A sturgeon cartilage bioactive peptide that promotes height growth and improves bone development, its preparation method and application

Abstract

This invention discloses an active peptide from sturgeon cartilage that promotes height growth and improves bone development, along with its preparation method and applications, belonging to the field of active peptide technology. Using sturgeon cartilage as raw material, this invention selects the polypeptide with the highest activity in promoting bone growth and development from different enzymatic hydrolysis combinations through MC3T3-E1 cell experiments, and verifies its positive effect using zebrafish experiments. Subsequently, two potential active peptide sequences from sturgeon cartilage with bone growth and development promotion functions are screened using mass spectrometry to identify their sequences and bioinformatics techniques such as molecular docking. The amino acid sequences of the active peptide segments are shown in SEQ ID NO.2 and SEQ ID NO.5. The sturgeon cartilage peptides prepared by this invention have activity in promoting bone growth and development, which can solve the problem of high-value utilization of sturgeon and can also be used as a functional factor in functional products, showing good application prospects.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a sturgeon cartilage bioactive peptide that promotes growth and improves bone development, as well as its preparation method and application. Background Technology

[0002] Sturgeon is an ancient and precious fish species with significant economic and ecological value. With the development of sturgeon farming, a large amount of sturgeon by-products have been generated, such as skin, bones, fins, and cartilage. Among these, cartilage contains abundant active ingredients such as collagen, chondroitin sulfate, and hyaluronic acid, possessing high research and development value. In recent years, extracting bioactive peptides with specific physiological functions from sturgeon cartilage has become a research hotspot. Compared to proteins, bioactive peptides are more easily absorbed by the gastrointestinal tract, thus exerting their biological activity in the human body and exhibiting higher bioavailability. They have potential application value in promoting bone growth and development.

[0003] Existing technologies for extracting bioactive peptides from sturgeon cartilage often suffer from low extraction efficiency and unclear active substances. In promoting bone growth and development, current technologies mainly focus on chemically synthesized small-molecule drugs and plant extracts. While these methods have some effect, they suffer from significant side effects and poor stability, failing to meet the demand for safe and effective products to promote bone growth and development. In summary, current technologies lack research and development on bioactive peptides from sturgeon cartilage, failing to fully realize their potential in promoting bone growth and development. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sturgeon cartilage active peptide that promotes height growth and improves bone development, as well as its preparation method and application.

[0005] According to a first aspect of the present invention, a sturgeon cartilage active peptide that promotes height growth and improves bone development is provided, wherein the sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP.

[0006] The specific sequences for GPSVSPTPG are shown in SEQ ID NO.1, LGGYGMMRM are shown in SEQ ID NO.2, PGAFMLG are shown in SEQ ID NO.3, PVPLP are shown in SEQ ID NO.4, QFELCFGGR are shown in SEQ ID NO.5, QLGP are shown in SEQ ID NO.6, QPAPLCT are shown in SEQ ID NO.7, QPSRYPA are shown in SEQ ID NO.8, SPTPGLC are shown in SEQ ID NO.9, and VPLP are shown in SEQ ID NO.10.

[0007] Furthermore, the sturgeon cartilage active peptide contains at least one of LGGYGMMRM or QFELCFGGR.

[0008] According to a second aspect of the present invention, a method for preparing sturgeon cartilage active peptides that promote height growth and improve bone development is provided, comprising the following steps:

[0009] S1. Sturgeon cartilage is pretreated and then homogenized with water to obtain mixture A;

[0010] S2. Add trypsin to the mixture A for the first isothermal enzymatic hydrolysis to obtain mixture B;

[0011] S3. Neutral protease and flavor protease are added to the mixture B for a second isothermal enzymatic hydrolysis, and the supernatant is obtained after cooling.

[0012] S4. Add activated carbon to the supernatant, filter through diatomaceous earth and spray dry to obtain sturgeon cartilage active peptides;

[0013] The prepared sturgeon cartilage active peptides contain at least one of GPSVSPTPG, LGGYGMMRM, PGAAMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC, or VPLP;

[0014] The specific sequences for GPSVSPTPG are shown in SEQ ID NO.1, LGGYGMMRM are shown in SEQ ID NO.2, PGAFMLG are shown in SEQ ID NO.3, PVPLP are shown in SEQ ID NO.4, QFELCFGGR are shown in SEQ ID NO.5, QLGP are shown in SEQ ID NO.6, QPAPLCT are shown in SEQ ID NO.7, QPSRYPA are shown in SEQ ID NO.8, SPTPGLC are shown in SEQ ID NO.9, and VPLP are shown in SEQ ID NO.10.

[0015] Furthermore, the conditions for the first isothermal enzymatic hydrolysis are: temperature 40-60℃, pH 6.5-8.5, time 1-3 h, and finally enzyme inactivation; the conditions for the second isothermal enzymatic hydrolysis are: temperature 40-60℃, pH 6.0-8.0, time 1-3 h, after which the pH is adjusted back to 7.0, and finally enzyme inactivation.

[0016] Furthermore, the mass ratio of sturgeon cartilage to water is 1:5 to 1:9, the amount of activated carbon added is 3% to 5% of the mass of sturgeon cartilage, and the activation conditions of the activated carbon are: temperature 50-60℃, time 0.5-1.5 h.

[0017] Furthermore, the amount of trypsin added is 10,000-14,000 U / g of sturgeon cartilage, the amount of neutral protease added is 5,000-7,000 U / g of sturgeon cartilage, and the amount of flavor protease added is 5,000-7,000 U / g of sturgeon cartilage.

[0018] Furthermore, the sturgeon cartilage active peptide contains at least one of LGGYGMMRM or QFELCFGGR.

[0019] According to a third aspect of the present invention, the application of the sturgeon cartilage active peptide as described herein in the preparation of functional products that promote bone growth and development is proposed.

[0020] According to a fourth aspect of the present invention, the application of sturgeon cartilage active peptides prepared by the method described herein in the preparation of functional products for promoting bone growth and development is proposed.

[0021] The beneficial effects of this invention are:

[0022] (1) This invention uses sturgeon cartilage as raw material and obtains sturgeon cartilage active peptides with bone growth and development function through enzymatic hydrolysis, effectively improving the utilization rate of sturgeon cartilage and the extraction efficiency of peptides. Simultaneously, the peptide with the highest bone growth and development activity was selected through cell experiments, and two potential sturgeon cartilage active peptide sequences with bone growth and development activity were screened using bioinformatics techniques such as mass spectrometry sequence identification and molecular docking. Their amino acid sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 5, where SEQ ID NO. 2 is specifically LGGYGMMRM and SEQ ID NO. 5 is specifically QFELCFGGR. These two peptide sequences not only have clear bone growth and development activity but are also derived from natural resources, exhibiting high safety and stability, providing new ideas and approaches for developing novel, safe, and effective products that promote bone growth and development.

[0023] (2) The present invention can obtain the desired sturgeon cartilage active peptides by simply combining mixing, pretreatment, enzymatic hydrolysis and filtration. It has low equipment requirements, simple and easy-to-operate process, facilitates large-scale industrial production, and is conducive to promoting the development of sturgeon farming industry.

[0024] (3) This invention uses bioinformatics technologies such as online peptide database retrieval and molecular docking to perform virtual screening of targeted peptides, which not only greatly shortens the research and development cycle and improves research and development efficiency, but also helps researchers better understand the interaction mechanism between peptide sequences and targets, providing theoretical support for further optimization of peptide sequences. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention.

[0026] Figure 1 This is a diagram illustrating the effect of sturgeon cartilage peptides on the viability of MC3T3-E1 cells in a specific embodiment of the present invention.

[0027] Figure 2 This is a diagram showing the effect of sturgeon cartilage peptides on the antioxidant activity index ROS in a specific embodiment of the present invention.

[0028] Figure 3 This is a diagram showing the effect of sturgeon cartilage peptides on the antioxidant-related index SOD in a specific embodiment of the present invention;

[0029] Figure 4 This is a graph showing the effect of sturgeon cartilage peptides on the content of osteogenic marker ALP in a specific embodiment of the present invention;

[0030] Figure 5This is a graph showing the effect of sturgeon cartilage peptides on the content of osteogenic marker OCN in a specific embodiment of the present invention;

[0031] Figure 6 This is a diagram illustrating the effect of sturgeon cartilage peptides on the mRNA expression of the osteogenic-related gene ALP in a specific embodiment of the present invention.

[0032] Figure 7 This is a diagram illustrating the effect of sturgeon cartilage peptides on the mRNA expression of the osteogenic-related gene OCN in a specific embodiment of the present invention.

[0033] Figure 8 This is a diagram showing the effect of sturgeon cartilage peptides on the mRNA expression of the osteogenic-related gene RUNX2 in a specific embodiment of the present invention;

[0034] Figure 9 This is a diagram illustrating the effect of sturgeon cartilage peptides on the mRNA expression of the osteogenic-related gene ERK in a specific embodiment of the present invention.

[0035] Figure 10 This is a typical image of zebrafish body length after processing sturgeon cartilage peptide samples in a specific embodiment of the present invention.

[0036] Figure 11 This is a typical fluorescence intensity diagram of zebrafish skull after processing sturgeon cartilage peptide samples in a specific embodiment of the present invention;

[0037] Figure 12 This is a diagram showing the molecular docking results of LGGYGMMRM and EGFR in a specific embodiment of the present invention;

[0038] Figure 13 This is a diagram showing the molecular docking results of QFELCFGGR and EGFR in a specific embodiment of the present invention;

[0039] Figure 14 This is a diagram showing the molecular docking results of LGGYGMMRM and BMP-2 in a specific embodiment of the present invention;

[0040] Figure 15 The diagram shows the molecular docking results of QFELCFGGR and BMP-2 in a specific embodiment of the present invention. Detailed Implementation

[0041] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0042] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0043] The first objective of this invention is to propose a sturgeon cartilage active peptide that promotes height growth and improves bone development. The sturgeon cartilage active peptide has a clear activity in promoting bone growth and development, and it is derived from natural resources, with high safety and good stability. This provides a new idea and approach for developing novel, safe, and effective products that promote bone growth and development.

[0044] The second objective of this invention is to provide a method for preparing sturgeon cartilage active peptides that promote growth and improve bone development. The preparation method has low equipment requirements, is simple and easy to operate, is suitable for large-scale industrial production, and is conducive to promoting the development of the sturgeon farming industry.

[0045] The third objective of this invention is to propose the application of the sturgeon cartilage active peptide as described above in the preparation of functional products that promote bone growth and development. The fourth objective of this invention is to propose the application of the sturgeon cartilage active peptide prepared by the method described above in the preparation of functional products that promote bone growth and development. This helps researchers better understand the interaction mechanism between peptide sequences and targets, providing theoretical support for further optimization of peptide sequences.

[0046] To achieve the above objectives, the present invention provides a sturgeon cartilage active peptide that promotes height growth and improves bone development, wherein the sturgeon cartilage active peptide contains at least one of GPSVSPTPG, LGGYGMMRM, PGAAMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP.

[0047] The specific sequences for GPSVSPTPG are shown in SEQ ID NO.1, LGGYGMMRM are shown in SEQ ID NO.2, PGAFMLG are shown in SEQ ID NO.3, PVPLP are shown in SEQ ID NO.4, QFELCFGGR are shown in SEQ ID NO.5, QLGP are shown in SEQ ID NO.6, QPAPLCT are shown in SEQ ID NO.7, QPSRYPA are shown in SEQ ID NO.8, SPTPGLC are shown in SEQ ID NO.9, and VPLP are shown in SEQ ID NO.10.

[0048] The sturgeon cartilage active peptide preferably contains at least one of LGGYGMMRM or QFELCFGGR.

[0049] This invention also provides a method for preparing sturgeon cartilage active peptides that promote height growth and improve bone development, comprising the following steps:

[0050] S1. Pretreat the sturgeon cartilage and then homogenize it with water to obtain a mixed solution A, where the mass ratio of the sturgeon cartilage to water is 1:5 - 1:9;

[0051] S2. Add trypsin to the mixed solution A for the first constant-temperature enzymatic hydrolysis to obtain a mixed solution B. The conditions for the first constant-temperature enzymatic hydrolysis are: temperature 40 - 60°C, pH 6.5 - 8.5, time 1 - 3 h, and finally inactivate the enzyme;

[0052] S3. Add neutral protease and flavor protease to the mixed solution A for the second constant-temperature enzymatic hydrolysis, and obtain the supernatant after cooling. The conditions for the second constant-temperature enzymatic hydrolysis are: temperature 40 - 60°C, pH 6.0 - 8.0, time 1 - 3 h. After the enzymatic hydrolysis, adjust the pH back to 7.0, and finally inactivate the enzyme;

[0053] S4. Add activated carbon to the supernatant, filter it through diatomaceous earth, and spray-dry it to obtain sturgeon cartilage active peptides. The addition amount of the activated carbon is 3% - 5% of the mass of the sturgeon cartilage, and the action conditions of the activated carbon are: temperature 50 - 60°C, time 0.5 - 1.5 h.

[0054] In the following specific examples and comparative examples, the addition amount of trypsin is 10000 - 14000 U / g of the sturgeon cartilage, the addition amount of neutral protease is 5000 - 7000 U / g of the sturgeon cartilage, and the addition amount of flavor protease is 5000 - 7000 U / g of the sturgeon cartilage.

[0055] In the following specific examples, the license number for the experimental animals used in the zebrafish test analysis is: SYXK (Zhe) 2022 - 0004. The feeding management meets the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC - 2025 - 12322 - 01.

[0056] Example 1

[0057] A sturgeon cartilage active peptide with the activity of promoting bone growth and development and its preparation method, including the following steps:

[0058] Step 1: Wash and drain the sturgeon cartilage (protein content 19.23%), cut it into small pieces, and homogenize it with water according to the material-liquid ratio of 1:7 (g:mL) to obtain a mixed solution.

[0059] Step 2: Pre-treat the mixture at 50°C with constant stirring for 30 min. Adjust the pH of the mixture to 7.5 with 1 M sodium hydroxide solution. After the pH stabilizes, add 12000 U / g of the sturgeon cartilage and enzymatically hydrolyze at 50°C for 2.0 h. During the enzymatic hydrolysis, continuously add 1 M sodium hydroxide solution to stabilize the pH of the solution at 7.5. Then, inactivate the enzyme at high temperature.

[0060] Step 3: Adjust the temperature of the mixture to 50℃, adjust the pH of the mixture to 7.0 with 1M hydrochloric acid solution, and after stabilization, add 6000 U / g of neutral protease and 6000 U / g of flavor protease of sturgeon cartilage. Enzymatically hydrolyze at 50℃ for 2.0 h. During the enzymatic hydrolysis, continuously add 1M sodium hydroxide solution to stabilize the pH of the solution at 7.0. Then, inactivate the enzyme at high temperature, cool, and centrifuge to collect the supernatant.

[0061] Step 4: After the enzymatic hydrolysis is completed, inactivate the enzyme at 85℃ for 20 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0062] Step 5: Add activated carbon to the supernatant at 4% of the weight of sturgeon cartilage, keep at a constant temperature of 55°C for 1.0 h, pass through diatomaceous earth, and then spray dry to obtain powdered sturgeon cartilage peptides.

[0063] Example 2

[0064] Step 1: Wash and drain sturgeon cartilage (protein content 19.23%), cut into small pieces, add water at a material-to-liquid ratio of 1:5 (g:mL) and homogenize to obtain a mixture.

[0065] Step 2: Pre-treat the mixture at 40℃ with constant stirring for 30 min. Adjust the pH of the mixture to 6.5 with 1 M sodium hydroxide solution. After the pH stabilizes, add 10000 U / g of the sturgeon cartilage and enzymatically hydrolyze at 40℃ for 1.0 h. During the enzymatic hydrolysis, continuously add 1 M sodium hydroxide solution to stabilize the pH of the solution at 6.5. Then, inactivate the enzyme at high temperature.

[0066] Step 3: Adjust the temperature of the mixture to 40℃, adjust the pH of the mixture to 6.0 with 1M hydrochloric acid solution, and after stabilization, add 5000 U / g of neutral protease and 5000 U / g of flavor protease of sturgeon cartilage. Enzymatically hydrolyze at 40℃ for 1.0 h. During the enzymatic hydrolysis, continuously add 1M sodium hydroxide solution to stabilize the pH of the solution at 6.0. Then, inactivate the enzyme at high temperature, cool, and centrifuge to collect the supernatant.

[0067] Step 4: After the enzymatic hydrolysis is completed, inactivate the enzyme at 80℃ for 10 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0068] Step 5: Add activated carbon to the supernatant at 4% of the weight of sturgeon cartilage, keep it at 50°C for 0.5 h, then pass it through diatomaceous earth and spray dry to obtain powdered sturgeon cartilage peptides.

[0069] Example 3

[0070] Step 1: Wash and drain sturgeon cartilage (protein content 19.23%), cut into small pieces, add water at a material-to-liquid ratio of 1:9 (g:mL) and homogenize to obtain a mixture.

[0071] Step 2: Pre-treat the mixture at 60℃ with constant stirring for 30 min. Adjust the pH of the mixture to 8.5 with 1 M sodium hydroxide solution. After the pH stabilizes, add 14000 U / g of the sturgeon cartilage and enzymatically hydrolyze at 60℃ for 3.0 h. During the enzymatic hydrolysis, continuously add 1 M sodium hydroxide solution to stabilize the pH of the solution at 8.5. Then, inactivate the enzyme at high temperature.

[0072] Step 3: Adjust the temperature of the mixture to 60℃, adjust the pH of the mixture to 8.0 with 1M hydrochloric acid solution, and after stabilization, add 7000 U / g of neutral protease and 7000 U / g of flavor protease of sturgeon cartilage. Enzymatically hydrolyze at 60℃ for 3.0 h. During the enzymatic hydrolysis, continuously add 1M sodium hydroxide solution to stabilize the pH of the solution at 8.0. Then, inactivate the enzyme at high temperature, cool, and centrifuge to collect the supernatant.

[0073] Step 4: After the enzymatic hydrolysis is completed, inactivate the enzyme at 90℃ for 30 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0074] Step 5: Add activated carbon to the supernatant at 5% of the weight of sturgeon cartilage, keep at a constant temperature of 60°C for 1.5 h, pass through diatomaceous earth, and then spray dry to obtain powdered sturgeon cartilage peptides.

[0075] Comparative Example 1

[0076] This comparative example involves a two-step hydrolysis process, employing alkaline protease, neutral protease, and flavor protease in a stepwise manner. The difference between this comparative example and Example 1 is that the stirring temperature in step two of this comparative example is 55°C, the pH of the mixture is adjusted to 8.5 using 1 M sodium hydroxide solution, the enzyme used is alkaline protease, and the hydrolysis conditions are: hydrolysis temperature 55°C, hydrolysis pH 8.5, and 1 M sodium hydroxide solution is continuously added dropwise during the hydrolysis process to stabilize the solution pH at 8.5. The remaining operations and processes are the same as in Example 1.

[0077] Comparative Example 2

[0078] This comparative example involves a two-step hydrolysis process, in which alkaline protease and neutral protease are used for stepwise enzymatic hydrolysis. The difference between this comparative example and Comparative Example 1 is that only neutral protease is used for enzymatic hydrolysis in step three of this comparative example, and the amount of neutral protease added is modified to 12000 U / g of the sturgeon cartilage. The remaining operations and processes are the same as those in Comparative Example 1.

[0079] Comparative Example 3

[0080] This comparative example involves a two-step hydrolysis process, using both acidic and neutral proteases. The difference between this comparative example and Comparative Example 2 is that the stirring temperature in step two of this comparative example is 37°C, the enzyme used is acidic protease, and the hydrolysis conditions are changed to: hydrolysis temperature 37°C, hydrolysis pH 2.5, and 1 M hydrochloric acid solution is continuously added dropwise during the hydrolysis process to stabilize the solution pH at 2.5. The remaining operations and processes are the same as in Comparative Example 2.

[0081] Comparative Example 4

[0082] This comparative example is a one-step hydrolysis process using alkaline protease. The difference between this comparative example and Example 1 is that step three in Example 1 is removed, and alkaline protease is used in step two. The hydrolysis conditions are modified as follows: hydrolysis temperature 55℃, hydrolysis pH 8.5, enzyme dosage 24000 U / g of the sturgeon cartilage, and hydrolysis time 4.0 h. The remaining operations and processes are the same as in Example 1.

[0083] Comparative Example 5

[0084] This comparative example is a one-step hydrolysis process using neutral protease. The difference between this comparative example and Example 1 is that step three in Example 1 is removed, and neutral protease is used in step two. The hydrolysis conditions are modified as follows: hydrolysis temperature 50℃, hydrolysis pH 7.0, enzyme dosage 24000 U / g of the sturgeon cartilage, and hydrolysis time 4.0 h. The remaining operations and processes are the same as in Example 1.

[0085] Comparative Example 6

[0086] This comparative example is a one-step hydrolysis process using acidic protease. The difference between this comparative example and Example 1 is that step three in Example 1 is removed, step two uses acidic protease, and the hydrolysis conditions are modified as follows: hydrolysis temperature 37℃, hydrolysis pH 2.5, enzyme dosage 24000 U / g of the sturgeon cartilage, hydrolysis time 4.0 h, and 1 M hydrochloric acid solution is continuously added during the hydrolysis process to stabilize the solution pH at 2.5. The remaining operations and processes are the same as in Example 1.

[0087] Example 4

[0088] This embodiment analyzes the MC3T3-E1 cell viability of sturgeon cartilage peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6.

[0089] The samples from Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6 were prepared into sturgeon cartilage active peptide solutions at different concentrations (0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL) and treated with these solutions for 24 h. Cell viability was measured using CCK8 assay. It was found that different concentrations of sturgeon cartilage active peptide solutions promoted cell viability to varying degrees, without significant inhibitory or toxic effects. At a drug concentration of 0.4 mg / mL, all different sample solutions significantly affected cell viability; therefore, a 0.4 mg / mL peptide solution was selected for subsequent experimental studies.

[0090] Healthy MC3T3-E1 cells were seeded into 96-well plates at a density of 5000 cells / well and cultured at 37°C with 5% CO2 for 24 h before being divided into groups. Both the model group and the experimental group were treated with 0.2 mmol / L H2O2 for 24 h to induce oxidative stress in the cells. Before adding H2O2, the original culture medium was aspirated, the cells were washed 1-2 times with PBS, and the H2O2 was prepared to the required concentration using serum-free medium. 100 μL of complete culture medium containing 0.2 mmol / L H2O2 was added to each well. The specific groupings are as follows:

[0091] Normal control group (MC3T3-E1+ complete culture medium without H2O2);

[0092] The blank control group (cell-free, with only complete culture medium added) is not shown in the figure.

[0093] Model group (MC3T3-E1+ culture medium);

[0094] Experimental group (MC3T3-E1 + different groups of peptides + culture medium).

[0095] The experimental group was incubated with 0.4 mg / mL sturgeon cartilage active peptide solution for 24 h as required, and cell viability was then measured using the CCK8 assay. The results showed that sturgeon cartilage active peptide promoted the proliferation of H2O2-treated MC3T3-E1 cells and significantly improved osteoblast activity.

[0096] Depend on Figure 1As can be seen, the cell viability of the model group was significantly reduced compared with the normal group (P < 0.001), indicating that the intervention of H2O2 on MC3T3-E1 cells was successful. Compared with the model group, the cell viability of Examples 1, 2, and 3 were significantly increased (P < 0.001), indicating that the peptide can effectively promote osteogenic differentiation of MC3T3-E1 cells. In addition, no significant change was observed in the cell viability of Comparative Example 5 compared with the model group (P < 0.05). This may be because different enzymatic hydrolysis processes resulted in inconsistencies in the enzyme cleavage sites during hydrolysis, leading to different peptide compositions and molecular structures, thus resulting in inconsistent effects on osteoblast activity.

[0097] Example 5

[0098] This embodiment analyzes the antioxidant activity of sturgeon cartilage peptides prepared in MC3T3-E1 cells according to Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6.

[0099] MC3T3-E1 cells with good growth were divided into groups of 1×10 5 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 h to allow cell adhesion. After cell adhesion, the culture medium was discarded, and 0.2 mmol / L H2O2 and 0.4 mg / mL peptide solution were added and incubated for another 24 h. ROS and SOD were measured according to the kit instructions, and the results are shown below. Figure 2 and Figure 3 As shown.

[0100] Depend on Figure 2 and Figure 3 It can be seen that sturgeon cartilage bioactive peptides can improve oxidative stress in MC3T3-E1 cells to some extent. Compared with the model group, the sturgeon cartilage bioactive peptides in the examples did not significantly reduce the ROS content of the cells, but the SOD content of the cells significantly increased (P < 0.05). The ROS content of comparative examples 5 and 6 was significantly reduced, and their SOD content was also significantly increased, just like the model group. However, the promotion of cell viability by these examples was not as high as that of the examples. Therefore, the optimal enzymatic hydrolysis process needs to be comprehensively considered based on the results of subsequent experiments.

[0101] Example 6

[0102] This embodiment analyzes the osteogenic markers of sturgeon cartilage peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6 above for MC3T3-E1 cells.

[0103] MC3T3-E1 cells with good growth were divided into groups of 1×105 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h to allow for cell adhesion. After cell adhesion, the culture medium was discarded, and 0.2 mmol / L H2O2 and 0.4 mg / mL peptide solution were added and incubated for another 24 h. Alkaline phosphatase (ALP) and osteocalcin (OCN) levels were measured according to the kit instructions. The results are shown below. Figure 4 and Figure 5 As shown.

[0104] ALP, secreted by osteoblasts, is a direct marker of bone formation. Increased ALP secretion directly indicates osteoblast proliferation or differentiation; children, due to their active bone growth, typically have higher ALP levels. OCN, synthesized by mature osteoblasts and osteocytes, is the most abundant non-collagenous protein in the bone matrix. OCN is associated with bone turnover, maintaining a normal bone mineralization rate, inhibiting cartilage mineralization, and suppressing abnormal hydroxyapatite crystal formation. ALP and OCN complement each other for a comprehensive assessment of bone formation: ALP reflects osteoblast number / early function, while OCN reflects mature osteoblast activity. Elevated levels of both during drug use indicate drug effectiveness; elevated OCN without elevated ALP may suggest an imbalance in bone turnover (e.g., a hypercatabolic state).

[0105] like Figure 4 and Figure 5 As shown, the model group cells had lower ALP and OCN levels compared to the normal group, indicating successful modeling. Compared to other experimental groups, the peptide samples of Example 1, Comparative Example 2, and Comparative Example 6 had a more significant positive effect on the ALP content of oxidative stress cells (P < 0.05); the peptide samples of Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 5 significantly increased the OCN content of oxidative stress cells (P < 0.05). A comprehensive analysis of the effects of peptides prepared by different enzymatic digestion processes on the viability, antioxidant capacity, and osteogenic markers of oxidative stress cells showed that the peptide samples of Example 1 had a more significant positive effect on osteogenic differentiation of MC3T3-E1 cells compared to other experimental groups, with Example 1 exhibiting the best effect.

[0106] Example 7

[0107] This embodiment analyzes osteogenic-related genes in MC3T3-E1 cells based on the sturgeon cartilage peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6.

[0108] Many genes are involved in the osteogenic differentiation of MC3T3-E1 cells, such as runt-related transcription factor 2 (Runx2), ALP, OCN, and EPK. To further verify the effects of peptides prepared by different enzymatic digestion processes on the osteogenic differentiation of MC3T3-E1 cells, the expression of osteogenic-related genes was analyzed. Detection of these gene expression using techniques such as Western blotting and polymerase chain reaction (PCR) can often serve as corroborating evidence to determine whether cells have formed osteogenic structures.

[0109] After coating with 0.1% gelatin and drying, the well-growing MC3T3-E1 cells were seeded into 12-well plates at a cell density of 5 × 10⁶ cells / well. 4 pcs / cm 2 After cell adhesion, the culture medium was discarded, and the cells were treated according to the experimental groups for 24 h. After the drug administration, differentiation was induced for 48 h. Total RNA was extracted from each group of cells, and the concentration and purity of RNA were measured. Using the extracted RNA as a template, RNA was reverse transcribed into cDNA. The obtained cDNA was stored at -20℃. The original solution was diluted 5 times for subsequent steps in the qPCR reaction. Real-time quantitative PCR was performed. The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃, 10 s, 60℃, 30 s, for a total of 40 cycles; melting curve: 95℃, 15 s, 60℃, 60 s, 95℃, 15 s. The Ct cycle threshold was obtained according to the two-step amplification program, and the relative expression of RNA in different groups was determined. Glycerol-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

[0110] The results are as follows Figure 6 , Figure 7 , Figure 8 and Figure 9 As shown, the expression of bone-related genes in each experimental group was upregulated to varying degrees compared with the model group, indicating that the addition of sturgeon cartilage bioactive peptides has a positive effect on osteogenic differentiation of MC3T3-E1 cells. Compared with other experimental groups, the gene expression levels in the examples did not show a significant advantage, but this does not mean that the positive effect of the examples on osteogenic differentiation of MC3T3-E1 cells was lower than that of other experimental groups.

[0111] Example 8

[0112] Therefore, this embodiment uses zebrafish experiments to analyze the sturgeon cartilage peptides prepared in Example 1 above, to further verify their positive effects on bone growth and development.

[0113] In this embodiment, the expression levels of the bmp2a, sp7, and gh1 genes in zebrafish were measured because they represent three key steps in the process of bone formation: bmp2a is a key signaling molecule that initiates bone development, sp7 is a core transcription factor that controls osteoblast differentiation, and gh1 is an important hormone that regulates linear bone growth.

[0114] Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). The samples were administered via water-soluble solution. A normal control group, a positive control group (Swisse "Calcium & Vitamin D" 500 μg / mL), a low-dose sturgeon chondroitin group (250 μg / mL), and a high-dose sturgeon chondroitin group (500 μg / mL) were established. After treatment at 28℃ for 3 days, samples from each group were collected. The body length, skull fluorescence intensity, and expression of osteogenic-related genes in the zebrafish were measured and analyzed. Statistical analysis of these indicators was used to evaluate the efficacy of the samples in promoting the growth and development of zebrafish. The results are shown in Tables 1, 2, and 3. Figure 10 and Figure 11 As shown in the figures, compared with the normal control group, the positive control group and the peptide sample group significantly promoted the body length growth of zebrafish, increased the fluorescence intensity of their skulls, and upregulated the expression of zebrafish osteogenic genes. This indicates that the addition of Swisse "Calcium & Vitamin D" and sturgeon cartilage active peptides has a positive effect on osteogenic differentiation in zebrafish, with the positive control group showing better results. Furthermore, the high-dose group of sturgeon cartilage peptides showed a better effect on promoting bone growth and development than the low-dose group.

[0115] Table 1 Results of sturgeon cartilage peptides promoting zebrafish body length growth (n = 10)

[0116] ;

[0117] Compared with the normal control group,

[0118] Table 2 Results of sturgeon chondroitin peptide promoting skeletal development in zebrafish (n = 10)

[0119] ;

[0120] Compared with the normal control group,

[0121] Table 3 Results of sturgeon cartilage peptide promoting the expression of osteogenic-related genes in zebrafish (n = 3)

[0122] ;

[0123] Compared with the normal control group, ,

[0124] Example 9

[0125] In this embodiment, mass spectrometry was used to identify peptide sequences, and PeptideRanker, ToxinPred, and BioPepDB were used to further screen for sturgeon cartilage bioactive peptides that can promote height growth and improve bone development. The specific experimental steps are as follows:

[0126] The liquid chromatography conditions are as follows:

[0127] Pre-column: PEPMAP NEO C18, 300 μm × 5 mm.

[0128] Analytical column: 150 μm id × 170 mm, packaging: Reprosil-Pur 120 C18-AQ 1.9 μm.

[0129] Mobile phase A is 0.1% FA; mobile phase B is 0.1% FA, 80% ACN; flow rate is 600 nL / min;

[0130] The analysis time for each component is 66 min.

[0131] The mass spectrometry conditions were as follows: full scan range 100-1500 m / z, first-stage mass spectrometry resolution set to 120000, AGC set to Standard, Maximum IT: 20 ms; second-stage mass spectrometry resolution set to Resolution: 15000, AGC set to Standard, Maximum IT: 22 ms, Cycle time: 2 s, peptide fragmentation collision energy set to 30, generating raw mass spectrometry data (.raw), ultimately obtaining more than 5000 peptide fragments.

[0132] Bioactivity scores were performed on 89 peptides with abundances higher than 115,000,000 identified by mass spectrometry. A score > 0.5 indicates reliable results, and a higher score indicates higher potential bioactivity. Toxicity prediction and innovation assessment were conducted, and the 10 peptides with the highest abundance were selected for further analysis, as shown in Table 4.

[0133] Table 4. Screening results of sturgeon cartilage peptides

[0134]

[0135] Example 10

[0136] Based on binding energy and peptide abundance, two sequences, QFELCFGGR and LGGYGMMRM, were selected from the peptides in Table 4 as characteristic peptide sequences in sturgeon cartilage peptides that promote bone growth and development. These two peptides were molecularly docked with receptor proteins using molecular docking software. The selected receptor proteins were EGFR (PDB: 1IVO) and BMP-2 (PDB: 3BMP). The results showed that both selected peptides could bind to the receptor proteins to a certain extent.

[0137] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that regulates cell proliferation and differentiation. Its signaling pathway is one of the main regulatory pathways of bone metabolism. EGFR can be activated by the EGF-related peptide growth factor family and various ligands, thereby promoting bone formation. In molecular docking, the EGF ligand structure was used as the docking site. After deleting the EGF structure, molecular docking was initiated to verify the binding affinity between sturgeon cartilage active peptides and the EGFR receptor. The results showed that both screened peptides could bind to the receptor protein EGFR to a certain extent. Figure 12 As shown, the main interactions between LGGYGMMRM and EGFR are van der Waals forces, C-H bonds, conventional hydrogen bonds, and alkylation. The maximum "-CDOCKERENERGY" value for LGGYGMMRM docking with EGFR is 98.3478 kcal / mol. Figure 13 As shown, QFELCFGGR and EGFR interact mainly through van der Waals forces, C-H bonds, alkylation, and conventional C-H bonds. The maximum "-CDOCKERENERGY" value when QFELCFGGR docks with EGFR is 115.803 kcal / mol.

[0138] Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor β (TGF-β) superfamily and can induce bone formation and regeneration. Studies have shown that bioactive peptides derived from fish bones can interact with BMP receptors, promoting osteoblast differentiation. Figure 14 As shown, LGGYGMMRM and BMP-2 mainly interact through van der Waals forces, C-H bonds, salt bridges, and conventional hydrogen bonds. The maximum "-CDOCKER ENERGY" value of LGGYGMMRM when docked with BMP-2 is 113.105 kcal / mol. Figure 15 As shown, QFELCFGGR and BMP-2 interact mainly through van der Waals forces, C-H bonds, attractive charges, conventional hydrogen bonds, and π-alkylation. The maximum "-CDOCKER ENERGY" value of QFELCFGGR when docked with BMP-2 is 131.779 kcal / mol.

[0139] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing sturgeon cartilage active peptides that promote height growth and improve bone development, characterized in that, Includes the following steps: S1. Sturgeon cartilage is pretreated and then homogenized with water to obtain mixture A; S2. Add trypsin to the mixture A for the first isothermal enzymatic hydrolysis to obtain mixture B; S3. Neutral protease and flavor protease are added to the mixture B for a second isothermal enzymatic hydrolysis, and the supernatant is obtained after cooling. S4. Add activated carbon to the supernatant, filter through diatomaceous earth and spray dry to obtain sturgeon cartilage active peptides; The conditions for the first isothermal enzymatic hydrolysis are: temperature 40-60℃, pH 6.5-8.5, time 1-3 h, and finally enzyme inactivation; the conditions for the second isothermal enzymatic hydrolysis are: temperature 40-60℃, pH 6.0-8.0, time 1-3 h, after which the pH is adjusted back to 7.0, and finally enzyme inactivation. The mass ratio of sturgeon cartilage to water is 1:5-1:9, the amount of activated carbon added is 3%-5% of the mass of sturgeon cartilage, and the activation conditions of the activated carbon are: temperature 50-60℃, time 0.5-1.5 h. The amount of trypsin added is 10,000-14,000 U / g of sturgeon cartilage, the amount of neutral protease added is 5,000-7,000 U / g of sturgeon cartilage, and the amount of flavor protease added is 5,000-7,000 U / g of sturgeon cartilage.

2. The preparation method according to claim 1, characterized in that, The sturgeon cartilage active peptides include at least one of GPSVSPTPG, LGGYGMMRM, PGAAMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC, or VPLP; The specific sequences for GPSVSPTPG are shown in SEQ ID NO.1, LGGYGMMRM are shown in SEQ ID NO.2, PGAFMLG are shown in SEQ ID NO.3, PVPLP are shown in SEQ ID NO.4, QFELCFGGR are shown in SEQ ID NO.5, QLGP are shown in SEQ ID NO.6, QPAPLCT are shown in SEQ ID NO.7, QPSRYPA are shown in SEQ ID NO.8, SPTPGLC are shown in SEQ ID NO.9, and VPLP are shown in SEQ ID NO.

10.

3. The application of a sturgeon cartilage active peptide prepared by the method described in claim 1 in the preparation of functional products for promoting bone growth and development.

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