Sturgeon cartilage active peptide with functions of promoting growth and improving bone development as well as preparation method and application of sturgeon cartilage active peptide

Abstract

The invention discloses sturgeon cartilage active peptide capable of promoting growth and improving bone development as well as a preparation method and application of the sturgeon cartilage active peptide, and belongs to the technical field of active peptide. According to the invention, sturgeon cartilage is taken as a raw material, polypeptide with the highest activity of promoting bone growth and development is selected from different enzymolysis combinations through MC3T3-E1 cell tests, and the positive effect of the polypeptide is verified through zebra fish tests; then, two potential sturgeon cartilage active peptide sequences with the function of promoting bone growth and development are screened out through bioinformatics technologies such as mass spectrum identification, molecular docking and the like, and the amino acid sequences of the sturgeon cartilage active peptide fragments are shown as SEQ ID NO.2 and SEQ ID NO.5. The sturgeon cartilage peptide prepared by the method disclosed by the invention has the activity of promoting bone growth and development, can solve the problem of high-value utilization of sturgeons, can also be applied to functional products as a functional factor, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active peptides, in particular to a sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development, and a preparation method and application thereof. BACKGROUND

[0002] Sturgeon is an ancient and precious fish with important economic and ecological value. With the development of sturgeon aquaculture industry, a large amount of sturgeon by-products such as fish skin, fish bone, fish fin and fish cartilage have been produced. Among them, fish cartilage contains rich active ingredients such as collagen, chondroitin sulfate and hyaluronic acid, and has high research and development value. In recent years, extracting active peptides with specific physiological functions from sturgeon cartilage has become a research hotspot. Compared with proteins, active peptides are more easily absorbed by the gastrointestinal tract and thus exert their biological activity in the human body, and have higher bioavailability. They have potential application value in promoting bone growth and development.

[0003] In the existing technology, the method for extracting active peptides from sturgeon cartilage often has problems such as low extraction efficiency and unclear active substances. In the aspect of promoting bone growth and development, the existing technology mainly focuses on chemical synthesis of small molecule drugs and plant extracts. Although these methods have certain effects, they have problems such as large side effects and poor stability, and cannot meet the needs of people for safe and effective products for promoting bone growth and development. In summary, the existing technology lacks research and development of sturgeon cartilage active peptides, and fails to fully exert their potential in promoting bone growth and development. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provide a sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development, and a preparation method and application thereof.

[0005] According to a first aspect of the present application, a sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development is provided, wherein the sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAFMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP. GPSVSPTPG has the specific sequence as shown in SEQ ID NO. 1, LGGYGMMRM has the specific sequence as shown in SEQ ID NO. 2, PGAAFMLG has the specific sequence as shown in SEQ ID NO. 3, PVPLP has the specific sequence as shown in SEQ ID NO. 4, QFELCFGGR has the specific sequence as shown in SEQ ID NO. 5, QLGP has the specific sequence as shown in SEQ ID NO. 6, QPAPLCT has the specific sequence as shown in SEQ ID NO. 7, QPSRYPA has the specific sequence as shown in SEQ ID NO. 8, SPTPGLC has the specific sequence as shown in SEQ ID NO. 9, and VPLP has the specific sequence as shown in SEQ ID NO. 10.

[0006] Further, the sturgeon cartilage active peptide comprises at least one of LGGYGMMRM or QFELCFGGR.

[0007] According to a second aspect of the present application, a preparation method of a sturgeon cartilage active peptide for promoting height growth and improving bone development is provided, comprising the following steps: S1, pretreating sturgeon cartilage and then homogenizing the pretreated sturgeon cartilage with water to obtain a mixed solution A; S2, adding trypsin to the mixed solution A to perform first constant-temperature enzymolysis, and obtaining a mixed solution B; S3, adding neutral protease and flavor protease to the mixed solution B to perform second constant-temperature enzymolysis, and obtaining a supernatant after cooling; S4, adding activated carbon to the supernatant, filtering through diatomite, and performing spray drying to obtain a sturgeon cartilage active peptide; The prepared sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAFMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP; GPSVSPTPG has the specific sequence as shown in SEQ ID NO. 1, LGGYGMMRM has the specific sequence as shown in SEQ ID NO. 2, PGAAFMLG has the specific sequence as shown in SEQ ID NO. 3, PVPLP has the specific sequence as shown in SEQ ID NO. 4, QFELCFGGR has the specific sequence as shown in SEQ ID NO. 5, QLGP has the specific sequence as shown in SEQ ID NO. 6, QPAPLCT has the specific sequence as shown in SEQ ID NO. 7, QPSRYPA has the specific sequence as shown in SEQ ID NO. 8, SPTPGLC has the specific sequence as shown in SEQ ID NO. 9, and VPLP has the specific sequence as shown in SEQ ID NO. 10.

[0008] Further, the first constant temperature enzymolysis condition is: temperature 40-60℃, pH 6.5-8.5, time 1-3 h, and finally enzyme is inactivated; the second constant temperature enzymolysis condition is: temperature 40-60℃, pH 6.0-8.0, time 1-3 h, and after the enzymolysis, the pH is adjusted to 7.0, and finally enzyme is inactivated.

[0009] Further, the mass ratio of the sturgeon cartilage to water is 1:5-1:9, the added amount of the activated carbon is 3%-5% of the mass of the sturgeon cartilage, and the action condition of the activated carbon is: temperature 50-60℃, time 0.5-1.5 h.

[0010] Further, the added amount of the trypsin is 10000-14000 U / g of the sturgeon cartilage, the added amount of the neutral protease is 5000-7000 U / g of the sturgeon cartilage, and the added amount of the flavourzyme is 5000-7000 U / g of the sturgeon cartilage.

[0011] Further, the sturgeon cartilage active peptide comprises at least one of LGGYGMMRM or QFELCFGGR.

[0012] According to a third aspect of the present application, the sturgeon cartilage active peptide as described is applied to the preparation of a functional product for promoting the growth and development of the skeleton.

[0013] According to a fourth aspect of the present application, the sturgeon cartilage active peptide prepared by the method as described is applied to the preparation of a functional product for promoting the growth and development of the skeleton.

[0014] The present application has the following beneficial effects: (1) The present application uses sturgeon cartilage as raw material, and through enzymolysis, a sturgeon cartilage active peptide with the function of promoting the growth and development of the skeleton is prepared, thereby effectively improving the utilization rate of the sturgeon cartilage and the extraction efficiency of the peptide. Meanwhile, through cell test, the peptide with the highest activity of promoting the growth and development of the skeleton is selected, and combining with mass spectrum identification, molecular docking and other bioinformatics technologies, two potential sturgeon cartilage active peptide sequences with the function of promoting the growth and development of the skeleton are screened out, the amino acid sequences of which are shown as SEQ ID NO. 2 and SEQ ID NO. 5, the SEQ ID NO. 2 is specifically LGGYGMMRM, and the SEQ ID NO. 5 is specifically QFELCFGGR. The two peptide sequences not only have the definite activity of promoting the growth and development of the skeleton, but also are derived from natural resources, have high safety and good stability, thereby providing a new idea and way for developing a new type, safe and effective product for promoting the growth and development of the skeleton.

[0015] (2) The scheme of the present application adopts simple operations such as mixing, pretreatment, enzymolysis and filtration to prepare the required sturgeon cartilage active peptide, has low requirement on equipment, simple process and easy operation, is convenient for large-scale industrial production, and is conducive to promoting the development of sturgeon breeding industry.

[0016] (3) The present application performs virtual screening of the directional binding polypeptide through bioinformatics technologies such as online polypeptide database retrieval and molecular docking, which not only greatly shortens the research and development period and improves the research and development efficiency, but also helps researchers to better understand the interaction mechanism of the peptide sequence and the target, and provides theoretical support for further optimization of the peptide sequence. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are included to provide a further understanding of the present application and are incorporated in and constitute a part of this application, illustrate embodiments of the present application and serve to explain the present application, and do not constitute improper limitations on the present application.

[0018] Figure 1 Figure for the influence of sturgeon cartilage peptide on the activity of MC3T3-E1 cells in the embodiment of the present application; Figure 2 Figure for the influence of sturgeon cartilage peptide on the ROS related index of antioxidant in the embodiment of the present application; Figure 3 Figure for the influence of sturgeon cartilage peptide on the SOD related index of antioxidant in the embodiment of the present application; Figure 4 Figure for the influence of sturgeon cartilage peptide on the ALP content of osteogenesis marker in the embodiment of the present application; Figure 5 Figure for the influence of sturgeon cartilage peptide on the OCN content of osteogenesis marker in the embodiment of the present application; Figure 6 Figure for the influence of sturgeon cartilage peptide on the mRNA expression of ALP of osteogenesis related gene in the embodiment of the present application; Figure 7 Figure for the influence of sturgeon cartilage peptide on the mRNA expression of OCN of osteogenesis related gene in the embodiment of the present application; Figure 8 Figure for the influence of sturgeon cartilage peptide on the mRNA expression of RUNX2 of osteogenesis related gene in the embodiment of the present application; Figure 9 Figure for the influence of sturgeon cartilage peptide on the mRNA expression of ERK of osteogenesis related gene in the embodiment of the present application; Figure 10 Typical figure of the body length of zebra fish after treatment of sturgeon cartilage peptide sample in the embodiment of the present application; Figure 11 Typical figure of the skull fluorescence intensity of zebra fish after treatment of sturgeon cartilage peptide sample in the embodiment of the present application; Figure 12 Figure is a molecular docking result diagram of LGGYGMMRM and EGFR in the embodiment of the present application; Figure 13 Figure is a molecular docking result diagram of QFELCFGGR and EGFR in the embodiment of the present application; Figure 14 Figure is a molecular docking result diagram of LGGYGMMRM and BMP-2 in the embodiment of the present application; Figure 15 Figure is a molecular docking result diagram of QFELCFGGR and BMP-2 in the embodiment of the present application. DETAILED DESCRIPTION

[0019] The present application will be further described in details below in conjunction with the accompanying drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related application, and are not a limitation on the application. In addition, it should be noted that only the parts related to the application are shown in the drawings for ease of description.

[0020] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in details below with reference to the accompanying drawings and embodiments.

[0021] The first object of the present application is to provide a sturgeon cartilage active peptide for promoting height growth and improving bone development, which has a clear activity of promoting bone growth and development, is derived from a natural resource, has high safety and good stability, and provides a new idea and approach for developing a new, safe and effective product for promoting bone growth and development.

[0022] The second object of the present application is to provide a preparation method of the sturgeon cartilage active peptide for promoting height growth and improving bone development, which has low equipment requirements, simple process and easy operation, is convenient for industrial large-scale production, and is conducive to promoting the development of sturgeon breeding industry.

[0023] The third object of the present application is to provide an application of the sturgeon cartilage active peptide as described in the preparation of a functional product for promoting bone growth and development. The fourth object of the present application is to provide an application of the sturgeon cartilage active peptide prepared by the method as described in the preparation of a functional product for promoting bone growth and development. This helps researchers better understand the interaction mechanism of peptide sequence and target, and provides theoretical support for further optimization of peptide sequence.

[0024] To achieve the above object, the application provides a sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development, wherein the sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAFMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP. The specific sequence of GPSVSPTPG is shown in SEQ ID NO. 1, the specific sequence of LGGYGMMRM is shown in SEQ ID NO. 2, the specific sequence of PGAAFMLG is shown in SEQ ID NO. 3, the specific sequence of PVPLP is shown in SEQ ID NO. 4, the specific sequence of QFELCFGGR is shown in SEQ ID NO. 5, the specific sequence of QLGP is shown in SEQ ID NO. 6, the specific sequence of QPAPLCT is shown in SEQ ID NO. 7, the specific sequence of QPSRYPA is shown in SEQ ID NO. 8, the specific sequence of SPTPGLC is shown in SEQ ID NO. 9, and the specific sequence of VPLP is shown in SEQ ID NO. 10.

[0025] Preferably, the sturgeon cartilage active peptide comprises at least one of LGGYGMMRM or QFELCFGGR.

[0026] The application further provides a preparation method of the sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development, comprising the following steps: S1, pretreating sturgeon cartilage and then homogenizing the sturgeon cartilage with water to obtain a mixed solution A, wherein the mass ratio of the sturgeon cartilage to water is 1:5-1:9; S2, adding trypsin to the mixed solution A to perform first constant-temperature enzymolysis, so as to obtain a mixed solution B, wherein the first constant-temperature enzymolysis is performed under the conditions of a temperature of 40-60 ℃, a pH of 6.5-8.5, a time of 1-3 h, and finally enzyme inactivation; S3, adding neutral protease and flavor protease to the mixed solution A to perform second constant-temperature enzymolysis, and then cooling to obtain a supernatant, wherein the second constant-temperature enzymolysis is performed under the conditions of a temperature of 40-60 ℃, a pH of 6.0-8.0, a time of 1-3 h, and finally enzyme inactivation after the pH is adjusted to 7.0; S4, adding activated carbon to the supernatant, filtering through diatomite, and spray drying to obtain the sturgeon cartilage active peptide, wherein the addition amount of the activated carbon is 3%-5% of the mass of the sturgeon cartilage, and the activated carbon is used under the conditions of a temperature of 50-60 ℃ and a time of 0.5-1.5 h.

[0027] The amount of trypsin added in the following specific examples and comparative examples is 10000-14000 U / g of the sturgeon cartilage, the amount of neutral protease added is 5000-7000 U / g of the sturgeon cartilage, and the amount of flavor protease added is 5000-7000 U / g of the sturgeon cartilage.

[0028] The experimental animal license number for zebrafish test analysis in the following specific examples is: SYXK (Zhejiang) 2022-0004, and the feeding and management conforms to the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC-2025-12322-01.

[0029] Example 1

[0030] A sturgeon cartilage active peptide with the activity of promoting bone growth and development and a preparation method thereof, comprising the following steps: Step one: wash the sturgeon cartilage (protein content 19.23%) and drain, cut into small pieces, and add water to homogenize according to the solid-liquid ratio of 1:7 (g:mL) to obtain a mixed solution.

[0031] Step two: pre-treat the mixed solution at 50°C for 30 min, adjust the pH of the mixed solution to 7.5 by 1 M sodium hydroxide solution, after the pH is stable, add 12000 U / g of the sturgeon cartilage of trypsin, and enzymolysis at 50°C for 2.0 h, constantly add 1 M sodium hydroxide solution to make the pH of the solution stable at 7.5 during the enzymolysis process, and then high-temperature enzyme inactivation.

[0032] Step three: adjust the temperature of the mixed solution to 50°C, adjust the pH of the mixed solution to 7.0 by 1 M hydrochloric acid solution, after the pH is stable, add 6000 U / g of the sturgeon cartilage of neutral protease and 6000 U / g of the sturgeon cartilage of flavor protease, and enzymolysis at 50°C for 2.0 h, constantly add 1 M sodium hydroxide solution to make the pH of the solution stable at 7.0 during the enzymolysis process, and then high-temperature enzyme inactivation, and after cooling, centrifuge to obtain the supernatant.

[0033] Step four: after the enzymolysis is completed, inactivate the enzyme at 85°C for 20 min, and after cooling, centrifuge at 5000 rpm for 5 min to obtain the supernatant.

[0034] Step five: add activated carbon at 4% of the mass of the sturgeon cartilage into the above supernatant, keep it at 55°C for 1.0 h, then pass through diatomite, and then spray dry to obtain the sturgeon cartilage peptide in powder form.

[0035] Example 2

[0036] Step one: sturgeon cartilage (protein content 19.23%) was washed and drained, cut into small pieces, and homogenized with water at a solid-liquid ratio of 1:5 (g:mL) to obtain a mixture.

[0037] Step two: the mixture was pre-treated at 40°C for 30 min, and the pH of the mixture was adjusted to 6.5 by 1 M sodium hydroxide solution. After the pH was stabilized, 10,000 U / g of the sturgeon cartilage of trypsin was added, and the mixture was enzymatically hydrolyzed at 40°C for 1.0 h. During the enzymatic hydrolysis, 1 M sodium hydroxide solution was continuously added to stabilize the pH of the solution at 6.5, and then the enzyme was inactivated at high temperature.

[0038] Step three: the temperature of the mixture was adjusted to 40°C, the pH of the mixture was adjusted to 6.0 by 1 M hydrochloric acid solution, and after stabilization, 5,000 U / g of the sturgeon cartilage of neutral protease and 5,000 U / g of the sturgeon cartilage of flavor protease were added. The mixture was enzymatically hydrolyzed at 40°C for 1.0 h. During the enzymatic hydrolysis, 1 M sodium hydroxide solution was continuously added to stabilize the pH of the solution at 6.0, and then the enzyme was inactivated at high temperature. After cooling, the supernatant was obtained by centrifugation.

[0039] Step four: after the enzymatic hydrolysis, the enzyme was inactivated at 80°C for 10 min. After cooling, the supernatant was obtained by centrifugation at 5,000 rpm for 5 min.

[0040] Step five: 4% of activated carbon based on the mass of the sturgeon cartilage was added to the above supernatant. After being kept at 50°C for 0.5 h, the mixture was filtered through diatomite, and then was spray-dried to obtain the sturgeon cartilage peptide in powder form.

[0041] Example 3

[0042] Step one: sturgeon cartilage (protein content 19.23%) was washed and drained, cut into small pieces, and homogenized with water at a solid-liquid ratio of 1:9 (g:mL) to obtain a mixture.

[0043] Step two: the mixture was pre-treated at 60°C for 30 min, and the pH of the mixture was adjusted to 8.5 by 1 M sodium hydroxide solution. After the pH was stabilized, 14,000 U / g of the sturgeon cartilage of trypsin was added, and the mixture was enzymatically hydrolyzed at 60°C for 3.0 h. During the enzymatic hydrolysis, 1 M sodium hydroxide solution was continuously added to stabilize the pH of the solution at 8.5, and then the enzyme was inactivated at high temperature.

[0044] Step three: the temperature of the mixture was adjusted to 60°C, the pH of the mixture was adjusted to 8.0 by 1 M hydrochloric acid solution, and after stabilization, 7,000 U / g of the sturgeon cartilage of neutral protease and 7,000 U / g of the sturgeon cartilage of flavor protease were added. The mixture was enzymatically hydrolyzed at 60°C for 3.0 h. During the enzymatic hydrolysis, 1 M sodium hydroxide solution was continuously added to stabilize the pH of the solution at 8.0, and then the enzyme was inactivated at high temperature. After cooling, the supernatant was obtained by centrifugation.

[0045] Step four: after the end of enzymatic hydrolysis, the enzyme was inactivated at 90°C for 30 min, and after cooling, the supernatant was obtained by centrifugation at 5000 rpm for 5 min.

[0046] Step five: activated carbon was added to the supernatant at 5% of the mass of the sturgeon cartilage, and after keeping at 60°C for 1.5 h, diatomite was passed through, and then spray drying was performed to obtain powdered sturgeon cartilage peptide.

[0047] Comparative Example 1 This comparative example is two-step hydrolysis, and alkaline protease, neutral protease and flavor protease are used for step-by-step enzymatic hydrolysis during the hydrolysis process. 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 mixed solution is adjusted to 8.5 by 1 M sodium hydroxide solution, the enzyme used is alkaline protease, the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature 55°C, enzymatic hydrolysis pH 8.5, and 1 M sodium hydroxide solution is continuously added during the enzymatic hydrolysis process to keep the solution pH at 8.5. The rest of the operation and process are the same as those of Example 1.

[0048] Comparative Example 2 This comparative example is two-step hydrolysis, and alkaline protease and neutral protease are used for step-by-step enzymatic hydrolysis during the hydrolysis process. 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 rest of the operation and process are the same as those of Comparative Example 1.

[0049] Comparative Example 3 This comparative example is two-step hydrolysis, and acid protease and neutral protease are used for enzymatic hydrolysis during the hydrolysis process. 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 acid protease, and the enzymatic hydrolysis conditions are modified to: enzymatic hydrolysis temperature 37°C, enzymatic hydrolysis pH 2.5, and 1 M hydrochloric acid solution is continuously added during the enzymatic hydrolysis process to keep the solution pH at 2.5. The rest of the operation and process are the same as those of Comparative Example 2.

[0050] Comparative Example 4 This comparative example is one-step hydrolysis, and alkaline protease is used for enzymatic hydrolysis during the hydrolysis process. 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, and the enzymatic hydrolysis conditions are modified to: enzymatic hydrolysis temperature 55°C, enzymatic hydrolysis pH 8.5, enzyme amount 24000 U / g of the sturgeon cartilage, and enzymatic hydrolysis time 4.0 h. The rest of the operation and process are the same as those of Example 1.

[0051] Comparative Example 5 The comparative example is one-step hydrolysis, and neutral protease is used in the hydrolysis process. The difference between the comparative example and example 1 is that step three in example 1 is removed, and neutral protease is used in step two. The enzyme hydrolysis conditions are modified as follows: enzyme hydrolysis temperature 50°C, enzyme hydrolysis pH 7.0, enzyme amount 24000 U / g of the sturgeon cartilage, enzyme hydrolysis time 4.0 h, and the rest of the operation and process are the same as those in example 1.

[0052] Comparative example 6 The comparative example is one-step hydrolysis, and acid protease is used in the hydrolysis process. The difference between the comparative example and example 1 is that step three in example 1 is removed, and acid protease is used in step two. The enzyme hydrolysis conditions are modified as follows: enzyme hydrolysis temperature 37°C, enzyme hydrolysis pH 2.5, enzyme amount 24000 U / g of the sturgeon cartilage, enzyme hydrolysis time 4.0 h, and 1 M hydrochloric acid solution is continuously added during the enzyme hydrolysis process to keep the solution pH at 2.5, and the rest of the operation and process are the same as those in example 1.

[0053] Example 4

[0054] In this example, the sturgeon cartilage peptide prepared in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5 and comparative example 6 is subjected to MC3T3-E1 cell viability analysis.

[0055] The samples in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5 and comparative example 6 are prepared into sturgeon cartilage active peptide solutions with 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 are used on MC3T3-E1 cells for 24 h, and CCK8 is used to determine the cell viability. It is found that the sturgeon cartilage active peptide solutions with different concentrations all promote the cell viability to different degrees, and no significant inhibition and toxic effects are observed. Among them, when the drug concentration is 0.4 mg / mL, the different sample solutions all have a significant effect on the cell viability, and therefore, the polypeptide solution with a concentration of 0.4 mg / mL is selected for subsequent test research.

[0056] The well-grown MC3T3-E1 cells were inoculated in a 96-well plate with a cell density of 5000 cells per well, and were placed in a 37°C, 5% CO2 incubator for 24 hours before grouping. The model group and the experimental group were intervened with 0.2 mmol / L H2O2 for 24 hours to cause oxidative stress in the cells. When the cells were treated with H2O2, the original culture medium was first removed, and the cells were washed with PBS for 1-2 times. The H2O2 was prepared into the required concentration with serum-free medium, and 100 μL of complete medium containing 0.2 mmol / L H2O2 was added to each well. The specific grouping is as follows: Normal control group (without H2O2, MC3T3-E1 + complete medium); Blank group (without cells, only complete medium), not shown in the figure; Model group (MC3T3-E1 + medium); Experimental group (MC3T3-E1 + different groups of polypeptides + medium).

[0057] The experimental group was incubated with 0.4 mg / mL sturgeon cartilage active peptide solution for 24 hours, and then the cell viability was determined by CCK8 method. It was found that the sturgeon cartilage active peptide could promote the proliferation of MC3T3-E1 cells intervened by H2O2, and significantly improve the activity of osteoblasts.

[0058] From Figure 1 It can be seen that compared with the normal group, the cell viability of the model group was significantly reduced (P < 0.001), indicating that the intervention of H2O2 on MC3T3-E1 cells was successful. Compared with the model group, the cell viability of the examples, comparative example 1 and comparative example 2 was significantly increased (P < 0.001), indicating that the polypeptides could effectively promote the osteogenic differentiation of MC3T3-E1 cells. In addition, compared with the cell viability of the model group, comparative example 5 did not show significant changes (P < 0.05), which may be due to the different enzyme digestion processes, resulting in inconsistent enzyme cleavage sites in the enzyme digestion process, different polypeptide compositions and molecular structures were obtained by hydrolysis, leading to inconsistent effects on the activity of osteoblasts.

[0059] Example 5

[0060] This example is directed to the MC3T3-E1 cell antioxidant analysis of the sturgeon cartilage peptides prepared in the above examples 1, 2, 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5 and comparative example 6.

[0061] The well-grown MC3T3-E1 cells were inoculated in a 96-well plate with a cell density of 5000 cells per well, and were placed in a 37°C, 5% CO2 incubator for 24 hours before grouping. The model group and the experimental group were intervened with 0.2 mmol / L H2O2 for 24 hours to cause oxidative stress in the cells. When the cells were treated with H2O2, the original culture medium was first removed, and the cells were washed with PBS for 1-2 times. The H2O2 was prepared into the required concentration with serum-free medium, and 100 μL of complete medium containing 0.2 mmol / L H2O2 was added to each well. The specific grouping is as follows: 5The MC3T3-E1 cells in good growth condition were planted in 96-well plates at a density of 1×10 5 cells per well, and were cultured in a cell incubator at 37°C and 5% CO2 for 24 h to wait for cell adhesion. After cell adhesion, the culture medium was discarded, and 0.2 mmol / L H2O2 and 0.4 mg / mL polypeptide solution were added for incubation for 24 h. According to the kit instructions, ROS and SOD were determined, and the results are shown in Figure 2 and Figure 3 .

[0062] As can be seen from Figure 2 and Figure 3 , the sturgeon cartilage active peptide can improve the oxidative stress of MC3T3-E1 cells to some extent. Among them, the sturgeon cartilage active peptide of the examples failed to significantly reduce the ROS content of the cells compared with the modeling group, but the SOD content of the cells was significantly increased (P < 0.05). The ROS content of Comparative Examples 5 and 6 was significantly reduced, and the SOD content thereof was also significantly increased compared with the modeling group, but the promotion of cell viability thereof did not reach the level of the examples, so the optimal enzymolysis process needs to be considered comprehensively according to the subsequent test results.

[0063] Example 6

[0064] This example is aimed at the MC3T3-E1 cell osteogenic marker analysis of the sturgeon cartilage peptides prepared in the above-mentioned Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6.

[0065] The MC3T3-E1 cells in good growth condition were planted in 96-well plates at a density of 1×10 5 cells per well, and were cultured in a cell incubator at 37°C and 5% CO2 for 24 h to wait for cell adhesion. After cell adhesion, the culture medium was discarded, and 0.2 mmol / L H2O2 and 0.4 mg / mL polypeptide solution were added for incubation for 24 h. According to the kit instructions, ROS and SOD were determined, and the results are shown in Figure 4 and Figure 5 .

[0066] ALP is secreted by osteoblasts and is a direct marker of bone formation. An increase in the secretion of ALP can directly indicate the proliferation or differentiation of osteoblasts. Since the skeleton of children is growing actively, children will have a higher ALP content. OCN is synthesized by mature osteoblasts and osteocytes and is the highest non-collagen protein in bone matrix. OCN is related to bone turnover, can maintain the normal mineralization rate of bone, inhibit the mineralization rate of cartilage, and inhibit the formation of abnormal hydroxyapatite crystals. ALP and OCN complement each other to comprehensively evaluate bone formation: ALP reflects the number / early function of osteoblasts, and OCN reflects the activity of mature osteoblasts. When using drugs, both can increase to indicate the effectiveness of the drug; if OCN increases but ALP does not, it may indicate an imbalance in bone turnover (such as a high catabolic state).

[0067] As shown in Figure 4 and Figure 5 , the cell content of ALP and OCN in the model group is lower than that in the normal group, indicating that the modeling is successful. Compared with other experimental groups, the polypeptide samples of Example, Comparative Example 2 and Comparative Example 6 have a more significant positive effect on the ALP content of oxidative stress cells (P < 0.05); the polypeptide samples of Example, Comparative Example 1, Comparative Example 2 and Comparative Example 5 significantly increase the OCN content of oxidative stress cells (P < 0.05). Comprehensive analysis of the effects of polypeptides prepared by different enzymolysis processes on the viability, antioxidant properties and osteogenic markers of oxidative stress cells shows that the polypeptide samples of Example have a more significant positive effect on the osteogenic differentiation of MC3T3-E1 cells compared with other experimental groups, and Example 1 shows a better effect.

[0068] Example 7

[0069] This example is directed to the MC3T3-E1 cell osteogenic related gene analysis of sturgeon cartilage peptides prepared in the above-mentioned Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6.

[0070] During the osteogenic differentiation of MC3T3-E1 cells, many genes are involved, such as runt-related transcription factor 2 (Runx2), ALP, OCN and EPK, etc. To further verify the effects of polypeptides prepared by different enzymolysis processes on the osteogenic differentiation of MC3T3-E1 cells, the expression of osteogenic related genes is analyzed and studied. The expression of these genes is often detected by Western blotting and polymerase chain reaction techniques, which can also be used as a kind of evidence to determine whether the cells are osteogenic.

[0071] Well-grown MC3T3-E1 cells were seeded in a 12-well plate coated with 0.1% gelatin and dried, and the cell density was 5 x 104 cells / cm2 2 After the cells adhered, the culture medium was discarded, and the cells were intervened for 24 h according to the experimental grouping. After the administration, the cells were induced to differentiate for 48 h. The total RNA of the cells in each group was extracted, and the concentration and purity of the RNA were measured. The RNA was reversely transcribed into cDNA, and the obtained cDNA was stored at -20℃. The original solution was diluted 5 times for subsequent steps. Real-time fluorescent quantitative PCR detection was performed. The reaction conditions were as follows: 95℃ pre-denaturation for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles; melting curve 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s. According to the two-step amplification program, the Ct cycle threshold value was obtained, and the relative expression of the RNA in different groups was calculated. Glycerol-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

[0072] The results are shown in Tables 1-3. Figure 6 、 Figure 7 、 Figure 8 and Figure 9 As shown in Tables 1-3, the expression of the bone-related genes in each experimental group was up-regulated to different degrees compared with the model group, indicating that the addition of sturgeon cartilage active peptide had a positive effect on the osteogenic differentiation of MC3T3-E1 cells. Compared with other experimental groups, the gene expression of the embodiment did not have obvious advantages, but this did not mean that the positive effect of the sample of the embodiment on the osteogenic differentiation of MC3T3-E1 cells was lower than that of the samples of other experimental groups.

[0073] Example 8

[0074] Therefore, the zebrafish test analysis was performed on the sturgeon cartilage peptide prepared in Example 1, to further verify the positive effect of the sturgeon cartilage peptide on the growth and development of the skeleton.

[0075] In this embodiment, the expression levels of bmp2a, sp7 and gh1 genes in zebrafish were determined, because they respectively represent three key links in the process of skeleton formation: bmp2a is a key signal molecule for starting skeleton development, sp7 is a core transcription factor for controlling osteoblast differentiation, and gh1 is an important hormone for regulating linear growth of the skeleton.

[0076] Randomly selected 3 dpf wild type AB strain zebrafish in beaker, each beaker (experimental group) were treated 30 zebrafish, water soluble sample. Set up normal control group, positive control group (Swisse "Calcium & Vitamin D" 500 μg / mL), sturgeon cartilage peptide low dose group (250 μg / mL) and sturgeon cartilage peptide high dose group (500 μg / mL). After 3 days of treatment at 28℃, the samples of each group were collected, and the body length of zebrafish, skull fluorescence intensity and the expression of osteogenesis related genes were determined and analyzed. The statistical analysis results of the above indexes were used to evaluate the growth and development promoting effect of the sample on zebrafish, and the results are shown in Tables 1, 2 and 3, Figure 10 and Figure 11 It can be seen from the chart that compared with the normal control group, the positive control group and the polypeptide sample group can significantly promote the body length growth of zebrafish, increase the skull fluorescence intensity and up-regulate the expression of zebrafish osteogenesis related genes, which shows that the addition of Swisse "Calcium & Vitamin D" and sturgeon cartilage active peptide has a positive effect on the osteogenesis differentiation of zebrafish, and the positive control group shows better effect. In addition, the sturgeon cartilage peptide high dose group shows better effect on promoting the growth and development of bone than the low dose group.

[0077] Table 1 Sturgeon cartilage peptide promotes the growth of zebrafish body length (n = 10) ; Compared with the normal control group,

[0078] Table 2 Sturgeon cartilage peptide promotes the growth of zebrafish bone (n = 10) ; Compared with the normal control group,

[0079] Table 3 Sturgeon cartilage peptide promotes the expression of zebrafish osteogenesis related genes (n = 3) ; Compared with the normal control group,

[0080] Example 9 In this embodiment, mass spectrometry was used to identify peptide sequences, PeptideRanker, ToxinPred and BioPepDB were used to further screen sturgeon cartilage active peptides capable of promoting height and improving bone development. The specific experimental steps are as follows: The liquid chromatography conditions are as follows: Pre-column: PEPMAP NEO C18, size 300 μm x 5 mm, Analytical column: 150 μm i.d. x 170 mm, packing Reprosil-Pur 120 C18-AQ 1.9 μm, Mobile phase A: 0.1% FA; mobile phase B: 0.1% FA, 80% ACN; flow rate: 600 nL / min; Each component analysis time is 66 min.

[0081] The mass spectrometry conditions are: full scan range 100-1500 m / z, first mass spectrometry resolution is set to 120000, AGC is Standard, Maximum IT: 20 ms; second mass spectrometry resolution is set to Resolution: 15000, AGC is Standard, Maximum IT: 22 ms Cycle time: 2 s, peptide fragment collision energy is set to 30, and mass spectrometry detection original data (.raw) is generated, finally more than 5000 polypeptide fragments are obtained.

[0082] PeptideRanker is used to score the biological activity of 89 peptides identified by mass spectrometry with an abundance higher than 115000000, when the score is > 0.5, the result is reliable, and the higher the score, the higher the potential biological activity; ToxinPred is used to predict the toxicity of 34 peptides with a score > 0.5; BioPepDB is used to assess the innovation of the peptides, and the 10 peptides with the highest abundance ratio are selected for subsequent analysis.

[0083] The final screened peptides are shown in Table 4, and the results show that these polypeptides are mainly composed of 3-9 amino acids, with a molecular weight of 424.2685-1154.518 Da, which is consistent with the conclusion that bioactive peptides usually contain 2-20 amino acid residues. Peptide Ranker is used to score these polypeptides, and 34 polypeptides with a score > 0.5 are selected, ToxinPred predicts that the 34 polypeptides have no toxicity, BioPepDB assesses that they all have innovation, and the 10 peptides with the highest abundance ratio are selected, as shown in Table 4.

[0084] Table 4: Screening results of sturgeon cartilage peptides

[0085] Example 10 According to the binding energy and peptide segment abundance, GPSVSPTPG and LGGYGMMRM were screened from the peptide segments in Table 4 as the characteristic peptide sequences with the effect of promoting the growth and development of bone in sturgeon cartilage peptides. The two peptide segments were subjected to molecular docking with the receptor protein by using a molecular docking software, and the receptor protein was selected as EGFR (PDB: 1IVO) and BMP-2 (PDB: 3BMP). The results showed that the two screened peptide segments could be combined and docked with the receptor protein to a certain extent.

[0086] The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that can regulate the proliferation and differentiation of cells. Its signal pathway is one of the main regulation pathways of bone metabolism. EGFR can be activated by the EGF-related peptide growth factor family and various ligands, thereby promoting bone formation. In the molecular docking, the EGF ligand structure was used as the docking site, and the EGF structure was deleted to verify the binding affinity between the sturgeon cartilage active peptide and the EGFR receptor. The results showed that the two screened peptide segments could be combined and docked with the receptor protein EGFR to a certain extent. As shown in FIG. 6, the main interactions between LGGYGMMRM and EGFR were van der Waals force, carbon-hydrogen bond, conventional hydrogen bond and alkylation, and the maximum "-CDOCKER ENERGY" value of LGGYGMMRM and EGFR docking was 98.3478 kcal / mol; as shown in FIG. 7, the main interactions between QFELCFGGR and EGFR were van der Waals force, carbon-hydrogen bond, alkylation and conventional carbon-hydrogen bond, and the maximum "-CDOCKER ENERGY" value of QFELCFGGR and EGFR docking was 115.803 kcal / mol. Figure 12 Figure 13 Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor β (TGF-β) superfamily, which can induce bone formation and regeneration. Studies have shown that active peptides derived from fish bone can interact with BMP receptors to promote osteoblast differentiation. As shown in FIG. 8, the main interactions between LGGYGMMRM and BMP-2 were van der Waals force, carbon-hydrogen bond, salt bridge and conventional hydrogen bond, and the maximum "-CDOCKER ENERGY" value of LGGYGMMRM and BMP-2 docking was 113.105 kcal / mol; as shown in FIG. 9, the main interactions between QFELCFGGR and BMP-2 were van der Waals force, carbon-hydrogen bond, attractive charge, conventional hydrogen bond and π-alkylation, and the maximum "-CDOCKER ENERGY" value of QFELCFGGR and BMP-2 docking was 131.779 kcal / mol.

[0087] Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor β (TGF-β) superfamily, which can induce bone formation and regeneration. Studies have shown that active peptides derived from fish bone can interact with BMP receptors to promote osteoblast differentiation. As shown in FIG. 8, the main interactions between LGGYGMMRM and BMP-2 were van der Waals force, carbon-hydrogen bond, salt bridge and conventional hydrogen bond, and the maximum "-CDOCKER ENERGY" value of LGGYGMMRM and BMP-2 docking was 113.105 kcal / mol; as shown in FIG. 9, the main interactions between QFELCFGGR and BMP-2 were van der Waals force, carbon-hydrogen bond, attractive charge, conventional hydrogen bond and π-alkylation, and the maximum "-CDOCKER ENERGY" value of QFELCFGGR and BMP-2 docking was 131.779 kcal / mol. Figure 14 Figure 15

[0088] ​​​The specific implementation of the present application is described above, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A sturgeon cartilage active peptide with the functions of promoting height growth and improving bone development, characterized in that, The sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAFMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP; The specific sequence of GPSVSPTPG is shown in SEQ ID NO. 1, the specific sequence of LGGYGMMRM is shown in SEQ ID NO. 2, the specific sequence of PGAAFMLG is shown in SEQ ID NO. 3, the specific sequence of PVPLP is shown in SEQ ID NO. 4, the specific sequence of QFELCFGGR is shown in SEQ ID NO. 5, the specific sequence of QLGP is shown in SEQ ID NO. 6, the specific sequence of QPAPLCT is shown in SEQ ID NO. 7, the specific sequence of QPSRYPA is shown in SEQ ID NO. 8, the specific sequence of SPTPGLC is shown in SEQ ID NO. 9, and the specific sequence of VPLP is shown in SEQ ID NO.

10.

2. The active peptide of sturgeon cartilage according to claim 1, characterized in that, The sturgeon cartilage active peptide comprises at least one of LGGYGMMRM or QFELCFGGR.

3. A method for preparing sturgeon cartilage active peptide for promoting height growth and improving bone development, characterized in that, The method comprises the following steps: S1, pretreating sturgeon cartilage and then adding water to homogenize to obtain a mixed solution A; S2, adding trypsin to the mixed solution A to perform first constant-temperature enzymolysis to obtain a mixed solution B; S3, adding neutral protease and flavor protease to the mixed solution B to perform second constant-temperature enzymolysis, and obtaining supernatant after cooling; S4, adding activated carbon to the supernatant, filtering through diatomite and spray drying to obtain a sturgeon cartilage active peptide; The prepared sturgeon cartilage active peptide comprises at least one of GPSVSPTPG, LGGYGMMRM, PGAAFMLG, PVPLP, QFELCFGGR, QLGP, QPAPLCT, QPSRYPA, SPTPGLC or VPLP; The specific sequence of GPSVSPTPG is shown in SEQ ID NO. 1, the specific sequence of LGGYGMMRM is shown in SEQ ID NO. 2, the specific sequence of PGAAFMLG is shown in SEQ ID NO. 3, the specific sequence of PVPLP is shown in SEQ ID NO. 4, the specific sequence of QFELCFGGR is shown in SEQ ID NO. 5, the specific sequence of QLGP is shown in SEQ ID NO. 6, the specific sequence of QPAPLCT is shown in SEQ ID NO. 7, the specific sequence of QPSRYPA is shown in SEQ ID NO. 8, the specific sequence of SPTPGLC is shown in SEQ ID NO. 9, and the specific sequence of VPLP is shown in SEQ ID NO.

10.

4. The method for preparing sturgeon cartilage active peptides according to claim 3, characterized in that, The first constant temperature enzymolysis condition is: temperature 40-60℃, pH 6.5-8.5, time 1-3 h, and finally enzyme is inactivated; the second constant temperature enzymolysis condition is: temperature 40-60℃, pH 6.0-8.0, time 1-3 h, and after the enzymolysis, the pH is adjusted to 7.0, and finally enzyme is inactivated.

5. The method for preparing sturgeon cartilage active peptides according to claim 3, characterized in that, The mass ratio of the sturgeon cartilage to water is 1:5-1:9, the added amount of the activated carbon is 3%-5% of the mass of the sturgeon cartilage, and the action condition of the activated carbon is: temperature 50-60℃, time 0.5-1.5 h.

6. The method for preparing sturgeon cartilage active peptides according to claim 3, characterized in that, The added amount of the trypsin is 10000-14000 U / g of the sturgeon cartilage, the added amount of the neutral protease is 5000-7000 U / g of the sturgeon cartilage, and the added amount of the flavourzyme is 5000-7000 U / g of the sturgeon cartilage.

7. The method for preparing sturgeon cartilage active peptides according to claim 3, characterized in that, The sturgeon cartilage active peptide comprises at least one of LGGYGMMRM or QFELCFGGR. 8.A use of the sturgeon cartilage active peptide according to any one of claims 1-2 in the preparation of a functional product for promoting the growth and development of bones. 9.A use of the sturgeon cartilage active peptide prepared by the method according to any one of claims 3-7 in the preparation of a functional product for promoting the growth and development of bones.

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