A method for preparing sturgeon cartilage peptides to increase bone density

Abstract

The present invention relates to the field of biological extraction, and a preparation method of sturgeon cartilage peptide for increasing bone density. The method comprises the following steps: a) pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage; b) enzymolyzing the pre-treated cartilage, adding a composite enzyme, adjusting the pH value and controlling the temperature, performing an enzymolysis reaction, and obtaining an enzymolysis solution; c) centrifuging the enzymolysis solution to remove insoluble matter and obtain a clear liquid; d) performing membrane separation on the clear liquid to remove macromolecular impurities and obtain a cartilage peptide concentrate; e) drying the cartilage peptide concentrate to obtain a sturgeon cartilage peptide powder. The preparation method comprises the steps of pre-treatment, enzymolysis, centrifugation, membrane separation and drying. The composite enzyme is compounded by protein glutaminase, Lys-C protease and alkaline protease. The method of the present invention is simple to operate, efficient, economical and environmentally friendly, can significantly improve the extraction rate, purity and biological activity of sturgeon cartilage peptide, reduce production costs, and meet market demand. It has been verified by experiments that the composition of the present invention can significantly improve bone density and effectively prevent and alleviate osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and in particular relates to a method for preparing sturgeon cartilage peptide for increasing bone density. Background Art

[0002] With the increasing prevalence of osteoporosis, improving bone density has become a research hotspot in this field. More and more research focuses on developing new preparation methods to more effectively improve bone density and prevent related diseases. However, the existing preparation methods for improving bone density still have some shortcomings in terms of ingredient compatibility, absorption efficiency and long-term safety, which affects their application effect.

[0003] After searching, a composition for increasing bone density with publication number CN104758360B was found. The patent discloses a composition including chondroitin sulfate, D-amino acid glucose hydrochloride, bone drynaria extract, seaweed, maca, hemp seed and vitamin C. The composition contains multiple effective ingredients, which can increase bone density, protect and repair joint cartilage tissue, and delay body aging. However, in this technical solution, the compatibility of multiple ingredients may have the problem of insufficient synergistic effect, resulting in limited overall effect of the composition. In addition, the plant extract components in the composition are relatively complex, which may affect its long-term stability and safety.

[0004] After searching, a composition and application for improving bone density with publication number CN116711862B was found. The patent discloses a composition including bovine collagen peptide, cartilage extract, eggshell membrane extract, turmeric, milk mineral salt and sodium hyaluronate. The composition can effectively improve bone density, prevent and relieve bone and joint pain through reasonable composition. However, in this technical solution, the extraction process of the cartilage extract and eggshell membrane extract used is relatively complicated, which may lead to high production costs. In addition, the synergistic mechanism of each component is not described in detail in the composition, which may affect the stability and reliability of its overall effect.

[0005] The above problems indicate that the existing bone density increasing compositions and preparation methods still have certain deficiencies in terms of ingredient compatibility, absorption efficiency, long-term stability and safety. Therefore, the present invention provides a preparation method of sturgeon cartilage peptide for increasing bone density, which improves absorption efficiency, enhances long-term stability and safety, thereby meeting the market demand for efficient and safe bone density increasing products. Summary of the invention

[0006] The purpose of the present invention is to provide a sturgeon cartilage peptide composition for increasing bone density and a preparation method thereof, which can effectively improve the extraction rate, purity and biological activity of sturgeon cartilage peptides, while reducing production costs and meeting market demand; the present invention also provides specific steps of the preparation method to ensure that the preparation process is simple, efficient, economical and environmentally friendly.

[0007] The method for preparing a sturgeon cartilage peptide composition for increasing bone density according to the present invention comprises the following steps: a) pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage; b) enzymatically hydrolyzing the pretreated cartilage, adding a complex enzyme, adjusting the pH value and controlling the temperature, performing an enzymatic reaction, and obtaining an enzymatic hydrolyzate; c) centrifuging the enzymatic hydrolyzate to remove insoluble matter to obtain a clear solution; d) subjecting the clear liquid to membrane separation to remove macromolecular impurities to obtain a cartilage peptide concentrate; e) drying the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder.

[0008] Specifically, the pretreatment includes washing, chopping and defatting to remove impurities and fat in the cartilage.

[0009] Specifically, the particle size of the cartilage after the pretreatment is between 0.8-1.2 mm.

[0010] The degreasing step uses 70% ethanol solution as a degreasing agent, the degreasing temperature is 35±5°C, the degreasing time is 1.5 hours, and the degreasing equipment is an ultrasonic assisted degreasing device (frequency 40kHz, power 200W). After the degreasing is completed, the fat residue is detected by Soxhlet extraction to ensure that the fat content is less than 0.5% (w / w) before the subsequent enzymatic hydrolysis step can be carried out.

[0011] Specifically, the complex enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of (1-3): (1-2): 1.

[0012] The protein glutaminase (purchased from Sigma-Aldrich, product number P8038, activity ≥5000 U / g), Lys-C protease (purchased from Thermo Fisher, product number 90307, ​​activity ≥2000 U / g), and alkaline protease (purchased from Novozymes, product number Alcalase 2.4L, activity ≥2.4 AU / g) are compounded according to the mass ratio. If other brands of enzyme preparations are used, the dosage needs to be adjusted through preliminary experiments to ensure that the total enzyme activity is equivalent to the above ratio.

[0013] Specifically, the pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0014] Specifically, the pH value of the enzymatic hydrolysis reaction is 7.0-8.0, the temperature is 35-45° C., and the time is 2-4 hours.

[0015] Specifically, the centrifugal separation conditions are: centrifugal speed 4000-6000rpm, time 10-20min.

[0016] Specifically, the membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Daltons and an operating pressure of 0.1-0.3 MPa; the ultrafiltration membrane is a polyethersulfone membrane.

[0017] Specifically, the drying is carried out by spray drying or freeze drying; spray drying: 40L / h, hot air inlet temperature 160°C, outlet temperature 80°C, residual moisture ≤5%; freeze drying: pre-freezing temperature -40°C (2h), primary drying -20°C (10h, vacuum degree 10Pa), analytical drying 25°C (5h, vacuum degree 5Pa), final moisture ≤3%.

[0018] The entire preparation process does not use complex equipment and only includes basic steps such as compounding, mixing, centrifugation and membrane separation. It is easy to operate and low in cost. At the same time, no harmful chemicals are used, which is environmentally friendly and suitable for large-scale production applications.

[0019] First, the fresh sturgeon cartilage is pretreated. The pretreatment includes cleaning, chopping and degreasing steps to remove impurities and fat in the cartilage to ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is: clean the fresh sturgeon cartilage with clean water to remove impurities on the surface; then chop the cleaned cartilage to make the cartilage particle size uniform; finally, put the chopped cartilage into the degreasing equipment, remove the fat in the cartilage with the degreasing agent, and obtain the pretreated cartilage.

[0020] Next, the pretreated cartilage is enzymatically hydrolyzed. The specific operation is as follows: the pretreated cartilage is added to the enzymatic hydrolysis reactor and the compound enzyme solution is added. The compound enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of (1-3): (1-2): 1. The pH value is adjusted to 7.0-8.0, the temperature is controlled to 35-45°C, and the enzymatic reaction time is 2-4h to obtain an enzymatic hydrolyzate.

[0021] After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is centrifuged. The specific operation is: pour the enzymatic hydrolyzate into a centrifuge, set the centrifugal speed to 4000-6000rpm, the centrifugal time to 10-20 minutes, remove the insoluble matter, and obtain a clear solution.

[0022] Next, the clear liquid is subjected to membrane separation. The specific operation is: the clear liquid is separated by an ultrafiltration membrane, the molecular weight cutoff of the ultrafiltration membrane is 5000-10000 Daltons, the operating pressure is 0.1-0.3 MPa, and the macromolecular impurities are removed to obtain a cartilage peptide concentrate.

[0023] Finally, the cartilage peptide concentrate is dried. The specific operation is: the cartilage peptide concentrate is sent to a spray dryer or a freeze dryer for drying to obtain sturgeon cartilage peptide powder.

[0024] Through the above steps, the extraction rate, purity and biological activity of sturgeon cartilage peptide can be effectively improved, while reducing the production cost. The compound enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of (1-3): (1-2): 1.

[0025] Protein glutaminase has strong protein hydrolysis ability and can effectively degrade the protein in cartilage; Lys-C protease can specifically hydrolyze the peptide bonds of protein and improve the purity of peptide segments; alkaline protease has high activity under alkaline conditions and can further improve the hydrolysis rate of protein. By compounding in proportion, efficient enzymatic hydrolysis effect can be achieved, and the extraction rate and purity of cartilage peptides can be improved.

[0026] The beneficial effects of the present invention are as follows: 1. The use of compound enzymes improves the enzymatic hydrolysis effect and increases the extraction rate and purity of sturgeon cartilage peptides: By compounding protein glutaminase, Lys-C protease and alkaline protease, efficient degradation of cartilage protein can be achieved, and the purity and extraction rate of peptides can be improved. In particular, alkaline protease has higher activity under alkaline conditions, which can further increase the hydrolysis rate of protein and improve the biological activity of cartilage peptides.

[0027] 2. The use of membrane separation technology effectively removes large molecular impurities and improves the purity of cartilage peptides: Ultrafiltration membrane separation can remove large molecular impurities in the enzymatic hydrolysate, retain small molecular cartilage peptides, and improve the purity of the product. In addition, membrane separation technology is simple to operate, low cost, and suitable for large-scale production applications.

[0028] 3. Optimized enzymatic conditions and purification steps improve the biological activity of cartilage peptides: By strictly controlling the pH value, temperature and time of the enzymatic hydrolysis reaction, the activity and stability of the enzyme can be ensured, and the biological activity of the cartilage peptide can be improved. At the same time, the optimized centrifugal separation and membrane separation steps can effectively remove insoluble matter and macromolecular impurities, further improving the purity and stability of the cartilage peptide.

[0029] 4. The choice of drying method ensures the quality and stability of the product: Spray drying or freeze drying can effectively remove moisture and maintain the biological activity and stability of chondroitin. In particular, freeze drying can maximize the retention of the active ingredients of chondroitin and extend the shelf life of the product.

[0030] 5. The preparation method of the present invention is simple, efficient, economical and environmentally friendly: The entire preparation process does not use complex equipment and only includes basic steps such as compounding, mixing, centrifugation and membrane separation. It is easy to operate and low in cost. At the same time, no harmful chemicals are used, which is environmentally friendly and suitable for large-scale production applications. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments. Those skilled in the art should recognize that the present invention covers all possible alternatives, improvements and equivalents within the scope of the claims.

[0032] The following examples were tested according to the following experimental scheme, and the following are the experimental conditions of all examples: In the degreasing of all the embodiments described below, a 70% ethanol solution was used as the degreasing agent, the degreasing temperature was 35±5° C., the degreasing time was 1.5 hours, and the degreasing equipment was an ultrasonic assisted degreasing device (frequency 40 kHz, power 200 W).

[0033] The protein glutaminase (purchased from Sigma-Aldrich, product number P8038, activity ≥5000 U / g), Lys-C protease (purchased from Thermo Fisher, product number 90307, ​​activity ≥2000 U / g), and alkaline protease (purchased from Novozymes, product number Alcalase 2.4L, activity ≥2.4 AU / g) described in the examples are compounded according to the mass ratio.

[0034] The following is the drying process of the embodiment Spray drying: 40L / h, hot air inlet temperature 160℃, outlet temperature 80℃, residual moisture ≤5%; Freeze drying: pre-freezing temperature -40℃ (2h), primary drying -20℃ (10h, vacuum degree 10Pa), analytical drying 25℃ (5h, vacuum degree 5Pa), final moisture ≤3%.

[0035] The preparation method of the sturgeon cartilage peptide composition for increasing bone density of the present invention comprises the following steps.

[0036] Embodiment 1;

[0037] a) Pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage. The pre-treatment includes cleaning, chopping and degreasing steps to remove impurities and fat in the cartilage to ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is: clean the fresh sturgeon cartilage with clean water to remove impurities on the surface; then chop the cleaned cartilage to make the cartilage particle size uniform, with a particle size between 0.8-1.2mm; finally, put the chopped cartilage into a degreasing device, remove the fat in the cartilage with a degreasing agent, and obtain pre-treated cartilage.

[0038] b) Enzymatically hydrolyzing the pretreated cartilage. The specific operation is as follows: adding the pretreated cartilage to the enzymatic hydrolysis reactor and adding the complex enzyme solution. The complex enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of 2:1.5:1. The pH value is adjusted to 7.5, the temperature is controlled to 40°C, and the enzymatic hydrolysis reaction time is 3 hours to obtain an enzymatic solution. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0039] c) centrifuging the enzymatic hydrolysate to remove insoluble matter and obtain a clear solution. The specific operation is: pouring the enzymatic hydrolysate into a centrifuge, setting the centrifugal speed to 5000 rpm, the centrifugal time to 15 minutes, removing insoluble matter, and obtaining a clear solution. The centrifugal speed regulator and the centrifugal time controller are used to control the speed and time of the centrifuge.

[0040] d) The clear liquid is subjected to membrane separation to remove macromolecular impurities to obtain a cartilage peptide concentrate. The specific operation is: the clear liquid is separated by an ultrafiltration membrane, the molecular weight cutoff of the ultrafiltration membrane is 7500 Daltons, the operating pressure is 0.2 MPa, the macromolecular impurities are removed, and the cartilage peptide concentrate is obtained. The membrane separation equipment is used to perform the membrane separation operation, and the operating pressure regulator is used to control the operating pressure.

[0041] e) Drying the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder. The specific operation is: sending the cartilage peptide concentrate into a freeze dryer for freeze drying to obtain sturgeon cartilage peptide powder.

[0042] Embodiment 2;

[0043] a) Pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage. The pre-treatment includes cleaning, chopping and degreasing steps to remove impurities and fat in the cartilage to ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is: clean the fresh sturgeon cartilage with clean water to remove impurities on the surface; then chop the cleaned cartilage to make the cartilage particle size uniform, with a particle size between 0.8-1.2mm; finally, put the chopped cartilage into a degreasing device, remove the fat in the cartilage with a degreasing agent, and obtain pre-treated cartilage.

[0044] b) Enzymatically hydrolyzing the pretreated cartilage. The specific operation is as follows: adding the pretreated cartilage to an enzymatic hydrolysis reactor and adding a composite enzyme solution. The composite enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of 1:1:1. The pH value is adjusted to 8.0, the temperature is controlled to 35°C, and the enzymatic hydrolysis reaction time is 2 hours to obtain an enzymatic hydrolyzate. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0045] c) centrifuging the enzymatic hydrolysate to remove insoluble matter and obtain a clear solution. The specific operation is: pouring the enzymatic hydrolysate into a centrifuge, setting the centrifugal speed to 4000 rpm, the centrifugal time to 10 minutes, removing insoluble matter, and obtaining a clear solution. The centrifugal speed regulator and the centrifugal time controller are used to control the speed and time of the centrifuge.

[0046] d) The clear liquid is subjected to membrane separation to remove macromolecular impurities to obtain a cartilage peptide concentrate. The specific operation is: the clear liquid is separated by an ultrafiltration membrane, the molecular weight cutoff of the ultrafiltration membrane is 5000 Daltons, the operating pressure is 0.1 MPa, the macromolecular impurities are removed, and the cartilage peptide concentrate is obtained. The membrane separation equipment is used to perform the membrane separation operation, and the operating pressure regulator is used to control the operating pressure.

[0047] e) Drying the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder. The specific operation is: sending the cartilage peptide concentrate into a freeze dryer for freeze drying to obtain sturgeon cartilage peptide powder.

[0048] Embodiment 3;

[0049] a) Pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage. The pre-treatment includes cleaning, chopping and degreasing steps to remove impurities and fat in the cartilage to ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is: clean the fresh sturgeon cartilage with clean water to remove impurities on the surface; then chop the cleaned cartilage to make the cartilage particle size uniform, with a particle size between 0.8-1.2mm; finally, put the chopped cartilage into a degreasing device, remove the fat in the cartilage with a degreasing agent, and obtain pre-treated cartilage.

[0050] b) Enzymatically hydrolyzing the pretreated cartilage. The specific operation is as follows: adding the pretreated cartilage to an enzymatic hydrolysis reactor and adding a composite enzyme solution. The composite enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of 3:2:1. The pH value is adjusted to 7.0, the temperature is controlled to 45°C, and the enzymatic hydrolysis reaction time is 4 hours to obtain an enzymatic hydrolyzate. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0051] c) centrifuging the enzymatic hydrolysate to remove insoluble matter and obtain a clear solution. The specific operation is: pouring the enzymatic hydrolysate into a centrifuge, setting the centrifugal speed to 6000 rpm, the centrifugal time to 20 minutes, removing insoluble matter, and obtaining a clear solution. The centrifugal speed regulator and the centrifugal time controller are used to control the speed and time of the centrifuge.

[0052] d) The clear liquid is subjected to membrane separation to remove macromolecular impurities to obtain a cartilage peptide concentrate. The specific operation is: the clear liquid is separated by an ultrafiltration membrane, the molecular weight cutoff of the ultrafiltration membrane is 10,000 Daltons, the operating pressure is 0.3 MPa, the macromolecular impurities are removed, and the cartilage peptide concentrate is obtained. The membrane separation equipment is used to perform the membrane separation operation, and the operating pressure regulator is used to control the operating pressure.

[0053] e) Drying the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder. The specific operation is: sending the cartilage peptide concentrate into a spray dryer for spray drying to obtain sturgeon cartilage peptide powder.

[0054] Comparative Example 1 No degreasing treatment was performed, and the remaining steps and conditions were the same as in Example 1.

[0055] Comparative Example 2 No composite enzyme was used, only protein glutaminase and alkaline protease were used, and enzymatic hydrolysis was performed at a mass ratio of 2:1. The remaining steps and conditions were the same as those in Example 1.

[0056] Comparative Example 3 Without ultrafiltration membrane separation, the clear liquid after centrifugation was directly dried, and the remaining steps and conditions were the same as those in Example 1.

[0057] Comparative Example 4 A single enzyme (protein glutaminase) was used for enzymatic hydrolysis, and the remaining steps and conditions were the same as those in Example 1.

[0058] Comparative Example 5 The enzymatic hydrolysis reaction time was shortened to 0.5 h, and the remaining steps and conditions were the same as those in Example 1.

[0059] In Comparative Example 1, the enzymatic efficiency was reduced due to the lack of degreasing. Mechanism analysis showed that the fat layer (thickness > 50 μm) would hinder the diffusion of enzyme molecules (confirmed by fluorescent labeling tracking experiments). In Comparative Example 3, after omitting membrane separation, HPLC detection showed that the product contained 12-15% of macromolecular proteins above 10,000 Da. These impurities would cause turbid precipitation during product storage (accelerated test 40°C / 75%RH).

[0060] The performance of the sturgeon cartilage peptide powder prepared in the examples and comparative examples was evaluated: (1) Detection of extraction rate, purity and biological activity: The extraction rate and purity of the sturgeon cartilage peptides prepared in each embodiment and comparative example were detected by high performance liquid chromatography (HPLC), and the biological activity was detected by in vitro cell experiments. Specific data are shown in Table 1.

[0061] (2) Stability test: The sturgeon cartilage peptide powder prepared in each embodiment and comparative example was stored at 40°C ± 2°C for 6 months in an aluminum foil bag (oxygen permeability ≤ 0.5 cc / m 2 ·day) to test its stability. The specific data are shown in Table 1.

[0062] Table 1 Extraction rate, purity and biological activity of sturgeon cartilage peptide Extraction rate (%) purity(%) Biological activity (unit / mg) Purity after 6 months (%) Example 1 93 95 210 92 Example 2 91 94 190 91 Example 3 88 93 189 90 Comparative Example 1 70 85 150 80 Comparative Example 2 65 80 130 75 Comparative Example 3 55 73 115 70 Comparative Example 4 60 82 140 78 Comparative Example 5 50 70 110 65 When comparative example 1 was not defatted, residual fat (>5%) wrapped the cartilage matrix, hindering the contact between the enzyme and the substrate, resulting in a 33% decrease in the extraction rate; In Comparative Example 3, when no membrane separation was performed, macromolecular impurities (such as collagen fragments, molecular weight>10 kDa) were mixed into the final product, reducing the purity to 73%. In order to conduct a bone density control test, tablets were prepared according to the following composition by weight: 200 parts of sturgeon cartilage peptide, 20 parts of sodium hyaluronate, 0.005 parts of vitamin D3, 5 parts of vitamin C, and 5 parts of sweetener, wherein erythritol was selected as the sweetener.

[0063] In order to verify the effect of the present invention, the following experiment was conducted: 100 osteoporosis patients were selected and divided into two groups, each with 50 people. The experimental group took the sturgeon cartilage peptide composition prepared by the present invention every day, and the control group took ordinary calcium supplements every day. After taking it for 6 months, the bone density of the two groups of patients was tested. The results showed that the bone density of the patients in the experimental group increased by an average of 15%, while the bone density of the patients in the control group only increased by 5%. This shows that the sturgeon cartilage peptide composition of the present invention has a significant effect of improving bone density and can effectively prevent and alleviate osteoporosis.

[0064] Period: Continuous administration for 6 months, with a follow-up period of 3 months (to monitor delayed effects); Primary endpoint: Change in lumbar spine (L1-L4) BMD (%); Secondary endpoints: Serum bone metabolism markers: β-collagen degradation product (β-CTX), osteocalcin (OC), total type I procollagen N-terminal peptide (tPINP); Safety indicators: liver and kidney function (ALT, Cr), gastrointestinal adverse reaction records.

[0065] Bone density improvement effect: Group Baseline bone density (g / cm²) 6-month bone density (g / cm²) Rate of change (%) p-value (vs baseline) p-value (between groups) Experimental Group 0.682±0.032 0.784±0.028 +15.0% <0.001 <0.01 Control group 0.679±0.029 0.713±0.031 +5.0% 0.032 Detection method: Dual-energy X-ray absorptiometry (DXA, Hologic Discovery Wi model), calibrated by the same equipment and operator.

[0066] Changes in bone metabolism markers: index Experimental group (Δ%) Control group (Δ%) Biological significance β-CTX -38.2% -12.1% Bone resorption inhibition (*p<0.05 vs control group) OC +25.6% +7.3% Bone formation promotion (*p<0.05 vs control group) tP +31.8% +9.4% Accelerates collagen synthesis Validity: The bone density of the experimental group increased by 3 times that of the control group (15% vs 5%), and the bone metabolism markers showed a two-way regulatory effect of "inhibiting bone resorption + promoting bone formation", confirming that the composition of the present invention has the unique advantage of multi-target intervention in osteoporosis; Follow-up data showed that the bone density of the experimental group continued to increase (+2.3%) three months after drug discontinuation, indicating that the therapeutic effect was sustained.

[0067] Security: The adverse reaction rate of the composition of the present invention is significantly lower than that of conventional calcium preparations (4% vs 10%), and is particularly suitable for long-term use; Low-dose vitamin D3 (0.005 parts) did not cause the risk of hypercalcemia (blood calcium level: 2.32mmol / L in the experimental group vs. 2.35mmol / L in the control group).

[0068] Comparative experiment: In order to prove the irreplaceability of the formula ratio, the following control experiments are added: Group Bone density improvement rate (%) Formula of the present invention 15.0 Sturgeon peptide alone 8.7 Vitamin C deficiency 9.7 By comparison, it can be seen that the method of the present invention has significant advantages in improving the extraction rate, purity and biological activity of sturgeon cartilage peptide, and the produced cartilage peptide powder has good stability.

[0069] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A method for preparing sturgeon cartilage peptide for increasing bone density, characterized in that: The following steps are involved: a) pre-treating fresh sturgeon cartilage to obtain pre-treated cartilage; b) enzymatically hydrolyzing the pretreated cartilage, adding a complex enzyme, adjusting the pH value and controlling the temperature, performing an enzymatic reaction, and obtaining an enzymatic hydrolyzate; c) centrifuging the enzymatic hydrolyzate to remove insoluble matter to obtain a clear solution; d) subjecting the clear liquid to membrane separation to remove macromolecular impurities to obtain a cartilage peptide concentrate; e) drying the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder; The complex enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease in a mass ratio of (1-3): (1-2): 1; The pH value of the enzymatic hydrolysis reaction is 7.0-8.0, the temperature is 35-45°C, and the time is 2-4h; The centrifugal separation conditions are: centrifugal speed 4000-6000rpm, time 10-20min.

2. The method for preparing a sturgeon cartilage peptide for increasing bone density according to claim 1, characterized in that: The pretreatment includes cleaning, chopping and degreasing.

3. The method for preparing a sturgeon cartilage peptide for increasing bone density according to claim 1, characterized in that: The pH regulator is sodium hydroxide or hydrochloric acid.

4. The method for preparing a sturgeon cartilage peptide for increasing bone density according to claim 1, characterized in that: The membrane separation adopts an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Daltons and an operating pressure of 0.1-0.3 MPa.

5. The method for preparing a sturgeon cartilage peptide for increasing bone density according to claim 1, characterized in that: The drying is carried out by spray drying or freeze drying.

6. The method for preparing a sturgeon cartilage peptide for increasing bone density according to claim 2, characterized in that: The degreasing step uses 70% ethanol solution as a degreasing agent, the degreasing temperature is 35±5° C., the degreasing time is 1.5 hours, and the degreasing equipment is an ultrasonic assisted degreasing device.