Osteoblast proliferation promoting peptide as well as preparation method and application thereof

Abstract

The invention discloses an osteoblast proliferation promoting peptide as well as a preparation method and application thereof, and belongs to the technical field of protein peptide preparation. The amino acid sequence of the osteoblast proliferation promoting peptide is shown as any one of SEQ ID NO.1-3. The invention further discloses a preparation method of the osteoblast proliferation promoting peptide. The invention also provides a preparation method of the osteoblast proliferation promoting peptide, which comprises the following steps: extracting collagen by using a protease-assisted acid method, and carrying out enzymolysis by using alkaline protease to obtain the osteoblast proliferation promoting peptide. The osteoblast proliferation promoting peptide provided by the invention can promote proliferation and expression of MC3T3-E1 cell differentiation and mineralization related genes through an MAPK pathway, so that osteoporosis is prevented and bone health is improved. And the method has a good application prospect in the fields of bone cell proliferation promoting related orthopaedic and dental medicines, health care products, functional foods and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of protein peptide preparation, and particularly to an osteoblast proliferation promoting peptide, a preparation method thereof, and an application thereof. Background Art

[0002] The protein content in the scales of Liza haematocheila is rich, and it is a protein raw material that can be developed and utilized. This protein is mainly collagen, and also contains a small amount of albumin, globulin, and elastin. The fish skin contains rich collagen, and extracting collagen from the fish skin has become the primary choice. The fish bone is mainly composed of organic matter and minerals. Among the organic matter, collagen is the main component, and its proportion in the organic matter can reach a relatively high level; in terms of minerals, the main component is hydroxyapatite, which is a calcium phosphate salt with an appropriate calcium-phosphorus ratio (1.67) and is easy to be absorbed by the human body. Collagen forms a fiber network structure in the form of collagen fibrils and collagen fibers. Collagen has a very wide range of applications in food, beauty, pharmaceuticals, and membrane materials. Collagen can be made into bioactive peptides (BPs) by methods such as enzymatic hydrolysis, chemical degradation, and microbial fermentation.

[0003] The application development of traditional livestock product-derived collagen peptides is restricted by religious beliefs and infectious diseases. However, the skin, bones, scales, etc. of aquatic animals are rich in collagen and are a safe source of collagen products. Therefore, many scholars at home and abroad have carried out research on extracting collagen from the skin, scales, and bones of aquatic animals, but there are few reports on extracting collagen from the processing by-products of Liza haematocheila. Liza haematocheila has delicate and delicious meat and a high protein content, and it is an economically edible fish distributed worldwide. Liza haematocheila is one of the important varieties of marine fishing, with an annual output of more than ten thousand tons. Due to its wide range of salinity and temperature adaptability, fast growth, and strong stress resistance, it can grow in both seawater and freshwater, and it is a breeding variety with great market development potential.

[0004] Bone metabolism of the body includes bone formation metabolism and bone resorption metabolism, which may be regulated by various external factors at the same time, and its related molecular mechanisms involve multiple aspects such as genes, hormones, paracrine factors, and signal pathways. Among them, the signal pathway plays an important regulatory role. Osteoblasts (OB) develop from bone marrow mesenchymal stem cells with multi-directional differentiation potential. Biochemically and histochemically, osteoblasts are rich in ALP, can synthesize COLI, secrete bone matrix, express osteocalcin gene, and can absorb and transport calcium ions, etc. Therefore, osteoblasts play an extremely important role in the process of bone formation, and promoting the proliferation and differentiation and maturation of osteoblasts has positive significance for preventing and treating osteoporosis.

[0005] Existing reports on the collagen of mullet scales, skin, and bones have involved the research on the extraction of mullet collagen and the enzymatic hydrolysis process of collagen peptides. However, most of them focus on the effect of hydrolysis degree on the yield of polypeptides, and have not involved the enzymatic preparation of specific bioactive functional peptides. There is also no systematic research on the identification of mullet scale, skin, and bone collagen peptides and their ability to promote the proliferation activity of osteoblasts (improve osteoporosis). Summary of the Invention

[0006] The purpose of the present invention is to provide a peptide that promotes osteoblast proliferation, its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The peptide that promotes osteoblast proliferation can promote the differentiation of MC3T3-E1 cells and the proliferation and expression of mineralization-related genes, so as to achieve the purpose of preventing osteoporosis and improving bone health.

[0007] To achieve the above purpose, the present invention provides the following solutions:

[0008] Technical solution 1: A peptide that promotes osteoblast proliferation, and its amino acid sequence is shown in any one of SEQ ID NO.1-3.

[0009] Furthermore, the peptide that promotes osteoblast proliferation is derived from mullet scales, mullet skin, and mullet bones; when the peptide that promotes osteoblast proliferation is derived from mullet scales, its amino acid sequence is shown in SEQ ID NO.1; when the peptide that promotes osteoblast proliferation is derived from mullet skin, its amino acid sequence is shown in SEQ ID NO.2; when the peptide that promotes osteoblast proliferation is derived from mullet bones, its amino acid sequence is shown in SEQ ID NO.3.

[0010] Among them, mullet scales: GGPGHQGPGGMPGER (SEQ ID NO.1); mullet skin: FDGLQ (SEQ ID NO.2); mullet bones: GHPGPKGMKG (SEQ ID NO.3).

[0011] Technical solution 2: The preparation method of the peptide that promotes osteoblast proliferation, which uses pepsin-assisted acid method to extract collagen, and then uses alkaline protease to enzymatically hydrolyze to obtain the peptide that promotes osteoblast proliferation.

[0012] (1) By introducing pepsin assistance in the acid extraction process, the present invention can not only quickly obtain complete collagen, but also effectively reduce environmental pollution, shorten the reaction time, and reduce energy consumption when using pepsin for extraction;

[0013] (2) By using alkaline protease as the hydrolyzing enzyme, while ensuring the degree of protein hydrolysis, the present invention obtains peptides with excellent osteoblast proliferation-promoting effects.

[0014] Further, the preparation of the collagen includes the following steps: washing the mullet scales, mullet skin or mullet bones, adding acetic acid solution with a concentration of 0.5 mol / L, with a material-liquid ratio of 1:20 - 1:55 g / mL, enzymatically hydrolyzing for 18 - 34 h, and the enzyme addition amount being 1 - 3.5%.

[0015] Further, when the collagen is derived from mullet scales or mullet skin, the preparation of the collagen includes the following steps: washing the mullet scales or mullet skin, adding acetic acid solution with a concentration of 0.5 mol / L, with a material-liquid ratio of 1:30 g / mL, enzymatically hydrolyzing for 24 h, and the enzyme addition amount being 2%; when the collagen is derived from mullet bones, the preparation of the collagen includes the following steps: washing and pulverizing the mullet bones, adding acetic acid solution with a concentration of 0.5 mol / L, with a material-liquid ratio of 1:45 g / mL, enzymatically hydrolyzing for 28 h, and the enzyme addition amount being 2.5%.

[0016] Further, the addition amount of the alkaline protease is 3 - 7%; the pH during enzymatic hydrolysis is 8 - 11, and the enzymatic hydrolysis temperature is 35 - 55 °C.

[0017] Further, when the collagen is derived from mullet scales, the addition amount of the alkaline protease is 5%; the pH during enzymatic hydrolysis is 9, and the enzymatic hydrolysis temperature is 45 °C; when the collagen is derived from mullet skin, the addition amount of the alkaline protease is 4%; the pH during enzymatic hydrolysis is 10, and the enzymatic hydrolysis temperature is 50 °C; when the collagen is derived from mullet bones, the addition amount of the alkaline protease is 4%; the pH during enzymatic hydrolysis is 9, and the enzymatic hydrolysis temperature is 45 °C.

[0018] Technical solution three: Application of the osteoblast proliferation peptide or the osteoblast proliferation peptide prepared by the preparation method in the preparation of products for promoting osteoblast proliferation.

[0019] Further, the promotion of osteoblast proliferation includes promoting the proliferation, differentiation and mineralization of MC3T3-E1 cells.

[0020] Technical solution four: Application of the osteoblast proliferation peptide or the osteoblast proliferation peptide prepared by the preparation method in the preparation of products for preventing and / or treating osteoporosis and improving bone health.

[0021] The product includes drugs.

[0022] The present invention discloses the following technical effects:

[0023] The present invention extracts collagen (PSC) from the scales, skin, and bones of lizardfish by pepsin-assisted acid method, effectively improving the yield of PSC, reaching over 32%, 36%, and 24% respectively, and the degree of hydrolysis can reach 23.57%, 25.41%, and 20.51%. The present invention selects alkaline protease as the hydrolytic enzyme. While ensuring the degree of protein hydrolysis, peptides GGPGHQGPGGMPGER, FDGLQ, and GHPGPKGMKG with excellent osteoblast proliferation-promoting effects are obtained. The verification results show that these peptides have good water solubility and ADMET properties, showing HIA+) and good Caco2-permeability, indicating that the drug has better absorption ability in the small intestine, is more conducive to human absorption and utilization, and can promote the differentiation of MC3T3-E1 cells and the proliferation and expression of mineralization-related genes through the MAPK pathway, thereby preventing osteoporosis and improving bone health. The development of lizardfish scale, skin, and bone collagen peptides and the study of their mechanism of action on bone health can not only meet the market and consumer demands, but also help the development of the aquatic product processing industry, and is more conducive to increasing fishermen's income and promoting the revitalization of rural industries. Brief Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 Effects of liquid-to-material ratio (A), enzymolysis time (B), and enzyme dosage (C) on the yield of PSC extracted from lizardfish scales;

[0026] Figure 2 Effects of liquid-to-material ratio (A), enzymolysis time (B), and enzyme dosage (C) on the yield of PSC extracted from lizardfish skin;

[0027] Figure 3 Effects of liquid-to-material ratio (A), enzymolysis time (B), and enzyme dosage (C) on the yield of PSC extracted from lizardfish bones;

[0028] Figure 4 Effects of enzyme dosage (A), pH (B), and enzymolysis temperature (C) on the degree of hydrolysis during the enzymolysis of lizardfish scale collagen;

[0029] Figure 5 Effects of enzyme dosage (A), pH (B), and enzymolysis temperature (C) on the degree of hydrolysis during the enzymolysis of lizardfish skin collagen;

[0030] Figure 6Effect of enzyme dosage (A), pH (B) and hydrolysis temperature (C) on the degree of hydrolysis during the enzymatic hydrolysis of pike bone collagen;

[0031] Figure 7 Mass spectrum of fish scale collagen peptide;

[0032] Figure 8 Structural formula of fish scale collagen peptide;

[0033] Figure 9 Mass spectrum of fish skin collagen peptide;

[0034] Figure 10 Structural formula of fish skin collagen peptide;

[0035] Figure 11 Mass spectrum of fish bone collagen peptide;

[0036] Figure 12 Structural formula of fish bone collagen peptide;

[0037] Figure 13 Study on the ability of GG-15 peptide to promote osteoblast proliferation;

[0038] Figure 14 Study on the ability of FDGLQ peptide to promote osteoblast proliferation;

[0039] Figure 15 Study on the ability of GG-10 peptide to promote osteoblast proliferation;

[0040] Figure 16 Study on the ability of GG-15 peptide to promote osteoblast differentiation; where A is the statistical result of ALP activity at different concentrations, and B is the ALP staining condition at different concentrations;

[0041] Figure 17 Study on the ability of FDGLQ peptide to promote osteoblast differentiation; where A is the statistical result of ALP activity at different concentrations, and B is the ALP staining condition at different concentrations;

[0042] Figure 18 Study on the ability of GG-10 peptide to promote osteoblast differentiation; where A is the statistical result of ALP activity at different concentrations, and B is the ALP staining condition at different concentrations;

[0043] Figure 19 Study on the ability of GG-15 peptide to promote osteoblast mineralization; where 0, 1, 10 and 50 are the peptide addition concentrations, A in the upper row is the magnification of 0 times, and B in the lower row is the magnification of 10 times;

[0044] Figure 20 Study on the ability of FDGLQ peptide to promote osteoblast mineralization; where 0, 1, 10 and 50 are the peptide addition concentrations, A in the upper row is the magnification of 0 times, and B in the lower row is the magnification of 10 times;

[0045] Figure 21 Study on the ability of GG-10 peptide to promote osteoblast mineralization; among them, 0, 1, 10, and 50 are the peptide addition concentrations, A in the upper row is the magnification factor of 0 times, and B in the lower row is the magnification factor of 10 times;

[0046] Figure 22 Effect of GG-15 peptide on the expression of related genes OCN (A), ALP (B), RUNX2 (E), BMP2 (F) and pathway genes ERK1 (C), JNK1 (D), P38 (G);

[0047] Figure 23 Amplification of proteins by GG-15 peptide (A) and expression of proteins ERK (B), p-ERK (C), BMP2 (D), RUNX2 (E) by GG-15 peptide;

[0048] Figure 24 Effect of FDGLQ peptide on the expression of related genes OCN (A), ALP (B), RUNX2 (E), BMP2 (F) and pathway genes ERK1 (C), JNK1 (D), P38 (G);

[0049] Figure 25 Amplification of proteins by FDGLQ peptide (A) and expression of proteins ERK (B), p-ERK (C), BMP2 (D), RUNX2 (E) by FDGLQ peptide;

[0050] Figure 26 Effect of GG-10 peptide on the expression of related genes OCN (A), ALP (B), RUNX2 (E), BMP2 (F) and pathway genes ERK1 (C), JNK1 (D), P38 (G);

[0051] Figure 27 Amplification of proteins by GG-10 peptide (A) and expression of proteins ERK (B), p-ERK (C), BMP2 (D), RUNX2 (E) by GG-10 peptide. Detailed implementation manners

[0052] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0055] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0056] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0057] Unless otherwise specified, the test methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained commercially.

[0058] Example 1

[0059] A method for preparing collagen peptide, comprising the following steps:

[0060] (1) Extracting collagen

[0061] (1) Take 10 g of mullet fish scales, wash them 3 times with tap water, and remove other impurities mixed in. Then place the fish scales in a cool place to air dry naturally. Add acetic acid solution with a concentration of 0.5 mol / L to the processed fish scales, with a material-liquid ratio of 1:20 - 1:40 (unit: g / mL), an enzymatic hydrolysis time of 18 - 30 (unit: h), and an enzyme addition amount (pepsin) of 1 - 3 (unit: %). Among them, the addition order of pepsin and acetic acid is to add acetic acid first and then pepsin. After the extraction time is reached, centrifuge to collect the supernatant, adjust it to 0.9 mol / L with NaCl, salt out and stand at 4 °C, then centrifuge, and freeze-dry the precipitate to obtain mullet fish scale collagen powder;

[0062] (2) Take 10 g of mullet fish skin, wash and drain it, cut it into small pieces of the same size, put them into a 500 mL beaker, add 0.1 mol / L NaOH solution to soak, stir continuously at low speed for 24 h, and change the NaOH solution every 4 h to remove non-collagen in the mullet fish skin. Then wash the fish skin repeatedly with distilled water until it is neutral, and then soak the fish skin with 10% n-butanol at a material-liquid ratio of 1:10 g / mL, continue to stir continuously at low speed for 24 h, and change the n-butanol solution every 4 h to remove the grease in the mullet fish skin. Finally, wash the mullet fish skin with distilled water until there is no smell of n-butanol. Add acetic acid solution with a concentration of 0.5 mol / L to the processed fish skin, with a material-liquid ratio of 1:20 - 1:40 (unit: g / mL), an enzymatic hydrolysis time of 20 - 28 (unit: h), and an enzyme addition amount (pepsin) of 1 - 3 (unit: %). After the extraction time is reached, centrifuge to collect the supernatant, adjust it to 0.9 mol / L with NaCl, salt out and stand at 4 °C, then centrifuge, and freeze-dry the precipitate to obtain mullet fish skin collagen powder;

[0063] (3) Take 10 g of mullet fish bones, wash and drain them, and powder them. Add 200 mL of 0.1 mol / L NaOH solution, stir continuously at low speed for 20 h, and change the NaOH solution every 4 h to remove non-collagen in the fish bones. Then wash repeatedly with distilled water at 4 °C until it is neutral. After draining, add 300 mL of 10% n-butanol, stir continuously at low speed for 24 h, and change the n-butanol every 4 h to remove the grease. Add 0.5 mol / L EDTA solution to decalcify for 48 h, and then wash repeatedly with distilled water at 4 °C and drain. Add acetic acid solution with a concentration of 0.5 mol / L to the processed fish bones, with a material-liquid ratio of 1:35 - 1:55 (unit: g / mL), an enzymatic hydrolysis time of 22 - 34 (unit: h), and an enzyme addition amount (pepsin) of 1.5 - 3.5 (unit: %). After the extraction time is reached, centrifuge to collect the supernatant, adjust it to 0.9 mol / L with NaCl, salt out and stand at 4 °C, then centrifuge, and freeze-dry the precipitate to obtain mullet fish bone collagen powder.

[0064] (II) Preparation of osteoblast proliferation-promoting peptides by enzymatic hydrolysis

[0065] Prepare the collagen powder from mullet scales into a 0.025 g / mL collagen solution, add alkaline protease for enzymatic hydrolysis. The addition amount of alkaline protease is 3-7%, the pH is 8-10, and the enzymatic hydrolysis temperature is 35-55 °C to obtain enzymatic hydrolysate 1;

[0066] Prepare the collagen powder from mullet skin into a 0.025 g / mL collagen solution, add alkaline protease for enzymatic hydrolysis. The addition amount of alkaline protease is 3-5%, the pH is 9-11, and the enzymatic hydrolysis temperature is 45-55 °C to obtain enzymatic hydrolysate 2;

[0067] Prepare the collagen powder from mullet bones into a 0.025 g / mL collagen solution, add alkaline protease for enzymatic hydrolysis. The addition amount of alkaline protease is 3-5%, the pH is 8-10, and the enzymatic hydrolysis temperature is 40-50 °C to obtain enzymatic hydrolysate 3.

[0068] During the extraction of collagen from mullet scales, skin, and bones, there are significant differences in the yields of the obtained collagen powder (PSC) by different extraction methods. The PSC yield is calculated by the following formula:

[0069]

[0070] In the formula: y is the PSC yield; m1 is the mass of PSC after freeze-drying / g; m2 is the mass of mullet scales, skin, or bones after treatment / g.

[0071] Calculated by the above formula, the yield of PSC extracted from mullet scales is 32.13 ± 0.21%. Test the effects of the material-liquid ratio, enzymatic hydrolysis time, and enzyme addition amount on the yield of PSC extracted from mullet scales. The results are as Figure 1 shown. When the enzymatic hydrolysis time and enzyme addition amount remain unchanged, Figure 1 A in shows the effect of the material-liquid ratio on the PSC yield. As the solution volume increases, the extraction rate gradually increases. As the solution volume continues to increase, the extraction rate begins to decrease. The collagen extraction rate reaches the highest under the condition of a material-liquid ratio of 1:30 g / ml. From Figure 1 B in, it can be seen that when the enzymatic hydrolysis time reaches 15 h, the extraction rate of collagen reaches more than 20%, and reaches 31.90 ± 0.11% at 24 h. As the time further increases, the collagen extraction rate begins to decline. Therefore, when the extraction time is 24 h, the extraction rate is the highest. As Figure 1As shown in C of Figure , when the solid-liquid ratio and enzymolysis time are fixed, it can be found that with the increase of enzyme dosage, the extraction rate gradually increases. When the enzyme dosage exceeds 2%, the extraction rate begins to decrease. The collagen extraction rate reaches the highest under the condition of 2% enzyme dosage. This is because pepsin can depolymerize the terminal peptides of collagen, increasing its solubility in aqueous solution, but has no effect on the main structure of collagen, and its optimal pH value is 2-4, so it is suitable for combination with the acid method. When the protein concentration is constant, pepsin can hydrolyze large protein molecules into small peptide fragments, increasing the collagen content; however, if the enzyme dosage is too high, over-enzymolysis will occur, causing small protein fragments to be further hydrolyzed into free amino acids, resulting in a significant decrease in collagen content.

[0072] Calculated by the above formula, the yield of PSC extracted from mullet skin is 36.92±1.05%. The effects of solid-liquid ratio, enzymolysis time, and enzyme dosage on the yield of PSC extracted from mullet skin were tested, and the results are as Figure 2 shown. When the enzymolysis time and enzyme dosage remain unchanged, Figure 2 A of Figure shows the effect of solid-liquid ratio on the yield of PSC; while the enzyme content has a greater impact on the collagen extraction rate. With the increase of enzyme dosage, the collagen extraction rate increases; when the enzyme dosage is 2%, the collagen extraction rate from mullet skin reaches the highest. On the premise that the other extraction conditions are the same, the effect of enzymolysis time on the collagen extraction rate is as Figure 2 shown in B of Figure . When the enzymolysis time is short, the contact between the enzyme and the substrate is insufficient, resulting in a low collagen extraction rate and certain losses; with the increase of enzymolysis time, the fish skin and the enzyme are fully contacted, increasing the collagen extraction rate. After that, if the enzymolysis time is extended further, over-enzymolysis of collagen will lead to a decrease in its extraction rate. As Figure 2 shown in C of Figure , when the solid-liquid ratio and enzymolysis time are fixed, it can be found that with the increase of enzyme dosage, the extraction rate gradually increases. When the enzyme dosage exceeds 2.5%, the extraction rate begins to decrease. This is because pepsin can depolymerize the terminal peptides of collagen, increasing its solubility in aqueous solution, but has no effect on the main structure of collagen, and its optimal pH value is 2-4, so it is suitable for combination with the acid method. When the protein concentration is constant, pepsin can hydrolyze large protein molecules into small peptide fragments, increasing the collagen content; however, if the enzyme dosage is too high, over-enzymolysis will occur, causing small protein fragments to be further hydrolyzed into free amino acids, resulting in a significant decrease in collagen content.

[0073] Calculated by the above formula, the yield of PSC extracted from mullet bone is 24.131±0.40%. The effects of solid-liquid ratio, enzymolysis time, and enzyme dosage on the yield of PSC extracted from mullet bone were tested, and the results are as Figure 3 shown. When the enzymolysis time and enzyme dosage remain unchanged, Figure 3A shows the effect of the solid-liquid ratio on the PSC yield. As the solution volume increases, the extraction rate gradually increases. As the solution volume continues to increase, the extraction rate begins to decrease. The collagen extraction rate reaches the highest under the condition of a solid-liquid ratio of 1:45 g / ml. On the premise that the degree of fish bone crushing is the same and the rest of the extraction conditions are consistent, the effect of the extraction time on the collagen extraction rate is as shown in Figure 3 Figure B. When the extraction time reaches 22 h, the extraction rate of collagen reaches more than 20%, and reaches 24.19% at 28 h. As the time further increases, the collagen extraction rate begins to decline because part of the collagen will be hydrolyzed when the enzyme acts for too long. As shown in Figure 3 Figure C, when the solid-liquid ratio and the enzymolysis time are fixed, it can be found that as the enzyme addition amount increases, the extraction rate gradually increases. When the enzyme addition amount exceeds 2.5%, the extraction rate begins to decrease. This is because pepsin can depolymerize the terminal peptides of collagen, increasing its solubility in aqueous solution, but has no effect on the main structure of collagen, and its optimal pH value is 2-4, so it is suitable for combination with the acid method. When the protein concentration is constant, pepsin can hydrolyze large protein molecules into small peptide fragments, increasing the collagen content; but if the enzyme addition amount is too high, over-enzymolysis will occur, and small molecular protein fragments will be continuously hydrolyzed into free amino acids, resulting in a significant decrease in the collagen content.

[0074] The degree of hydrolysis (DH) is the degree of hydrolysis of proteins during the protein hydrolysis process, that is, the ratio of the number of peptide bonds broken to the total number of proteins, and is used to express the effect of protein hydrolysis. In this invention, the formaldehyde titration method is used to measure the degree of hydrolysis, and the degree of hydrolysis of the collagen in the scales of mullet fish is calculated according to the following formula.

[0075]

[0076] In the formula: C1—the molar concentration of the sodium hydroxide solution; C2—the mass concentration of the porcine skin collagen solution; V0—the volume of sodium hydroxide consumed in the blank experiment; V1—the volume of sodium hydroxide consumed in titrating the sample; V2—the volume of the selected sample hydrolysis solution; h tot —is the number of millimoles of peptide bonds per gram of the original protein (mmol / g).

[0077] After calculation, the corresponding DH values in the scales, skin and bones of mullet fish are 23.45±0.33%, 25.41±0.55% and 20.51±0.27% respectively.

[0078] By single-factor variable research, the effects of pH, enzyme addition amount and enzymolysis temperature on the degree of hydrolysis during the enzymolysis process of mullet fish scale collagen were studied. The results are as shown in Figure 4 Figure. The difference in the enzymolysis pH value will affect the spatial structure of the enzyme and even inactivate the enzyme. At the same time, the pH value affects the binding state of the substrate and the enzyme, and thus the degree of protein hydrolysis is also affected. As shown byFigure 4 As can be seen from A, when the enzyme addition amount is less than 5%, the overall hydrolysis degree increases. After the enzyme addition amount exceeds 5%, the hydrolysis degree shows no obvious change. This is because the enzyme and the substrate have fully reacted and the hydrolysis is basically complete, so it tends to be flat. From Figure 4 As can be seen from B, the hydrolysis degree shows an upward trend with the increase of pH value and reaches the maximum at pH 9.0, and then decreases significantly with the further increase of pH value. This may be because excessive pH destroys the structure of pepsin and affects the reaction between the enzyme and the substrate. As Figure 4 As shown in C, the influence of the enzymatic hydrolysis temperature on the hydrolysis degree shows a trend of first increasing and then slowly decreasing. The hydrolysis degree reaches the maximum at 45 °C, and then decreases when the temperature is higher than 45 °C. This may be because as the temperature gradually increases, the enzyme activity weakens, and the number of generated polypeptides also decreases.

[0079] The effects of pH, enzyme addition amount and enzymatic hydrolysis temperature on the hydrolysis degree during the enzymatic hydrolysis of pike skin collagen were studied by single-factor variable method. The results are as Figure 5 shown. The difference in the pH value for enzyme cleavage affects the spatial structure of the enzyme and even inactivates the enzyme. At the same time, the pH value affects the binding state between the substrate and the enzyme, and thus affects the degree of protein hydrolysis. As Figure 5 As can be seen from A, when the enzyme addition amount is less than 4%, the overall hydrolysis degree increases. After the enzyme addition amount exceeds 4%, the hydrolysis degree shows no obvious change. This is because the enzyme and the substrate have fully reacted and the hydrolysis is basically complete, so it tends to be flat. From Figure 5 As can be seen from B, the hydrolysis degree shows an upward trend with the increase of pH value and reaches the maximum at pH 10.0, and then decreases significantly with the further increase of pH value. This may be because excessive pH destroys the structure of pepsin and affects the reaction between the enzyme and the substrate. As Figure 5 As shown in C, the influence of the enzymatic hydrolysis temperature on the hydrolysis degree shows a trend of first increasing and then slowly decreasing. The hydrolysis degree reaches the maximum at 50 °C, and then decreases when the temperature is higher than 50 °C. This may be because as the temperature gradually increases, the enzyme activity weakens, and the number of generated polypeptides also decreases.

[0080] The effects of pH, enzyme addition amount and enzymatic hydrolysis temperature on the hydrolysis degree during the enzymatic hydrolysis of pike bone were studied by single-factor variable method. The results are as Figure 6 shown. The difference in the pH value for enzyme cleavage affects the spatial structure of the enzyme and even inactivates the enzyme. At the same time, the pH value affects the binding state between the substrate and the enzyme, and thus affects the degree of protein hydrolysis. As Figure 6 As can be seen from A, when the enzyme addition amount is less than 4%, the overall hydrolysis degree increases. After the enzyme addition amount exceeds 4%, the hydrolysis degree shows no obvious change. This is because the enzyme and the substrate have fully reacted and the hydrolysis is basically complete, so it tends to be flat. From Figure 6As can be seen from B of [the relevant content], the degree of hydrolysis shows an upward trend with the increase of pH value, reaching the maximum at pH 9.0, and decreasing significantly with the further increase of pH value. This may be because excessive pH destroys the structure of pepsin and affects the reaction between the enzyme and the substrate. As Figure 6 As shown in C of [the relevant content], the influence of enzymatic hydrolysis temperature on the degree of hydrolysis shows a trend of first increasing and then slowly decreasing. The degree of hydrolysis reaches the maximum at an enzymatic hydrolysis temperature of 45 °C, and the degree of hydrolysis decreases after the temperature is greater than 45 °C. This may be because as the temperature gradually increases, the activity of the enzyme also weakens, and the number of generated polypeptides also decreases.

[0081] Example 2

[0082] The enzymatic hydrolysates 1-3 prepared in Example 1 were respectively analyzed and identified for their amino acid sequences by liquid chromatography-mass spectrometry, and peptides GGPGHQGPGGMPGER (fish scale, SEQ ID NO.1), FDGLQ (fish skin, SEQ ID NO.2), and GHPGPKGMKG (fish bone, SEQ ID NO.3) were obtained. The mass spectrum and structural formula of fish scale collagen peptide are shown in Figure 7 and 8 ; The mass spectrum and structural formula of fish skin collagen peptide are shown in Figure 9 and 10 ; The mass spectrum and structural formula of fish bone collagen peptide are shown in Figure 11 and 12 ; The specific method is as follows:

[0083] 1. Pretreatment: Take an appropriate amount of the sample and dissolve it with 50 mM NH4HCO3, then add DTT solution to make its concentration 10 mmol / L, and reduce it in a water bath at 56 °C for 1 h. Add IAM solution to make its final concentration 55 mmol / L, and react in the dark for 40 min. Use a self-packed desalting column for desalting, and evaporate the solvent in a vacuum centrifugal concentrator at 45 °C;

[0084] 2. LC-MS / MS detection:

[0085] 1) Liquid chromatography conditions: Analytical column: 150 μm i.d.×150 mm, packed with Acclaim PepMapRPLC C18, 1.9 μm, Mobile phase A: 0.1% formic acid; Mobile phase B: 0.1% formic acid, 80% ACN; Flow rate: 600 nL / min; Analysis time for each component: 66 min;

[0086] 2) Mass spectrometry conditions:

[0087] Primary mass spectrometry parameters: Resolution: 70,000; AGC target: 3e6; Maximum IT: 100 ms; Scan range: 100 to 1500 m / z;

[0088] MS / MS parameters: Resolution: 17,500; AGC target: 1e5; Maximum IT: 50 ms; TopN: 20; NCE / stepped NCE: 28.

[0089] 3. Database search: The raw mass spectrometry files were used to search the target protein database by Byonic.

[0090] The scale, skin, and bone collagen peptides prepared in Example 1 were analyzed and identified by LC-MS / MS respectively. It was found that 1449, 855, and 418 peptide sequences were obtained by pepsin digestion of PSC.

[0091] To predict the properties of the peptide sequences, the online tool NovoPro (https: / / www.novopro.cn / tools / ) was first used to simplify the Simplified Molecular Input Line Entry Specification (SMILES) format of the peptides, and the bioactivity, water solubility, toxicity, and ADMET of 1449, 855, and 418 peptide sequences were predicted. Generally, bioactive peptides are defined as peptides with a PeptideRanker score greater than 0.5 within the range of 0 to 1, which are considered to have potential bioactivity, and the higher the value, the greater the bioactivity; then, through the score (>100) and abundance (>0.01%), 17, 13, and 5 data were obtained respectively (Tables 1 - 3); finally, by docking with epidermal growth factor EGFR, peptides GGPGHQGPGGMPGER (scale), FDGLQ (skin), and GHPGPKGMKG (bone) with high bioactivity, good water solubility, good ADME properties, high abundance, high score, and potential to promote osteoblast proliferation were screened out.

[0092] Table 1

[0093]

[0094] Table 2

[0095]

[0096]

[0097] Table 3

[0098]

[0099] As can be obtained from Tables 1 - 3, the peptide sequences GGPGHQGPGGMPGER, FDGLQ, and GHPGPKGMKG simultaneously possess good water solubility and ADMET properties, showing HIA+ and good Caco2 - permeability. This indicates that the drugs GGPGHQGPGGMPGER, FDGLQ, and GHPGPKGMKG have good absorption ability in the small intestine and potential osteoblast proliferation - promoting ability.

[0100] Example 3

[0101] Effects of peptides GGPGHQGPGGMPGER (GG - 15), FDGLQ, and GHPGPKGMKG (GG - 10) on the proliferation, differentiation, and mineralization of MC3T3 - E1 cells:

[0102] MC3T3 - E1 mouse embryonic osteoblasts have strong osteogenic differentiation ability and can differentiate into mature osteocytes expressing bone - specific genes, which is a mature model for studying osteocyte biology and metabolism. During the research process, corresponding experiments were carried out by synthesizing peptides GG - 15, FDGLQ, and GG - 10. The synthesized GG - 15, FDGLQ, and GG - 10 were from Xi'an Navi Biotechnology Co., Ltd. KRBH solution (7.54 g of NaCl, 0.36 g of KCl, 0.15 g of MgSO4, 0.16 g of KH2PO4, 0.28 g of CaCl2, 0.42 g of NaHCO3, 1.00 g of BSA, 2.38 g of HEPES, 1 L of deionized water. After mixing with a magnetic stirrer, adjust the pH to 7.4, filter and sterilize, and store at 4°C for later use).

[0103] (1) Effects of peptides GG - 15, FDGLQ, and GG - 10 on the proliferation of MC3T3 - E1 cells

[0104] Inoculate the MC3T3 - E1 cell suspension into a 96 - well plate (1×10 5 cells per well), culture for 24 hours, and add synthetic peptides GG - 15, FDGLQ, and GG - 10 with different concentrations (1 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) respectively. Set three replicates for each group and culture for 24 h. After 24 h, add 10 μL of CCK - 8 solution to each well, incubate in an incubator for 3 h, and detect the OD value of each well of cells at 450 nm using a microplate reader. Calculate the cell proliferation rate according to the following formula:

[0105]

[0106] After treating MC3T3-E1 cells with samples GG-15, FDGLQ, and GG-10 at different concentrations for 24 hours, the cell proliferation rate was detected. As Figures 13 - 15 can be seen, MC3T3-E1 cells grew well at concentrations ranging from 0 to 50 μg / mL, showing a promoting proliferative activity. However, when the concentration was 100 μg / mL and 200 μg / mL, the cell activity decreased. There were extremely significant differences between the 10 μg / mL and 200 μg / mL groups and the Control group (P<0.001), indicating that the cell survival rate was the highest at 10 μg / mL, and 200 μg / mL had a negative impact on cell activity. When the concentrations were 1 μg / mL and 50 μg / mL, there were no significant differences compared with the Control group, suggesting that it had little effect on cell survival. Therefore, 1 μg / mL, 10 μg / mL, and 50 μg / mL were used as the sample concentrations for subsequent experiments.

[0107] (2) Effects of peptides GG-15, FDGLQ, and GG-10 on the differentiation of MC3T3-E1 cells

[0108] Seed the cells in a 6-well plate (1×10 4 cells per well), and after overnight culture, change to osteogenic induction medium (containing peptides at different concentrations (0, 1, 10, 50 μg / mL)) for culture. Continuously treat with osteogenic induction medium containing different concentrations of peptides for 7 days, and change the medium every 2 days. After 7 days, remove the medium and wash with distilled water. Prepare the ALP staining working solution according to the kit instructions, add 200 μL to each well, incubate in a 37°C incubator for 15 - 20 min, and wash with distilled water. Stain with methyl green counterstain solution for 3 - 5 min, wash slightly with distilled water, and observe under a microscope. Culture as above, and after 7 days, discard the old medium. Operate according to the ALP activity detection kit.

[0109] During bone repair and formation, osteoblasts undergo proliferation, differentiation, and mineralization stages. The activity of Akaie phos phase (ALP) reflects the degree of osteoblast differentiation. The higher the ALP activity, the later the osteoblast differentiation stage. Figures 16 - 18 Shows the effects of GG-15, FDGLQ, and GG-10 on the differentiation of MC3T3-E1 cells. It can be seen that as the concentration of peptide GG-15 increased, the ALP activity gradually increased and the ALP staining was significantly enhanced. When the concentration of peptide GG-15 was 50 μg / mL, the ALP activity reached the maximum and the ALP staining was more obvious, indicating that peptides GGPGHQGPGGMPGER, FDGLQ, and GHPGPKGMKG promoted the differentiation of MC3T3-E1 cells.

[0110] (3) Effects of peptides GG-15, FDGLQ, and GG-10 on the mineralization of MC3T3-E1 cells

[0111] Seed the cells in a 96-well plate (1×10 4 cells per well), and after culturing for 24 h, replace the medium with osteogenic induction medium (containing peptides at different concentrations (0, 1, 10, 50 μg / mL)) for culture. After 21 days of culture, aspirate the culture medium, add 1 mL of 4% paraformaldehyde to each well, fix at room temperature for 30 min, discard the supernatant, wash 3 times with PBS, add 0.1% alizarin red staining solution and stain in the dark for 30 min, then aspirate the staining solution. Observe under a microscope and take pictures for archiving.

[0112] Figures 19 - 21 They are the cell mineralization results corresponding to peptides GG-15, FDGLQ, and GG-10 respectively. The calcium deposition reflects the mineralization level of osteoblasts. Higher calcium deposition indicates a higher degree of osteoblast mineralization. The results show that the staining intensity of mineralized calcium nodules in cells treated with peptides GG-15, FDGLQ, and GG-10 is significantly greater than that of cells with a peptide concentration of 0. It indicates that peptides GG-15, FDGLQ, and GG-10 have a significant promoting effect on the mineralization of MC3T3-E1 cells.

[0113] (4) Effects of peptides GG-15, FDGLQ, and GG-10 on cell differentiation, pathway genes, and proteins

[0114] Seed the cells in a 6-well plate (1×10 4(After overnight culture, the medium was replaced with osteogenic induction medium (containing peptides at different concentrations (0, 1, 10, 50 μg / mL)) for culture. The cells were divided into a control group (Control) and a model group, and the intervention continued for 7 days. The medium was changed every 2 days. After 7 days, the culture medium in the culture plate was completely aspirated with a micropipette, 1 mL of PBS solution pre-cooled at 4°C was added, and the plate was gently shaken for washing. The PBS was completely aspirated with a micropipette, 1 mL of RNA extraction solution was added, and the mixture was gently shaken and vortexed to lyse the cells. The liquid was transferred into a 1.5 mL centrifuge tube, 100 μL of chloroform substitute was added, the centrifuge tube was inverted for 15 s, thoroughly mixed, and allowed to stand for 3 min. Centrifugation was performed at 12,000 rpm for 10 min at 4°C. 400 μL of the supernatant was transferred to a new centrifuge tube, 550 μL of isopropanol was added, the mixture was inverted and mixed, and placed at -20°C for 15 min. Centrifugation was performed at 12,000 rpm for 10 min at 4°C, and the white precipitate was RNA. The liquid was aspirated, 1 mL of 75% ethanol was added, the precipitate was inverted and mixed for washing, and centrifugation was performed at 12,000 rpm for 5 min at 4°C. The liquid was completely aspirated, the centrifuge tube was placed on the laminar flow bench and blown for 3 - 5 min, and 15 μL of RNA lysate was added to dissolve the RNA. The concentration and purity of RNA were detected using Nanodrop 2000. RNA with too high a concentration was diluted at an appropriate ratio to a final concentration of 200 ng / μL for reverse transcription. Reverse transcription was completed on a conventional PCR instrument and amplification was completed on a fluorescence quantitative PCR instrument.)

[0115] (The differentiation and mineralization of MC3T3-E1 cells are crucial in the process of bone formation. In this invention, the expressions of two related genes ALP and RUNX2 involved in osteoblast differentiation and the expressions of genes OCN and BMP2 related to bone matrix secretion were analyzed. The effects of osteogenic peptides GG-15, FDGLQ, and GG-10 on the differentiation of MC3T3-E1 cells, MAPK pathway genes, and proteins are shown in Figures 22 - 27As shown. The results showed that osteogenic peptides GG-15, FDGLQ, and GG-10 significantly promoted the expression of ALP gene, indicating that peptide GG-15 might promote the differentiation of osteoblasts, which was consistent with the previously observed elevation. The gene and protein expressions of RUNX2 and BMP2 were also significantly increased, further indicating that peptides GG-15, FDGLQ, and GG-10 promoted the differentiation and mineralization of MC3T3-E1 cells. Peptides GG-15, FDGLQ, and GG-10 significantly affected the gene and protein expressions of ERK1 and had a significant promoting effect on the expression of p-ERK1 / 2 protein, which indicated that the treatment with peptides GG-15, FDGLQ, and GG-10 could also activate the ERK1 / 2 pathway in osteoblasts, and peptide GG-15 up-regulated the expressions of p38 and JNK2 genes. ERK1 was involved in regulating cell proliferation and differentiation, p38 was involved in managing inflammation and apoptosis, and the JNK family played a crucial role in the cellular stress response. Therefore, peptides GG-15, FDGLQ, and GG-10 might affect the proliferation of MC3T3-E cells through the MAPK pathway. From the perspective of gene and protein expressions, peptides GG-15, FDGLQ, and GG-10 mainly promoted cell proliferation and differentiation by enhancing the expressions of ERK1 and JNK1. Subsequently, they induced the changes of osteoblasts towards bone morphogenesis through the expression levels of ALP and BMP2, thereby preventing osteoporosis and improving bone health.

[0116] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An osteoblast proliferation-promoting peptide, characterized in that, The amino acid sequence is as shown in any one of SEQ ID NO.1-3.

2. The osteoblast proliferation-promoting peptide according to claim 1, characterized in that, The osteoblast proliferation-promoting peptide is derived from the scales, skin and bones of mullet; when the osteoblast proliferation-promoting peptide is derived from mullet scales, its amino acid sequence is as shown in SEQ ID NO.1; when the osteoblast proliferation-promoting peptide is derived from mullet skin, its amino acid sequence is as shown in SEQ ID NO.2; when the osteoblast proliferation-promoting peptide is derived from mullet bones, its amino acid sequence is as shown in SEQ ID NO.

3.

3. The preparation method of the osteoblast proliferation-promoting peptide according to claim 1 or 2, characterized in that, Collagen is extracted by pepsin-assisted acid method and then enzymatically hydrolyzed by alkaline protease to obtain the osteoblast proliferation-promoting peptide.

4. The preparation method according to claim 3, characterized in that, The preparation of the collagen comprises the following steps: Wash the scales, skin or bones of mullet, add acetic acid solution with a concentration of 0.5 mol / L, the material-liquid ratio is 1:20-1:55 g / mL, enzymatically hydrolyze for 18-34 h, and the enzyme addition amount is 1-3.5%.

5. The preparation method according to claim 3, characterized in that, When the collagen is derived from mullet scales or skin, the preparation of the collagen comprises the following steps: Wash the scales or skin of mullet, add acetic acid solution with a concentration of 0.5 mol / L, the material-liquid ratio is 1:30 g / mL, enzymatically hydrolyze for 24 h, and the enzyme addition amount is 2%; when the collagen is derived from mullet bones, the preparation of the collagen comprises the following steps: Wash the mullet bones and powder them, add acetic acid solution with a concentration of 0.5 mol / L, the material-liquid ratio is 1:45 g / mL, enzymatically hydrolyze for 28 h, and the enzyme addition amount is 2.5%.

6. The preparation method according to claim 3, wherein The addition amount of the alkaline protease is 3-7%; the pH during enzymatic hydrolysis is 8-11, and the enzymatic hydrolysis temperature is 35-55 °C.

7. The preparation method according to claim 3, characterized in that, When the collagen is derived from mullet scales, the addition amount of the alkaline protease is 5%, the pH during enzymatic hydrolysis is 9, and the enzymatic hydrolysis temperature is 45 °C; when the collagen is derived from mullet skin, the addition amount of the alkaline protease is 4%, the pH during enzymatic hydrolysis is 10, and the enzymatic hydrolysis temperature is 50 °C; when the collagen is derived from mullet bones, the addition amount of the alkaline protease is 4%, the pH during enzymatic hydrolysis is 9, and the enzymatic hydrolysis temperature is 45 °C.

8. Use of the osteoblast proliferation-promoting peptide according to claim 1 or 2 or the osteoblast proliferation-promoting peptide prepared by the preparation method according to any one of claims 3-7 in the preparation of a product for promoting osteoblast proliferation.

9. The application according to claim 8, wherein The promotion of osteoblast proliferation includes promoting the proliferation, differentiation and mineralization of MC3T3-E1 cells.

10. Use of the osteoblast proliferation-promoting peptide according to claim 1 or 2 or the osteoblast proliferation-promoting peptide prepared by the preparation method according to any one of claims 3-7 in the preparation of a product for preventing and / or treating osteoporosis.

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