, and uses alkaline protease and collagenase II for matrix-directed enzymatic digestion and gradient digestion. Following this, non-denatured type II collagen is separated by three-phase centrifugation and purified using biomimetic H-column chromatography. This method yields non-denatured type II collagen with high extraction rate, high purity, and high structural integrity, and also contains a certain amount of mucopolysaccharides. Mucopolysaccharides are an important component of the bone matrix, promoting increased bone density and possessing significant practical value. Detailed Implementation

[0065] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0066] Example 1

[0067] A method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity includes the following steps:

[0068] S1, Decalcification: Prepare clean chicken breast cartilage and cut it into fragments ranging from 1mm to 5mm in size. The cartilage is soaked in EDTA-citric acid buffer solution pre-cooled to 4℃ at a mass ratio of 1.5:18. The EDTA-citric acid buffer solution is changed every 8 hours. The stirring speed is 120 rpm and the soaking time is 48 hours. The cartilage is then removed and rinsed with purified water to obtain decalcified cartilage.

[0069] The EDTA-citric acid buffer solution has a concentration of 0.5 mol / L and contains 1 mmol / L β-mercaptoethanol, with a pH of 6.8.

[0070] S2, Degreasing: The decalcified cartilage was degreased using supercritical CO2 extraction. The decalcified cartilage was placed in the extraction vessel of the supercritical CO2 extraction equipment, and the extraction temperature was set to 35℃, the pressure to 11MPa, and the extraction time to 35min. After degreasing was completed, the cartilage was removed to obtain the degreased cartilage.

[0071] S3, Matrix-directed enzymatic hydrolysis: Prepare a Tris-HCl buffer containing alkaline protease and collagenase II; add defatted cartilage to Tris-HCl buffer at a mass ratio of 1:12 and perform gradient digestion at 37°C: first shake at 55 rpm for 2 h, then let stand for 13 h, then add 0.0005 g / mL proteinase K inhibitor and continue shaking at 55 rpm for 3 h to obtain an enzymatic hydrolysate containing non-denatured type II collagen;

[0072] The Tris-HCl buffer contains 5 mmol / L CaCl2, 2 mmol / L ZnCl2, 0.002 g / mL trehalose, 0.001 g / mL alkaline protease and 0.0006 g / mL collagenase II, and the pH of the Tris-HCl buffer is 7.4.

[0073] S4, Three-phase centrifugal separation:

[0074] S4.1 Initial centrifugation to remove residue: The enzymatic hydrolysate was centrifuged at 3200g for 22 minutes at 5°C to separate the contents and obtain supernatant A containing collagen and impurity proteins.

[0075] S4.2 Secondary centrifugation to precipitate impurity proteins: Add 0.12 g / mL PEG8000 to the supernatant A, and then centrifuge at 9000 g for 35 min to selectively precipitate impurity proteins. PEG8000 can change the physical properties of the solution, causing impurity proteins to precipitate, while collagen remains in the supernatant, resulting in supernatant B.

[0076] S4.3 Filtration: The supernatant B is filtered through a 0.22μm ceramic membrane pre-cooled to 3℃ to remove residual microparticle impurities. The filtrate is collected to obtain a relatively pure collagen solution.

[0077] S5, Biomimetic Fiber Column Chromatography Purification: The filtered collagen solution was loaded into an HA column, and then linear gradient elution was performed sequentially using NaCl aqueous solutions with concentrations of 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, and 0.35 mol / L. Each gradient eluted one HA column volume, and the elution flow rate was controlled at 1.2 mL / min. The temperature was maintained at 4℃ throughout the entire biomimetic fiber column chromatography purification process. The eluents of the 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L NaCl aqueous solutions were collected and combined to obtain the purified solution.

[0078] The HA column is a hydroxyapatite column containing 3 wt% hyaluronic acid to simulate the extracellular matrix (ECM) environment of chondrocytes. The hyaluronic acid is added as follows: hydroxyapatite is added to 7 times its weight of deionized water, which contains 3% hyaluronic acid (based on the weight of the hydroxyapatite). After thorough mixing, the deionized water is evaporated to remove the excess water, resulting in hydroxyapatite containing 3 wt% hyaluronic acid, which is then placed into the HA column. The HA column is equilibrated with a 0.05 mol / L Tris-HCl buffer containing 0.13 mol / L NaCl and 1.8 mmol / L CaCl2, with a pH of 7.1, before use.

[0079] S6, Dynamic pH Stabilization Treatment: The pH of the purification solution was immediately adjusted to 5.8 with 0.1 mol / L acetate buffer; and 3% of the volume of glycerol was added as a cryoprotectant; then it was slowly mixed in laminar flow mode using a microfluidic device with a channel diameter of 200 μm to avoid local pH abrupt changes, and the target protein solution was obtained.

[0080] The pH value of the 0.1 mol / L acetate buffer is 4.8, and the 0.1 mol / L acetate buffer is pre-cooled to 5°C;

[0081] S7, Vacuum freeze drying: The target protein solution is pre-frozen to -80℃. After complete freezing, it is sublimated at a vacuum of 0.01mBar and a temperature of -45℃ for 48 hours to remove the water and obtain a dried, non-denatured type II collagen product.

[0082] In this embodiment, the dried, non-denatured type II collagen product is rehydrated and reconstituted using PBS containing 5 mmol / L CaCl2 at pH 7.2 to restore it to its usable state.

[0083] Example 2

[0084] A method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity includes the following steps:

[0085] S1, Decalcification: Prepare clean shark fin cartilage and cut it into fragments ranging from 1mm to 5mm in size. The cartilage is soaked in EDTA-citric acid buffer solution pre-cooled to 3°C at a mass ratio of 1:15. The EDTA-citric acid buffer solution is changed every 6 hours. The stirring speed is 100 rpm and the soaking time is 48 hours. The cartilage is then removed and rinsed with purified water to obtain decalcified cartilage.

[0086] The EDTA-citric acid buffer solution has a concentration of 0.5 mol / L and contains 1 mmol / L β-mercaptoethanol, with a pH of 6.7.

[0087] S2, Degreasing: The decalcified cartilage is degreased using supercritical CO2 extraction technology; the decalcified cartilage is placed in the extraction vessel of the supercritical CO2 extraction equipment, the extraction temperature is set to 34℃, the pressure is 10MPa, and the extraction time is 30min. After degreasing is completed, the cartilage is removed to obtain degreased cartilage.

[0088] S3, Matrix-directed enzymatic hydrolysis: Prepare a Tris-HCl buffer containing alkaline protease and collagenase II; add defatted cartilage to Tris-HCl buffer at a mass ratio of 1:10 and perform gradient digestion at 38°C: first shake at 50 rpm for 2 h, then let stand for 12 h, then add 0.0004 g / mL proteinase K inhibitor and continue shaking at 50 rpm for 2 h to obtain an enzymatic hydrolysate containing non-denatured type II collagen;

[0089] The Tris-HCl buffer contains 5 mmol / L CaCl2, 2 mmol / L ZnCl2, 0.001 g / mL trehalose, 0.001 g / mL alkaline protease and 0.0005 g / mL collagenase II, and the pH of the Tris-HCl buffer is 7.3.

[0090] S4, Three-phase centrifugal separation:

[0091] S4.1 Initial centrifugation to remove residue: Centrifuge the enzymatic hydrolysate at 3000g for 20 minutes at 4℃ to separate it and obtain supernatant A containing collagen and impurity proteins.

[0092] S4.2 Secondary centrifugation to precipitate impurity proteins: Add 0.1 g / mL PEG8000 to the supernatant A, and then centrifuge at 8000 g for 30 min to selectively precipitate impurity proteins. PEG8000 can change the physical properties of the solution, causing impurity proteins to precipitate, while collagen remains in the supernatant, resulting in supernatant B.

[0093] S4.3 Filtration: The supernatant B is filtered through a 0.22μm ceramic membrane pre-cooled to 2℃ to remove residual microparticle impurities. The filtrate is collected to obtain a relatively pure collagen solution.

[0094] S5, Biomimetic Fiber Column Chromatography Purification: The filtered collagen solution was loaded into an HA column, and then linear gradient elution was performed sequentially using NaCl aqueous solutions with concentrations of 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, and 0.35 mol / L. Each gradient eluted one HA column volume, and the elution flow rate was controlled at 1 mL / min. The temperature was maintained at 4℃ throughout the entire biomimetic fiber column chromatography purification process. The eluents of the 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L NaCl aqueous solutions were collected and combined to obtain the purified solution.

[0095] The HA column is a hydroxyapatite column containing 2 wt% hyaluronic acid to simulate the extracellular matrix (ECM) environment of chondrocytes. The hyaluronic acid is added as follows: hydroxyapatite is added to 6 times its mass of deionized water, which contains 2% hyaluronic acid (based on the mass of the hydroxyapatite). After thorough mixing, the deionized water is evaporated to remove the excess water, resulting in hydroxyapatite containing 2 wt% hyaluronic acid, which is then placed into the HA column. The HA column is equilibrated with a 0.05 mol / L Tris-HCl buffer containing 0.12 mol / L NaCl and 1.5 mmol / L CaCl2, with a pH of 7.0, before use.

[0096] S6, Dynamic pH Stabilization Treatment: The pH of the purification solution was immediately adjusted to 5.5 with 0.1 mol / L acetate buffer; and 3% of the volume of glycerol was added as a cryoprotectant; then it was slowly mixed in laminar flow mode using a microfluidic device with a channel diameter of 200 μm to avoid local pH abrupt changes, and the target protein solution was obtained.

[0097] The pH value of the 0.1 mol / L acetate buffer is 4.8, and the 0.1 mol / L acetate buffer is pre-cooled to 4°C;

[0098] S7, Vacuum freeze drying: The target protein solution is pre-frozen to -80℃. After complete freezing, it is sublimated at a vacuum of 0.01mBar and a temperature of -45℃ for 48 hours to remove the water and obtain a dried, non-denatured type II collagen product.

[0099] In this embodiment, the dried, non-denatured type II collagen product is rehydrated and reconstituted using PBS containing 5 mmol / L CaCl2 at pH 7.2 to restore it to its usable state.

[0100] Example 3

[0101] A method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity includes the following steps:

[0102] S1, Decalcification: Prepare clean chicken breast cartilage and cut it into fragments ranging from 1mm to 5mm in size. The cartilage is soaked in EDTA-citric acid buffer solution pre-cooled to 4℃ at a mass ratio of 2:20. The EDTA-citric acid buffer solution is changed every 8 hours. The stirring speed is 150 rpm and the soaking time is 56 hours. The cartilage is then removed and rinsed with purified water to obtain decalcified cartilage.

[0103] The EDTA-citric acid buffer solution has a concentration of 0.6 mol / L and contains 1.5 mmol / L β-mercaptoethanol, with a pH of 6.9.

[0104] S2, Degreasing: The decalcified cartilage is degreased using supercritical CO2 extraction technology; the decalcified cartilage is placed in the extraction vessel of the supercritical CO2 extraction equipment, the extraction temperature is set to 36℃, the pressure is 12MPa, and the extraction time is 40min. After degreasing is completed, the cartilage is removed to obtain degreased cartilage.

[0105] S3, Matrix-directed enzymatic hydrolysis: Prepare a Tris-HCl buffer containing alkaline protease and collagenase II; add defatted cartilage to Tris-HCl buffer at a mass ratio of 1:15 and perform gradient digestion at 40°C: first shake at 60 rpm for 2.5 h, then let stand for 14 h, then add 0.0006 g / mL proteinase K inhibitor and continue shaking at 60 rpm for 4 h to obtain an enzymatic hydrolysate containing undenatured type II collagen;

[0106] The Tris-HCl buffer contains 6 mmol / L CaCl2, 3 mmol / L ZnCl2, 0.003 g / mL trehalose, 0.002 g / mL alkaline protease and 0.0008 g / mL collagenase II, and the pH of the Tris-HCl buffer is 7.5.

[0107] S4, Three-phase centrifugal separation:

[0108] S4.1 Initial centrifugation to remove residue: Centrifuge the enzymatic hydrolysate at 3500g for 25 minutes at 6℃ to separate it and obtain supernatant A containing collagen and impurity proteins.

[0109] S4.2 Secondary centrifugation to precipitate impurity proteins: Add 0.15 g / mL PEG8000 to the supernatant A, and then centrifuge at 10000 g for 40 min to selectively precipitate impurity proteins. PEG8000 can change the physical properties of the solution, causing impurity proteins to precipitate, while collagen remains in the supernatant, resulting in supernatant B.

[0110] S4.3 Filtration: The supernatant B is filtered through a 0.22μm ceramic membrane pre-cooled to 4℃ to remove residual microparticle impurities. The filtrate is collected to obtain a relatively pure collagen solution.

[0111] S5, Biomimetic Fiber Column Chromatography Purification: The filtered collagen solution was loaded into an HA column, and then linear gradient elution was performed sequentially using NaCl aqueous solutions with concentrations of 0.15 mol / L, 0.20 mol / L, 0.25 mol / L, 0.30 mol / L, and 0.35 mol / L. Each gradient elution consisted of 1.5 HA column volumes, and the elution flow rate was controlled at 1.5 mL / min. The temperature was maintained at 5℃ throughout the entire biomimetic fiber column chromatography purification process. The eluents of the 0.20 mol / L, 0.25 mol / L, and 0.30 mol / L NaCl aqueous solutions were collected and combined to obtain the purified solution.

[0112] The HA column is a hydroxyapatite column containing 4 wt% hyaluronic acid to simulate the extracellular matrix (ECM) environment of chondrocytes. The hyaluronic acid is added as follows: hydroxyapatite is added to 8 times its weight of deionized water, which contains 4% hyaluronic acid (based on the weight of the hydroxyapatite). After thorough mixing, the deionized water is evaporated to remove the excess water, resulting in hydroxyapatite containing 4 wt% hyaluronic acid, which is then placed into the HA column. The HA column is equilibrated with a 0.06 mol / L Tris-HCl buffer containing 0.15 mol / L NaCl and 2 mmol / L CaCl2, with a pH of 7.2, before use.

[0113] S6, Dynamic pH Stabilization Treatment: The pH of the purification solution was immediately adjusted to 6.0 with 0.12 mol / L acetate buffer; and 3.5% of the volume of glycerol was added as a cryoprotectant; then it was slowly mixed in laminar flow mode using a microfluidic device with a channel diameter of 200 μm to avoid local pH abrupt changes, and the target protein solution was obtained.

[0114] The pH value of the 0.12 mol / L acetate buffer is 4.9, and the 0.12 mol / L acetate buffer is pre-cooled to 6°C;

[0115] S7, Vacuum freeze drying: The target protein solution is pre-frozen to -80℃. After complete freezing, it is sublimated for 50 hours under a vacuum of 0.01 mBar and a temperature of -45℃ to remove the water and obtain a dried, non-denatured type II collagen product.

[0116] In this embodiment, the dried, non-denatured type II collagen product is rehydrated and reconstituted using PBS containing 5 mmol / L CaCl2 at pH 7.2 to restore it to its usable state.

[0117] In the above embodiments, the alkaline protease was obtained from Wuxi Laisheng Technology Co., Ltd., model 37071; collagenase II was obtained from Clostridium histolyticum, from Aibisin (Shanghai) Biotechnology Co., Ltd.; and the proteinase K inhibitor was obtained from Beijing Biolab Technology Co., Ltd.

[0118] Comparative Example 1

[0119] In S1, β-mercaptoethanol is not added to the EDTA-citric acid buffer; other parameters and methods are the same as in Example 1.

[0120] Comparative Example 2

[0121] In S3, no alkaline protease was added to the Tris-HCl buffer; other parameters and methods were the same as in Example 1.

[0122] Comparative Example 3

[0123] In S3, collagenase II was not added to the Tris-HCl buffer; other parameters and methods were the same as in Example 1.

[0124] Comparative Example 4

[0125] In S3, no proteinase K inhibitor was added; other parameters and methods were the same as in Example 1.

[0126] Comparative Example 5

[0127] In S3, no CaCl2, ZnCl2, or trehalose were added to the Tris-HCl buffer; other parameters and methods were the same as in Example 1.

[0128] Comparative Example 6

[0129] In S3, no hyaluronic acid is added to the HA column; other parameters and methods are the same as in Example 1.

[0130] The extraction yield, purity, triple helix structure retention integrity, and bone density increase effect of the non-denatured type II collagen in the above embodiments and comparative examples were tested.

[0131] 1. Extraction yield detection:

[0132] Using an electronic balance with an accuracy of 0.0001g, the mass of the prepared dried, non-denatured type II collagen product was accurately weighed and recorded as M1. Based on the experimental records, the initial mass of cartilage raw material, M0, was obtained. The extraction rate % was calculated using the formula: Extraction Rate % = (M1 / M0) × 100%. The results are shown in Table 1 below.

[0133] 2. Purity detection (High Performance Liquid Chromatography, HPLC):

[0134] The dried, non-denatured type II collagen product was reconstituted with PBS containing 5 mmol / L CaCl2 at pH 7.2 to prepare a solution with a concentration of 1 mg / mL. The solution was filtered through a 0.45 μm microporous membrane, and 10 μL was injected into the HPLC system. A C18 column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm was used. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The flow rate was set at 1.0 mL / min, the column temperature was maintained at 30 °C, and gradient elution was performed. The absorbance was measured at 214 nm. The sample purity was calculated based on the standard curve by comparing the chromatographic peaks with those of the standard. The results are shown in Table 1 below.

[0135] High-performance liquid chromatography (HPLC) for the determination of mucopolysaccharide content: An amino-bonded phase column, Waters XBridgeAmide column (4.6 × 250 mm, 5 μm), was selected; an acetonitrile-water system was used with an initial ratio of acetonitrile:water = 70:30 (v / v), isocratic elution for 30 min, followed by linear elution to acetonitrile:water = 50:50 (v / v) within 10 min, and then held for 5 min; the flow rate was set to 1.0 mL / min; the temperature was maintained at 35 °C; an evaporative light scattering detector (ELSD) was used, the drift tube temperature was set to 70 °C, and the gas (nitrogen) flow rate was 2.5 L / min. The injection volume was 20 μL. The dried, non-denatured type II collagen products obtained in each example and comparative example were rehydrated and reconstituted with PBS containing 5 mmol / L CaCl2 at pH 7.2. An appropriate amount of solution was taken, and an appropriate amount of acid (0.5 mol / L sulfuric acid) was added for hydrolysis at 80 °C for 2 h to hydrolyze the polysaccharides into monosaccharides or oligosaccharides. After hydrolysis, the solution was neutralized to neutral with alkali (1 mol / L NaOH), and then filtered through a 0.22 μm filter membrane. The filtrate was used as the sample for testing. The detection results are shown in Table 1 below.

[0136] 3. Detection of the integrity of the triple helix structure (circular dichroism spectroscopy, CD):

[0137] Principle: The secondary structure of proteins produces a characteristic circular dichroism signal in the far ultraviolet region (190-250nm). Collagen with a triple helix structure has a specific absorption peak in this region. By detecting the position and intensity of these absorption peaks, the retention of the triple helix structure can be determined.

[0138] Methods: The rehydrated collagen solution was diluted with PBS to a protein concentration of approximately 0.1 mg / mL. An appropriate amount of solution was placed in a quartz cuvette with a path length of 0.1 cm and scanned using a circular dichroism spectrometer. The scanning range was set to 190-250 nm, the scanning speed to 100 nm / min, the bandwidth to 1 nm, and the averaging time to 1 s. Using PBS as a blank control, after subtracting the background signal, the integrity of the triple helix structure was calculated by comparing the intensity of the characteristic absorption peak of the triple helix structure (around 220 nm) with the CD spectrum of standard triple helix collagen. The results are shown in Table 1 below.

[0139] 4. Add bone mineral density test: Select a bone mineral density of 0.190 g / cm³. 3 -0.198g / cm 3 Several Kunming mice (weighing 18g-22g) were randomly divided into four groups: Examples 1-3, Comparative Examples 1-6, and a blank control group, with 10 mice in each group. The prepared non-denatured type II collagen product was reconstituted into a 50wt% concentration using PBS containing 5 mmol / L CaCl2 at pH 7.2 and administered daily by gavage at a dose of 20 mg / kg body weight. The blank control group received an equal volume of physiological saline. This treatment continued for 8 weeks. In vivo bone mineral density (BMD) of the mouse femur was measured using dual-energy X-ray absorptiometry (DXA). The BMD of the blank control group was 0.196 g / cm³. 3 The average results are shown in Table 1 below.

[0140] Table 1 Detection Data Results

[0141] Sample Extraction rate (%) purity(%) Total mucopolysaccharides (%) The triple helix structure retains integrity (%) <![CDATA[Bone density g / cm 2 > Example 1 15.54 72.57 12.46 93.9 0.242 Example 2 14.86 71.81 11.38 93.3 0.237 Example 3 16.29 73.26 13.11 94.5 0.254 Comparative Example 1 13.24 70.12 10.37 89.7 0.229 Comparative Example 2 5.68 40.75 8.58 57.4 0.203 Comparative Example 3 4.54 35.16 5.06 51.0 0.206 Comparative Example 4 12.12 68.34 9.13 88.1 0.215 Comparative Example 5 10.37 65.58 8.92 88.6 0.212 Comparative Example 6 14.15 70.16 10.59 91.8 0.228

[0142] The results above show that the target proteins prepared in Examples 1 to 3 have high extraction rates and purity, and the triple helix structure is better preserved.

[0143] Comparative Example 1: EDTA-citrate buffer without the addition of β-mercaptoethanol. β-mercaptoethanol is a strong reducing agent. During decalcification, proteins in cartilage tissue are easily oxidized, leading to partial protein denaturation. The absence of β-mercaptoethanol prevents effective inhibition of the oxidation reaction, altering protein structure and affecting subsequent enzymatic digestion and extraction steps. This not only results in a reduced extraction rate because some target proteins are oxidized and difficult to extract, but also decreased purity due to increased impurities; the triple helix structure is also damaged by oxidation, reducing its integrity and affecting the bone density increase effect.

[0144] Comparative Example 2: Tris-HCl buffer without added alkaline protease. Alkaline protease assists collagenase II in more comprehensive breakdown of the cartilage matrix. Without alkaline protease, collagenase II alone is insufficient to fully break down the complex cartilage matrix, resulting in incomplete cartilage tissue breakdown. This directly leads to a significant decrease in extraction rate, as most collagen remains trapped in the undigested matrix; simultaneously, there are more impurities, resulting in a significant decrease in purity; and the damage to the triple helix structure due to mechanical factors during extraction is also relatively increased, leading to reduced integrity of the triple helix structure, decreased stability, and impact on bone density enhancement. Furthermore, the synergistic breakdown and protection of alkaline protease with metal ion chelation effectively preserves the integrity of the target protein.

[0145] Comparative Example 3: Tris-HCl buffer without collagenase II. Collagenase II can recognize and cleave specific peptide bonds in type II collagen molecules, causing the intercellular matrix in cartilage tissue to break down, thereby separating cells from the extracellular matrix and facilitating the extraction of non-denatured type II collagen from cartilage. Without collagenase II, it is almost impossible to effectively extract the target collagen from cartilage, affecting the bone density increase effect. Therefore, the extraction rate is extremely low, and the purity is also at a low level due to the presence of a large number of impurities; the integrity of the triple helix structure is also greatly reduced due to improper operation and impurity interference during the extraction process.

[0146] Comparative Example 4: No proteinase K inhibitor added. Proteases degrade undenatured type II collagen. During enzymatic hydrolysis, without a proteinase K inhibitor, the proteases will continue to act on the extracted collagen, leading to over-hydrolysis. This results in a decreased extraction rate because some collagen is destroyed; reduced purity due to degradation products becoming impurities; and damage to the triple helix structure due to degradation, reducing its integrity and affecting the bone density increase effect.

[0147] Comparative Example 5: Tris-HCl buffer without the addition of CaCl2, ZnCl2, and trehalose. Trehalose plays a role in protecting protein structure. Without trehalose, the triple helix structure of collagen is more easily damaged during enzymatic hydrolysis, leading to decreased integrity and stability of the triple helix structure and affecting the bone density increase effect. CaCl2 and ZnCl2 can synergistically stabilize the conformation of collagen, while trehalose protects hydrogen bonds through water molecule replacement, maintaining the stability of collagen. Low-temperature enzymatic hydrolysis reduces the risk of thermal denaturation.

[0148] Comparative Example 6: HA column without added hyaluronic acid. Hyaluronic acid in the HA column mimics the extracellular matrix (ECM) environment of chondrocytes, which helps with the specific binding and purification of collagen. Without hyaluronic acid, the selectivity of the HA column for collagen decreases, the removal of impurities is less effective, resulting in a slight decrease in extraction rate and purity; the integrity of the triple helix structure is also affected, impacting the bone density increase effect.

Claims

1. A method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity, characterized in that, Includes the following steps: S1, Decalcification: Prepare chicken breast cartilage or shark fin cartilage, soak it in EDTA-citric acid buffer, take it out, rinse it, and obtain decalcified cartilage; The concentration of the EDTA-citric acid buffer is 0.5 mol / L to 0.6 mol / L, and it contains 1 mmol / L to 1.5 mmol / L β-mercaptoethanol, with a pH of 6.7 to 6.

9. S2, Degreasing: Decalcified cartilage is degreased by supercritical CO2 extraction and removed to obtain degreased cartilage; S3, Matrix-directed enzymatic hydrolysis: The defatted cartilage was added to Tris-HCl buffer containing alkaline protease and collagenase II and digested in a gradient manner. Specifically, the cartilage was first shaken, then allowed to stand, and then proteinase K inhibitor was added and shaken again to obtain an enzymatic hydrolysate containing undenatured type II collagen. The Tris-HCl buffer contains 5 mmol / L–6 mmol / L CaCl2, 2 mmol / L–3 mmol / L ZnCl2, 0.001 g / mL–0.003 g / mL trehalose, 0.001 g / mL–0.002 g / mL alkaline protease, and 0.0005 g / mL–0.0008 g / mL collagenase II, with a pH of 7.3–7.

5. S4, Three-phase centrifugal separation: S4.1 Initial centrifugation to remove residue: Centrifuge to separate the residue and obtain supernatant A; S4.2 Secondary centrifugation to precipitate impurity proteins: Add PEG to the supernatant A, centrifuge to separate the precipitate, and obtain supernatant B; S4.3 Filtration: Filter the supernatant B and take the filtrate to obtain a collagen solution; S5, Biomimetic fiber column chromatography purification: The collagen solution is loaded into the HA column and eluted with NaCl aqueous solution using a linear gradient elution method. The eluents of different concentrations of NaCl aqueous solution are collected and combined to obtain the purified solution. The HA column is a hydroxyapatite column and contains 2wt% to 4wt% hyaluronic acid to simulate the extracellular matrix (ECM) environment of chondrocytes. S6, Dynamic pH Stabilization Treatment: Adjust the pH of the purification solution; add glycerol; mix in laminar flow mode using a microfluidic device to obtain the target protein solution; S7, dried, yields undenatured type II collagen.

2. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S1, the chicken breast cartilage or shark fin cartilage is cut into fragments with a size range of 1mm to 5mm; the soaking process is stirred at a speed of 100rpm to 150rpm.

3. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S2, the degreasing method includes: placing the decalcified cartilage into the extraction vessel of a supercritical CO2 extraction device, setting the extraction temperature to 34℃~36℃, the pressure to 10MPa~12MPa, and the extraction time to 30min~40min.

4. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S4, three-phase centrifugal separation includes: S4.1 Initial centrifugation to remove residue: Centrifuge the enzymatic hydrolysate at 3000g to 3500g for 20 to 25 minutes at 4℃ to 6℃ to separate the residue and obtain supernatant A containing collagen and impurity proteins. S4.2 Secondary centrifugation to precipitate impurity proteins: Add 0.1 g / mL to 0.15 g / mL PEG8000 to the supernatant A, and then centrifuge at 8000 g to 10000 g for 30 min to 40 min to separate the precipitate and obtain supernatant B; S4.3 Filtration: The supernatant B is filtered through a 0.22μm ceramic membrane to remove residual microparticle impurities, resulting in a collagen solution.

5. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 4, characterized in that, In S4.3, the ceramic membrane is pre-cooled to 2℃~4℃.

6. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S5, the HA column is equilibrated with an equilibration buffer before use. The equilibration buffer is a 0.05 mol / L to 0.06 mol / L Tris-HCl buffer containing 0.12 mol / L to 0.15 mol / L NaCl and 1.5 mmol / L to 2 mmol / L CaCl2, with a pH of 7.0 to 7.

2. The elution flow rate is controlled at 1 mL / min to 1.5 mL / min, and the temperature is maintained at 4℃ to 5℃ throughout the entire process of biomimetic fiber column chromatography purification.

7. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S6, the pH of the purification solution is adjusted using 0.1 mol / L to 0.12 mol / L acetate buffer. The pH of the 0.1 mol / L to 0.12 mol / L acetate buffer is 4.8 to 4.

9. The 0.1 mol / L to 0.12 mol / L acetate buffer is pre-cooled to 4°C to 6°C.

8. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S6, the amount of glycerol added is 3% to 3.5% of the volume of the purified liquid; the channel diameter of the microfluidic device is 200 μm.

9. The method for preparing non-denatured type II collagen that increases bone density and improves bone elasticity according to claim 1, characterized in that, In S7, drying is vacuum freeze-drying. The vacuum freeze-drying steps include: pre-freezing the target protein solution to -80°C, and after complete freezing, sublimating it at a vacuum of 0.01 mBar and a temperature of -45°C for 48h to 50h to remove the water and obtain a dried, non-denatured type II collagen product.