Preparation method of sturgeon cartilage peptide for increasing 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) pretreating fresh sturgeon cartilage to obtain pretreated cartilage; b) enzymatically hydrolyzing the pretreated cartilage, adding a composite enzyme, adjusting the pH value and controlling the temperature to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate; c) centrifugally separating the enzymatic hydrolysate to remove insoluble substances to obtain a supernatant; d) subjecting the supernatant 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 preparation method comprises steps such as pretreatment, enzymatic hydrolysis, centrifugal separation, membrane separation and drying. The composite enzyme is compounded from protein glutaminase, Lys-C protease and alkaline protease. The method of the present invention is simple, 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 demands. Through experimental verification, the composition of the present invention can significantly increase bone density and effectively prevent and alleviate osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological extraction, and specifically relates to a preparation method of 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 increase bone density and prevent related diseases. However, the existing preparation methods for increasing bone density still have some deficiencies in ingredient compatibility, absorption efficiency, and long-term safety, which affect their application effects.

[0003] After retrieval, a composition for increasing bone density with the publication number of CN104758360B is disclosed. This patent discloses a composition including chondroitin sulfate, D-glucosamine hydrochloride, Drynaria rhizome extract, seaweed, maca, hemp seed, and vitamin C. This composition contains multiple active ingredients, can increase bone density, protect and repair articular cartilage tissue, and delay the aging of the body. However, in this technical solution, there may be a problem of insufficient synergistic effect in the compatibility of multiple ingredients, resulting in limited overall effect of the composition. In addition, the plant extract components in this composition are relatively complex, which may affect its long-term stability and safety.

[0004] After retrieval, a composition for increasing bone density and its application with the publication number of CN116711862B is disclosed. This patent discloses a composition including bovine bone collagen peptide, cartilage extract, eggshell membrane extract, turmeric, milk mineral salt, and sodium hyaluronate. Through reasonable ingredient compatibility, this composition can effectively increase bone density and prevent and relieve osteoarticular pain. However, in this technical solution, the extraction processes of the cartilage extract and eggshell membrane extract used are relatively complex, which may lead to higher production costs. In addition, the synergistic action mechanism of each ingredient in this composition is not described in detail, which may affect the stability and reliability of its overall effect.

[0005] The above problems indicate that the existing compositions and preparation methods for increasing bone density still have certain deficiencies in 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, so as to meet the market demand for high-efficiency and safe bone density increasing products. Summary of the Invention

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

[0007] A preparation method of a sturgeon cartilage peptide composition for increasing bone density according to the present invention comprises the following steps:

[0008] a) Pretreat fresh sturgeon cartilage to obtain pretreated cartilage;

[0009] b) Enzymatically hydrolyze the pretreated cartilage, add a complex enzyme, adjust the pH value and control the temperature to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate;

[0010] c) Centrifuge and separate the enzymatic hydrolysate to remove insoluble substances to obtain a supernatant;

[0011] d) Carry out membrane separation on the supernatant to remove macromolecular impurities to obtain a cartilage peptide concentrate;

[0012] e) Dry the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder.

[0013] Specifically, the pretreatment includes washing, chopping and degreasing to remove impurities and fat in the cartilage.

[0014] Specifically, the particle size of the pretreated cartilage is between 0.8 - 1.2 mm.

[0015] In the degreasing step, a 70% ethanol solution is used as the 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 40 kHz, power 200 W). After degreasing, the fat residue is detected by the Soxhlet extraction method to ensure that the fat content is lower than 0.5% (w / w) before proceeding to the subsequent enzymatic hydrolysis step.

[0016] Specifically, the complex enzyme is prepared by compounding protein glutaminase, Lys-C protease and alkaline protease according to a mass ratio of (1 - 3):(1 - 2):1.

[0017] 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 brand 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.

[0018] Specifically, the pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0019] 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 h.

[0020] Specifically, the conditions for centrifugal separation are: the centrifugal speed is 4000 - 6000 rpm, and the time is 10 - 20 min.

[0021] Specifically, the membrane separation uses an ultrafiltration membrane with a molecular weight cut-off of 5000 - 10000 Da, and the operating pressure is 0.1 - 0.3 MPa; the ultrafiltration membrane is a polyethersulfone membrane.

[0022] Specifically, the drying is carried out by spray drying or freeze drying; Spray drying: 40 L / h, the hot air inlet temperature is 160 °C, the outlet temperature is 80 °C, and the residual moisture ≤ 5%; Freeze drying: the pre-freezing temperature is -40 °C (2 h), the primary drying is at -20 °C (10 h, vacuum degree 10 Pa), the analytical drying is at 25 °C (5 h, vacuum degree 5 Pa), and the final moisture ≤ 3%.

[0023] The entire preparation process does not use complex equipment and only includes basic steps such as compounding, mixing, centrifugation, and membrane separation. The operation is simple and the cost is low. At the same time, no harmful chemicals are used, which is environmentally friendly and suitable for large-scale production applications.

[0024] First, the fresh sturgeon cartilage is pretreated. The pretreatment includes cleaning, chopping, and degreasing steps to remove impurities and fat in the cartilage and ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is as follows: Wash the fresh sturgeon cartilage with clean water to remove surface impurities; then chop the cleaned cartilage to make the cartilage particle size uniform; finally, put the chopped cartilage into a degreasing device and remove the fat in the cartilage with a degreasing agent to obtain pretreated cartilage.

[0025] Next, the pretreated cartilage is enzymatically hydrolyzed. The specific operation is as follows: Add the pretreated cartilage to an enzymatic hydrolysis reaction kettle and add a compound enzyme solution. The compound enzyme is prepared by compounding protein glutaminase, Lys-C protease, and alkaline protease according to the mass ratio of (1 - 3):(1 - 2):1. Adjust the pH value to 7.0 - 8.0, control the temperature at 35 - 45 °C, and the enzymatic hydrolysis reaction time is 2 - 4 h to obtain an enzymatic hydrolysate.

[0026] After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is centrifugally separated. The specific operation is as follows: Pour the enzymatic hydrolysate into a centrifuge, set the centrifugal speed to 4000 - 6000 rpm, and the centrifugation time is 10 - 20 minutes to remove insoluble substances and obtain a clear liquid.

[0027] Next, the clear liquid is subjected to membrane separation. The specific operation is as follows: The clear liquid is separated through an ultrafiltration membrane. The cut-off molecular weight of the ultrafiltration membrane is 5,000 - 10,000 Daltons, and the operating pressure is 0.1 - 0.3 MPa. Macromolecular impurities are removed to obtain a chondro peptide concentrate.

[0028] Finally, the chondro peptide concentrate is dried. The specific operation is as follows: The chondro peptide concentrate is fed into a spray dryer or a freeze dryer for drying to obtain sturgeon chondro peptide powder.

[0029] Through the above steps, the extraction rate, purity, and biological activity of sturgeon chondro peptide can be effectively improved, while the production cost is reduced. The composite enzyme is prepared by compounding protein glutaminase, Lys-C protease, and alkaline protease according to a mass ratio of (1 - 3):(1 - 2):1.

[0030] Protein glutaminase has strong protein hydrolysis ability and can effectively degrade proteins in cartilage; Lys-C protease can specifically hydrolyze peptide bonds of proteins to improve the purity of peptide segments; alkaline protease has high activity under alkaline conditions and can further increase the hydrolysis rate of proteins. Through proportional compounding, an efficient enzymatic hydrolysis effect can be achieved, and the extraction rate and purity of chondro peptide can be improved.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The use of the composite enzyme improves the enzymatic hydrolysis effect and increases the extraction rate and purity of sturgeon chondro peptide:

[0033] By compounding protein glutaminase, Lys-C protease, and alkaline protease, efficient degradation of proteins in cartilage can be achieved, and the purity and extraction rate of peptide segments can be improved. In particular, alkaline protease has high activity under alkaline conditions and can further increase the hydrolysis rate of proteins and improve the biological activity of chondro peptide.

[0034] 2. The use of membrane separation technology effectively removes macromolecular impurities and improves the purity of chondro peptide:

[0035] Through ultrafiltration membrane separation, macromolecular impurities in the enzymatic hydrolysate can be removed, and small molecule chondro peptides can be retained, improving the purity of the product. In addition, the membrane separation technology is simple to operate and has low cost, making it suitable for large-scale production applications.

[0036] 3. The optimized enzymatic hydrolysis conditions and purification steps improve the biological activity of chondro peptide:

[0037] 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 chondro peptide can be improved. At the same time, the optimized centrifugal separation and membrane separation steps can effectively remove insoluble substances and macromolecular impurities, further improving the purity and stability of chondro peptide.

[0038] 4. The choice of drying method ensures the quality and stability of the product:

[0039] Spray drying or freeze drying can be used to effectively remove moisture and maintain the biological activity and stability of chondro-peptide. In particular, the freeze drying method can maximize the retention of the active ingredients of chondro-peptide and extend the shelf life of the product.

[0040] 5. The preparation method of the present invention is simple, efficient, economical and environmentally friendly:

[0041] The entire preparation process does not use complex equipment and only includes basic steps such as compounding, mixing, centrifugation and membrane separation. The operation is simple and the cost is low. At the same time, no harmful chemicals are used, which is environmentally friendly and suitable for large-scale production applications. Detailed implementation manners

[0042] The present invention will be 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 alternative solutions, improvement solutions and equivalent solutions within the scope of the claims.

[0043] The following examples were tested according to the following experimental protocols. The following are the experimental conditions for all examples:

[0044] For degreasing in all the following examples, 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).

[0045] The protein glutaminase (purchased from Sigma-Aldrich, catalog number P8038, activity ≥ 5000 U / g), Lys-C protease (purchased from Thermo Fisher, catalog number 90307, activity ≥ 2000 U / g), and alkaline protease (purchased from Novozymes, catalog number Alcalase 2.4L, activity ≥ 2.4 AU / g) described in the examples were compounded according to the mass ratio.

[0046] The following is the drying process of the examples

[0047] Spray drying: 40 L / h, hot air inlet temperature 160°C, outlet temperature 80°C, moisture residue ≤ 5%;

[0048] Freeze drying: pre-freezing temperature -40°C (2 h), primary drying -20°C (10 h, vacuum degree 10 Pa), analytical drying 25°C (5 h, vacuum degree 5 Pa), final moisture ≤ 3%.

[0049] A preparation method of a sturgeon cartilage peptide composition for increasing bone density according to the present invention comprises the following steps.

[0050] Example 1;

[0051] a) Pretreat fresh sturgeon cartilage to obtain pretreated cartilage. The pretreatment includes cleaning, chopping, and degreasing steps to remove impurities and fat in the cartilage and ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is as follows: Wash the fresh sturgeon cartilage with clean water to remove surface impurities; then chop the washed cartilage to make the cartilage particle size uniform, with the particle size between 0.8 - 1.2 mm; finally, put the chopped cartilage into a degreasing device and remove the fat in the cartilage through a degreasing agent to obtain pretreated cartilage.

[0052] b) Enzymatically hydrolyze the pretreated cartilage. The specific operation is as follows: Add the pretreated cartilage to an enzymatic hydrolysis reaction kettle and add a compound enzyme solution. The compound enzyme is prepared by compounding protein glutaminase, Lys - C protease, and alkaline protease in a mass ratio of 2:1.5:1. Adjust the pH value to 7.5, control the temperature at 40 °C, and the enzymatic hydrolysis reaction time is 3 h to obtain an enzymatic hydrolysate. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0053] c) Centrifuge the enzymatic hydrolysate to remove insoluble substances and obtain a clear liquid. The specific operation is as follows: Pour the enzymatic hydrolysate into a centrifuge, set the centrifugation speed at 5000 rpm, and the centrifugation time at 15 minutes to remove insoluble substances and obtain a clear liquid. The centrifugation speed regulator and centrifugation time controller are used to control the speed and time of the centrifuge.

[0054] d) Perform membrane separation on the clear liquid to remove macromolecular impurities and obtain a cartilage peptide concentrate. The specific operation is as follows: Separate the clear liquid through an ultrafiltration membrane. The cut - off molecular weight of the ultrafiltration membrane is 7500 Dalton, and the operating pressure is 0.2 MPa to remove macromolecular impurities and obtain a cartilage peptide concentrate. The membrane separation device is used for membrane separation operations, and the operating pressure regulator is used to control the operating pressure.

[0055] e) Dry the cartilage peptide concentrate to obtain sturgeon cartilage peptide powder. The specific operation is as follows: Feed the cartilage peptide concentrate into a freeze - dryer for freeze - drying to obtain sturgeon cartilage peptide powder.

[0056] Example 2;

[0057] a) Pre-treat the 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 and ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is as follows: Clean the fresh sturgeon cartilage with clean water to remove surface impurities; then chop the cleaned cartilage to make the cartilage particle size uniform, with the particle size between 0.8 - 1.2 mm; finally, put the chopped cartilage into a degreasing device and remove the fat in the cartilage through a degreasing agent to obtain pre-treated cartilage.

[0058] b) Enzymatically hydrolyze the pre-treated cartilage. The specific operation is as follows: Add the pre-treated cartilage to an enzymatic hydrolysis reaction kettle and add a complex enzyme solution. The complex enzyme is prepared by compounding protein glutaminase, Lys-C protease, and alkaline protease in a mass ratio of 1:1:1. Adjust the pH value to 8.0, control the temperature at 35 °C, and the enzymatic hydrolysis reaction time is 2 h to obtain an enzymatic hydrolysate. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0059] c) Centrifuge the enzymatic hydrolysate to remove insoluble substances and obtain a clear liquid. The specific operation is as follows: Pour the enzymatic hydrolysate into a centrifuge, set the centrifugation speed at 4000 rpm, and the centrifugation time is 10 minutes to remove insoluble substances and obtain a clear liquid. The centrifugation speed regulator and centrifugation time controller are used to control the speed and time of the centrifuge.

[0060] d) Perform membrane separation on the clear liquid to remove macromolecular impurities and obtain a concentrated cartilage peptide solution. The specific operation is as follows: Separate the clear liquid through an ultrafiltration membrane. The cut-off molecular weight of the ultrafiltration membrane is 5000 Daltons, and the operating pressure is 0.1 MPa to remove macromolecular impurities and obtain a concentrated cartilage peptide solution. The membrane separation equipment is used for membrane separation operations, and the operating pressure regulator is used to control the operating pressure.

[0061] e) Dry the concentrated cartilage peptide solution to obtain sturgeon cartilage peptide powder. The specific operation is as follows: Feed the concentrated cartilage peptide solution into a freeze dryer for freeze-drying to obtain sturgeon cartilage peptide powder.

[0062] Example 3;

[0063] a) Pre-treat the 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 and ensure the smooth progress of the subsequent enzymatic hydrolysis process. The specific operation is as follows: Clean the fresh sturgeon cartilage with clean water to remove surface impurities; then chop the cleaned cartilage to make the cartilage particle size uniform, with the particle size between 0.8 - 1.2 mm; finally, put the chopped cartilage into a degreasing device and remove the fat in the cartilage through a degreasing agent to obtain pre-treated cartilage.

[0064] b) Enzymatically hydrolyze the pretreated cartilage. The specific operation is as follows: Add the pretreated cartilage into an enzymatic hydrolysis reactor and add a complex enzyme solution. The complex enzyme is prepared by mixing protein glutaminase, Lys-C protease, and alkaline protease in a mass ratio of 3:2:1. Adjust the pH value to 7.0, control the temperature at 45 °C, and the enzymatic hydrolysis reaction time is 4 h to obtain an enzymatic hydrolysate. The pH regulator is sodium hydroxide or hydrochloric acid, which is used to adjust the pH value of the enzymatic hydrolysis reaction.

[0065] c) Centrifuge the enzymatic hydrolysate to remove insoluble substances and obtain a clear liquid. The specific operation is as follows: Pour the enzymatic hydrolysate into a centrifuge, set the centrifugation speed at 6000 rpm, and the centrifugation time at 20 minutes to remove insoluble substances and obtain a clear liquid. The centrifugation speed regulator and centrifugation time controller are used to control the speed and time of the centrifuge.

[0066] d) Perform membrane separation on the clear liquid to remove macromolecular impurities and obtain a concentrated cartilage peptide solution. The specific operation is as follows: Separate the clear liquid through an ultrafiltration membrane. The cut-off molecular weight of the ultrafiltration membrane is 10,000 Daltons, and the operating pressure is 0.3 MPa to remove macromolecular impurities and obtain a concentrated cartilage peptide solution. The membrane separation equipment is used for membrane separation operations, and the operating pressure regulator is used to control the operating pressure.

[0067] e) Dry the concentrated cartilage peptide solution to obtain sturgeon cartilage peptide powder. The specific operation is as follows: Feed the concentrated cartilage peptide solution into a spray dryer for spray drying to obtain sturgeon cartilage peptide powder.

[0068] Comparative Example 1

[0069] Do not perform degreasing treatment, and the remaining steps and conditions are the same as those in Example 1.

[0070] Comparative Example 2

[0071] Do not use the complex enzyme, only use protein glutaminase and alkaline protease, and perform enzymatic hydrolysis according to a mass ratio of 2:1. The remaining steps and conditions are the same as those in Example 1.

[0072] Comparative Example 3

[0073] Do not perform ultrafiltration membrane separation, directly dry the clear liquid after centrifugation, and the remaining steps and conditions are the same as those in Example 1.

[0074] Comparative Example 4

[0075] Use a single enzyme (protein glutaminase) for enzymatic hydrolysis, and the remaining steps and conditions are the same as those in Example 1.

[0076] Comparative Example 5

[0077] Shorten the enzymatic hydrolysis reaction time to 0.5 h, and the remaining steps and conditions are the same as those in Example 1.

[0078] In Comparative Example 1, non - degreasing led to a decrease in enzymatic hydrolysis efficiency. Mechanistic analysis showed that the fat layer (when the thickness > 50 μm) would hinder the diffusion of enzyme molecules (confirmed by fluorescence - labeled tracking experiments). After omitting membrane separation in Comparative Example 3, HPLC detection showed that the product contained 12 - 15% of macromolecular proteins with a molecular weight above 10,000 Da, and these impurities would cause turbidity and precipitation during the storage of the product (accelerated test at 40 °C / 75% RH).

[0079] Perform performance evaluations on the sturgeon cartilage peptide powders prepared in the examples and comparative examples:

[0080] (1) Detect the extraction rate, purity, and biological activity: Detect the extraction rate and purity of the sturgeon cartilage peptides prepared in each example and comparative example by high - performance liquid chromatography (HPLC), and detect the biological activity through in vitro cell experiments. The specific data are shown in Table 1.

[0081] (2) Stability test: Store the sturgeon cartilage peptide powders prepared in each example and comparative example at 40 °C ± 2 °C for 6 months, packaged in aluminum foil bags (oxygen transmission rate ≤ 0.5 cc / m 2 ·day) to detect their stability. The specific data are shown in Table 1.

[0082] Table 1 Extraction rate, purity, and biological activity of sturgeon cartilage peptides

[0083] Extraction rate (%) Purity (%) Biological activity (units / mg) Purity after 6 months (%) Example 1 93 95 210 92 Example 2 91 94 190 91 Example 3 88 93 189 90 Control Example 1 70 85 150 80 Control Example 2 65 80 130 75 Control Example 3 55 73 115 70 Control Example 4 60 82 140 78 Control Example 5 50 70 110 65

[0084] When there was no degreasing in Comparative Example 1, the 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;

[0085] When there was no membrane separation in Comparative Example 3, macromolecular impurities (such as collagen fragments, molecular weight > 10 kDa) were mixed into the final product, reducing the purity to 73%.

[0086] 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 D 3 , 5 parts of vitamin C, 5 parts of sweetener, and the sweetener was selected as erythritol.

[0087] To verify the effect of the present invention, the following experiment was carried out: 100 osteoporosis patients were selected and divided into two groups of 50 people each. The experimental group took the sturgeon cartilage peptide composition prepared by the present invention daily, and the control group took ordinary calcium supplements daily. After continuous administration for 6 months, the bone density of the two groups of patients was detected. 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 indicates that the sturgeon cartilage peptide composition of the present invention has a significant effect of increasing bone density and can effectively prevent and relieve osteoporosis.

[0088] Duration: Continuous administration for 6 months, follow-up period of 3 months (to monitor the delayed effect);

[0089] Primary endpoint: The change rate of lumbar spine (L1-L4) bone mineral density (%);

[0090] Secondary endpoints:

[0091] Serum bone metabolism markers: β-Collagen degradation products (β-CTX), osteocalcin (OC), total procollagen type I N-terminal peptide (tPINP);

[0092] Safety indicators: Liver and kidney functions (ALT, Cr), recording of gastrointestinal adverse reactions.

[0093] Effect of bone mineral density improvement:

[0094] Group Baseline bone mineral density (g / cm²) Bone mineral density after 6 months (g / cm²) Change rate (%) 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

[0095] Detection method: Dual-energy X-ray absorptiometry (DXA, Hologic Discovery Wi type), calibrated by the same device and the same operator.

[0096] Changes in bone metabolism markers:

[0097] 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) tPINP +31.8% +9.4% Accelerated bone collagen synthesis

[0098] Efficacy:

[0099] The bone mineral density increase in the experimental group was 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 the unique advantage of the composition of the present invention in multi-target intervention for osteoporosis;

[0100] Data during the follow-up period showed that the bone mineral density in the experimental group still maintained an increasing trend (+2.3%) 3 months after drug withdrawal, indicating the persistence of the efficacy.

[0101] Safety:

[0102] The adverse reaction rate of the composition of the present invention was significantly lower than that of conventional calcium agents (4% vs 10%), and it was especially suitable for long-term use;

[0103] Low-dose vitamin D3 (0.005 parts) did not cause the risk of hypercalcemia (serum calcium level: 2.32 mmol / L in the experimental group vs 2.35 mmol / L in the control group).

[0104] Comparative experiment:

[0105] To prove the irreplaceability of the formula ratio, the following control experiment was supplemented:

[0106] Group Bone mineral density increase rate (%) Formula of the present invention 15.0 Sturgeon peptide used alone 8.7 Absence of vitamin C 9.7

[0107] It can be seen by comparison that the method of the present invention has significant advantages in improving the extraction rate, purity and biological activity of sturgeon cartilage peptides, and at the same time, the produced cartilage peptide powder has good stability.

[0108] In addition to the above-mentioned embodiments, the present invention can also have other implementation manners. Any technical solutions formed by equivalent replacement or equivalent transformation fall 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; The pretreatment includes a degreasing step, wherein the degreasing step uses a 70% ethanol solution as a degreasing agent, the degreasing temperature is 35±5°C, the degreasing time is 1.5 hours, the degreasing equipment is an ultrasonic assisted degreasing device, the ultrasonic frequency is 40kHz, and the power is 200W; 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.

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

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 drying is carried out by spray drying or freeze drying.