Active peptide for promoting bone growth and preparation method and application thereof

Abstract

The application discloses an active peptide for promoting bone growth, and a preparation method and application thereof. The active peptide contains amino acid sequences GPAGPLGPV, LGVPLPL, FLPEPPQEE and SFLPEPPQE, and is derived from an enzymatic product of animal bone collagen. The average molecular weight of the enzymatic product is 500-1000 Da, and the mass percentage of components with a molecular weight less than 1000 Da is greater than 60%. The preparation method comprises the following steps: sequentially performing acid treatment, alkali treatment, extraction, filtration and ion exchange on animal bone, and then performing enzymolysis on the animal bone under the action of a protease to obtain an enzymolysis solution, wherein the protease is selected from alkaline protease, trypsin and papain; and the enzymolysis solution is separated and purified, wherein the separation and purification comprises ultrafiltration membrane grading and chromatographic separation, the cut-off molecular weight of the ultrafiltration membrane is 3kDa, and the chromatographic separation comprises gel chromatography and reverse-phase high performance liquid chromatography separation. The low-molecular active peptide obtained by the application can promote the proliferation and / or differentiation of osteoblasts, improve the alkaline phosphatase activity, has a small molecular weight and is easy to be absorbed and utilized by the body, has a wide raw material source, has a simple and controllable preparation process, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of bio-food technology, and in particular to an active peptide that promotes bone growth, its preparation method, and its application. Background Technology

[0002] Bone tissue is a crucial foundation for maintaining the structural integrity and motor function of the human body. Its growth, development, and remodeling depend on the dynamic balance between osteoblasts and osteoclasts. With the aging population and changing lifestyles, the incidence of osteoporosis, osteopenia, and osteodystrophy is rising annually, becoming a significant issue affecting human health. Currently, clinical and nutritional interventions mainly rely on calcium supplements, vitamin D, and hormones, but these methods generally suffer from limited absorption and utilization, high risk of side effects, and limited mechanisms of action, making it difficult to effectively promote bone formation and achieve precise regulation of bone metabolism. Therefore, developing safe and efficient novel bone health promoting factors is of great significance.

[0003] Collagen is a major component of the extracellular matrix of bone tissue, with type I collagen accounting for over 90% of the organic components of bone and playing a crucial role in bone mineralization, cell adhesion, and proliferation. In recent years, low-molecular-weight peptides obtained from enzymatic hydrolysis of collagen have become a research hotspot due to their good solubility, easy absorption, and high bioavailability. Existing studies have shown that collagen peptides can promote osteoblast proliferation, increase alkaline phosphatase activity, and regulate the expression of related genes, demonstrating a potential to promote bone formation to some extent.

[0004] Although existing research and related patents have touched upon the application of collagen peptides in bone health, there are still significant shortcomings overall. On the one hand, most technical solutions still focus on collagen protein hydrolysates or low-molecular-weight peptide mixtures, which have complex compositions, unclear active ingredients, and lack the isolation and identification of specific functional peptide sequences, making it difficult to establish a clear structure-activity relationship. On the other hand, existing research mostly focuses on evaluating macroscopic indicators such as cell proliferation or enzyme activity, lacking in-depth analysis of their mechanisms of action, especially at the level of key regulatory targets, such as signaling pathways closely related to cell proliferation and differentiation, such as epidermal growth factor receptor (EGFR), for which systematic research on the interaction between small molecule peptides derived from bone collagen and these pathways is still lacking. In addition, the active peptides screened in existing reports are mostly relatively large polypeptide fragments with high molecular weights, which have certain limitations in terms of in vivo absorption efficiency and bioavailability. High-purity, structurally well-defined small molecule functional peptide products are still relatively scarce, restricting their further application in functional foods and precision nutrition.

[0005] Therefore, there is an urgent need to develop a functional bioactive peptide derived from bone collagen, with a well-defined structure, small molecular weight, and a clear mechanism of action, and to establish a systematic technical system for its preparation, separation and purification, structural identification, and functional evaluation, so as to overcome the problems of unclear active ingredients, unclear mechanisms of action, and limited bioavailability in existing technologies, thereby providing new technical support for the development of bone health functional products. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an active peptide that promotes bone growth, as well as a method for preparing the active peptide and its application. The active peptide contains low-molecular-weight active peptides that can promote the proliferation and / or differentiation of osteoblasts, and the preparation method is simple and controllable, with readily available raw materials, which is expected to realize the high-value utilization of bone collagen resources.

[0007] This invention is achieved through the following technical solution: An active peptide that promotes bone growth, the active peptide containing the amino acid sequence GPAGPLGPV (Gly-Pro-Ala-Gly-Pro-Leu-Gly-Pro-Val) as shown in SEQ ID NO: 1, the amino acid sequence LGVPLPL (Leu-Gly-Val-Pro-Leu-Pro-Leu) as shown in SEQ ID NO: 2, the amino acid sequence FLPEPPQEE (Phe-Leu-Pro-Glu-Pro-Pro-Gln-Glu-Glu) as shown in SEQ ID NO: 3, and the amino acid sequence SFLPEPPQE (Ser-Phe-Leu-Pro-Glu-Pro-Pro-Gln-Glu) as shown in SEQ ID NO: 4.

[0008] Furthermore, the active peptide is derived from the enzymatic hydrolysis product of animal bone collagen, and the average molecular weight of the enzymatic hydrolysis product is 500-1000 Da, wherein the mass percentage of the component with a molecular weight less than 1000 Da is greater than 60%.

[0009] A method for preparing the above-mentioned bioactive peptide that promotes bone growth includes the following steps: Animal bones are subjected to acid treatment and alkali treatment in sequence, and then extracted, filtered, ion exchanged and dried to obtain bone collagen. The collagen protein was enzymatically hydrolyzed under the action of protease to obtain collagen protein hydrolysate; The collagen protein hydrolysate is separated and purified to obtain the active peptide. Animal bone can be bovine bone, pig bone, sheep bone, etc.

[0010] Furthermore, the protease is selected from one or more of alkaline protease, trypsin, and papain, and the enzymatic hydrolysis conditions are: pH 7.0-9.0, temperature 45-60℃, and hydrolysis time 2-4h.

[0011] Furthermore, the method for enzymatically hydrolyzing the collagen protein under the action of proteases is as follows: The collagen protein was added to deionized water at a material-to-liquid ratio of 1:20 (g / mL), the pH was adjusted to 8.0, alkaline protease was added at 2% of the substrate mass, and the mixture was enzymatically hydrolyzed at 50°C for 3 hours. Then adjust the pH to 7.5, add trypsin at 1% of the substrate mass, and continue enzymatic hydrolysis for 2 hours; After enzymatic hydrolysis, the system is heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, it is centrifuged, and the supernatant is collected to obtain the collagen protein hydrolysate.

[0012] Furthermore, the separation and purification includes ultrafiltration membrane fractionation and chromatographic separation, wherein the ultrafiltration membrane has a molecular weight cutoff of 3 kDa, and the chromatographic separation includes gel chromatography and reversed-phase high-performance liquid chromatography.

[0013] Furthermore, the method for separating and purifying the collagen protein hydrolysate is as follows: The collagen protein hydrolysate was fractionated by ultrafiltration membrane, and the permeate with a molecular weight of less than 3kDa was collected. The permeate was then freeze-dried to obtain crude peptide powder. Crude peptide powder was dissolved in deionized water and separated by Sephadex G-15 gel chromatography with distilled water as the mobile phase and a flow rate of 0.5 mL / min. The components corresponding to each elution peak at a wavelength of 220 nm were collected. Each component was initially screened for activity, and the components with activity higher than the set value were further separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic column was a C18 column, and the mobile phase was 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B). Gradient elution was used, and single peak components were collected and lyophilized for storage.

[0014] Furthermore, the method of sequentially treating animal bones with acid and then with alkali is as follows: The dried bone granules are placed in food-grade hydrochloric acid with a mass concentration of 3%-6%, and the material-to-liquid ratio is 1:8-10 (g / mL). After soaking at a temperature of 10-15℃ for 3-7 days, the granules are washed with water until the pH reaches 3.5-4.5. Then immerse it in lime water with a mass concentration of 1.5%-4.0%, with a material-to-liquid ratio of 1:6-8 (g / mL). Soak it at a temperature of 8-12℃ for 25-45 days, and then wash it with water until the pH reaches 6.0-6.5.

[0015] Furthermore, the method for obtaining collagen protein through sequential extraction, filtration, ion exchange, and drying is as follows: After alkali treatment, bone particles are added to deionized water at a material-to-liquid ratio of 1:1.5-2 (g / mL), with pH controlled at 4.5-5.5. Extraction is carried out by distillation at 55-90℃ under normal pressure to obtain a dilute gel solution with a mass concentration of 4%-5%. The diluted filtrate was then filtered through cotton cake. The resulting clear and transparent filtrate was then desalted sequentially through a strong acid cation exchange column and a strong base anion exchange column. The filtrate temperature was 30-40℃, the flow rate was 1.5-3.0 BV / h, and the final ash content was controlled to be ≤0.5%, conductivity ≤50μS / cm, and pH 4.8-5.2. After vacuum concentration and drying, collagen is obtained.

[0016] The application of the above-mentioned active peptide in the preparation of a product for promoting bone growth, wherein the product is a functional food, a special medical food, or a nutritional supplement.

[0017] This invention obtains an active peptide composition through specific raw material processing, enzymatic hydrolysis, purification, and separation processes. Molecular docking studies of low-molecular-weight active peptides (GPAGPLGPV, LGVPLPL, FLPEPPQEE, SFLPEPPQE) with defined amino acid sequences from this active peptide composition have revealed that these active peptides can stably bind to the epidermal growth factor receptor (EGFR), promoting osteoblast proliferation and / or differentiation, and increasing alkaline phosphatase activity. This results in their role in promoting bone formation, improving bone density, and preventing osteoporosis. Furthermore, the prepared active peptides have small molecular weights, simple structures, good water solubility, and potentially high bioavailability, making them easier for the body to absorb and utilize. They can be used in products that promote bone growth, such as functional foods, medical foods, or nutritional supplements. The raw materials are widely available, and the preparation process is controllable, making them suitable for industrial production. They have promising application prospects in the development of bone health functional foods and related products.

[0018] This invention overcomes the limitation of unclear active ingredients in traditional collagen peptides, helping to establish a clear structure-function relationship and improve the controllability and stability of the product. At the same time, this invention combines molecular docking technology and cell experiments to reveal the interaction mechanism between active peptides and EGFR receptors at the molecular level, clarifying that it may promote osteoblast proliferation and differentiation by regulating related signaling pathways, making up for the lack of clear mechanism of action in the prior art and enhancing the scientific nature and innovation of the technical solution. Attached Figure Description

[0019] Figure 1 This is a molecular weight distribution diagram of the collagen protein hydrolysate obtained in Example 1 of the present invention.

[0020] Figure 2 Mass spectra of four peptides: GPAGPLGPV (A), LGVPLPL (B), FLPEPPQEE (C), and SFLPEPPQE (D).

[0021] Figure 3 Global and detailed images of the docking of four peptides, GPAGPLGPV (A), LGVPLPL (B), FLPEPPQEE (C), and SFLPEPPQE (D), with EGFR. Detailed Implementation

[0022] Example 1: Extraction and enzymatic hydrolysis of collagen Animal bones were subjected to acid treatment and alkali treatment in sequence, followed by extraction, filtration, ion exchange and drying to obtain bone collagen. The bone collagen was then enzymatically hydrolyzed under the action of proteases to obtain bone collagen hydrolysate.

[0023] Specifically, the steps include the following: Add dried bone particles (bovine bones) to food-grade hydrochloric acid with a mass concentration of 3%-6% at a material-to-liquid ratio of 1:8-10 (g / mL), soak at a temperature of 10-15℃ for 3-7 days, and then wash with water until the pH reaches 3.5-4.5. Soak the mixture in lime water with a mass concentration of 1.5%-4.0% (pH 12.0-12.5), at a material-to-liquid ratio of 1:6-8 (g / mL), at a temperature of 8-12℃ for 25-45 days, and then wash it with water until the pH reaches 6.0-6.5. After alkali treatment, bone particles are added to deionized water at a material-to-liquid ratio of 1:1.5-2 (g / mL), and the pH is controlled at 4.5-5.5. The mixture is then extracted by distillation at atmospheric pressure at a temperature of 55-90℃ to obtain a dilute gel solution with a mass concentration of 4%-5%. The diluted filtrate was then filtered using cotton cake. The resulting clear and transparent filtrate was then desalted sequentially through a strongly acidic cation exchange column and a strongly basic anion exchange column. The filtrate temperature was 30-40℃, and the flow rate was 1.5-3.0 BV / h. The final ash content was controlled to be ≤0.5%, conductivity ≤50μS / cm, and pH 4.8-5.2. Cotton cake, as a fibrous filter medium, can effectively filter and remove impurities from the filtrate. After vacuum concentration and drying, collagen is obtained; Add collagen to deionized water at a material-to-liquid ratio of 1:20 (g / mL), adjust the pH to 8.0, add alkaline protease at 2% of the substrate mass, and enzymatically hydrolyze at 50℃ for 3 hours. Then adjust the pH to 7.5, add trypsin at 1% of the substrate mass, and continue enzymatic hydrolysis for 2 hours; After enzymatic hydrolysis, the system is heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, it is centrifuged, and the supernatant is collected to obtain the collagen protein hydrolysate.

[0024] The molecular weight and molecular weight distribution of collagen protein hydrolysate were determined using the following method: The collagen protein hydrolysate was dissolved in deionized water at a concentration of 2-5 mg / mL and filtered through a 0.45 μm filter to remove particulate impurities. Analysis was performed using a gel filtration chromatography (GFC) system with a TSKG2000SWXL or similar column. The mobile phase was acetonitrile / water / trifluoroacetic acid = 45 / 55 / 0.1 (V / V / V), the flow rate was 0.5 mL / min, and the detection wavelength was 220 nm. A standard curve was plotted using the molecular weights of standard proteins or peptides (such as cytochrome C, aprotinin, acetaminophen-acetaminophen-tyrosine-arginine, acetaminophen-acetaminophen-acetaminophen, etc.) as molecular weight versus retention time, and the molecular weight distribution range was calculated based on the retention times of the sample peaks.

[0025] The molecular weight distribution spectrum of the obtained enzyme hydrolysate is as follows: Figure 1 The results are shown in Table 1.

[0026] Table 1. Molecular weight distribution of collagen protein hydrolysate obtained in Example 1

[0027] As shown in Table 1, the molecular weight distribution of the collagen enzymatic hydrolysate is predominantly low molecular weight peptides, with components having a molecular weight less than 1000 Da accounting for approximately 63.05%. This indicates that the hydrolysate exhibits significant small molecular weight characteristics and is mainly enriched in the low molecular weight oligopeptide region. Gel permeation chromatography (GPC) analysis confirmed that the average molecular weight of the hydrolysate is 500-1000 Da.

[0028] Example 2: Isolation and purification of bioactive peptides The enzymatic hydrolysate obtained in Example 1 was fractionated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. Permeate with a molecular weight less than 3 kDa was collected. This permeate was lyophilized to obtain crude peptide powder. The crude peptide powder was dissolved in deionized water and separated by Sephadex G-15 gel chromatography with distilled water as the mobile phase at a flow rate of 0.5 mL / min. The fractions corresponding to each elution peak were collected based on absorbance (220 nm). Initial activity screening was performed on each fraction, and fractions with activity higher than a set value were further separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column and a mobile phase of 0.1% trifluoroacetic acid aqueous solution (A) and acetonitrile (B). Gradient elution was used, and single peak fractions were collected and lyophilized for storage.

[0029] Example 3: Identification of bioactive peptides The fractions obtained after separation and purification in Example 2 were sequenced using nano-LC-MS / MS. After chromatographic separation, the fractions were analyzed by secondary mass spectrometry. The raw data were then subjected to database matching and de novo sequencing analysis. The specific method is as follows: (1) The lyophilized active peptide powder obtained by RP-HPLC purification was dissolved in 0.1% formic acid aqueous solution at a concentration of 0.1 mg / mL, and impurities were removed by filtration through a 0.22 μm filter membrane. Subsequently, the sample was injected into a nano-RP-HPLC system for separation. The chromatographic column was a C18 nanocolumn (75 μm × 150 mm, 3 μm particle size). The mobile phase system was A phase 0.1% formic acid aqueous solution and B phase 0.1% formic acid acetonitrile solution. Gradient elution was performed from 5% B to 35% B at a flow rate of 300 nL / min. The eluted peptide fragments were directly introduced into a high-resolution tandem mass spectrometer and scanned in the range of 350-2000 m / z in positive ion mode. The selected peptide ions were fragmented by HCD, and secondary mass spectrometry (MS / MS) information was obtained.

[0030] (2) Import the raw data obtained from mass spectrometry into Proteome Discoverer or Mascot software for analysis. First, search the database (select the animal collagen database, allowing modifications such as hydroxyproline) to match known peptide sequences. For unmatched peptides, use de novo sequencing to deduce the sequence. Set the parent ion mass deviation to ±10ppm and the fragment ion deviation to ±0.02Da to ensure accurate and reliable sequence identification.

[0031] A total of 2151 peptides were identified using the above method. Peptides with an ALC score greater than 95% were screened, and their activity was further predicted using Peptide Ranker with a threshold of 0.8, resulting in 21 peptides with high potential bioactivity. Based on this, the toxicity, solubility, sensitization, and intestinal absorption characteristics of these peptides were comprehensively evaluated using tools such as ToxinPred, INNOVAGEN, Allergen FP, and ADMET Lab 3.0. Finally, four short peptides with excellent overall performance were selected: GPAGPLGPV, LGVPLPL, FLPEPPQEE, and SFLPEPPQE, for subsequent functional studies. Their corresponding mass spectra are shown below. Figure 2 As shown.

[0032] Example 4: Molecular docking analysis Molecular docking analysis of the above four bioactive peptides with epidermal growth factor receptor (EGFR) was performed using molecular docking software. The results are shown in Table 2.

[0033] The molecular docking analysis method is as follows: Molecular docking analysis was performed on the screened bioactive peptides and epidermal growth factor receptor (EGFR). First, the EGFR crystal structure was obtained from a protein database and subjected to dehydration, hydrogenation, and energy optimization to determine the active site. The target peptide molecule underwent conformation optimization and energy minimization. Flexible molecular docking was then performed between the peptide and receptor within a designated binding pocket. Binding energy and interaction modes were calculated using software such as AutoDock or Discovery Studio, and hydrogen bonds, hydrophobic interactions, and other non-covalent interactions were analyzed. The most stable binding conformation was screened using a scoring function, and the binding ability of the peptide to EGFR was evaluated based on binding energy and binding mode, providing a molecular mechanism basis for the in vitro bone-promoting function of the bioactive peptides.

[0034] Table 2. Molecular docking results of four bioactive peptides with EGFR

[0035] As shown in Table 2, the molecular docking results indicate that all four bioactive peptides can form stable binding bonds with the epidermal growth factor receptor (EGFR), with all binding energies being negative (-7.284 to -8.571 kJ / mol), indicating that the binding process is thermodynamically spontaneous. Among them, GPAGPLGPV (-8.571 kJ / mol) and SFLPEPPQE (-8.464 kJ / mol) exhibit lower binding energies, indicating higher binding stability with EGFR, while LGVPLPL has a relatively higher binding energy (-7.284 kJ / mol), indicating slightly weaker binding ability.

[0036] From the perspective of interaction types, hydrogen bonds play a dominant role in stabilizing peptide-receptor complexes. SFLPEPPQE forms the most hydrogen bonds (13), followed by FLPEPPQEE (11), while GPAGPLGPV and LGVPLPL form 6 and 3 hydrogen bonds respectively, indicating that the former two are more spatially conformationally favorable for forming multi-site binding with EGFR, thereby improving complex stability. In addition, SFLPEPPQE also forms a salt bridge (ARG84), further enhancing its binding strength with EGFR. In contrast, although GPAGPLGPV has fewer hydrogen bonds, its binding energy is the lowest, which may be related to the key site hydrogen bonds (such as TYR251, ARG285, etc.) and hydrophobic interactions formed.

[0037] Hydrophobic interactions also play a crucial role in stabilizing the complex. All four peptides interact with hydrophobic amino acid residues in EGFR (such as LEU, PHE, ALA, and TYR). GPAGPLV and SFLPEPPQE involve multiple hydrophobic interaction sites and interact with key residues (such as TYR251 and ALA265), further enhancing binding stability. Notably, TYR251 and ALA265 appear repeatedly in the binding of multiple peptides, suggesting they may be key binding sites for EGFR.

[0038] SFLPEPPQE and GPAGPLGPV exhibited strong overall advantages in binding energy, number of hydrogen bonds, and interaction type, suggesting a higher affinity for EGFR, potentially leading to more effective regulation of related signaling pathways and thus promoting osteoblast proliferation and differentiation. FLPEPPQEE followed, while LGVPLPL showed relatively weaker affinity. These results are consistent with the bioactivity of different peptides observed in subsequent cell experiments, providing molecular-level support for their mechanisms of action.

[0039] Example 5: Evaluation of osteoblast proliferation-promoting activity Four short peptides, GPAGPLGPV, LGVPLPL, FLPEPPQEE, and SFLPEPPQE, were synthesized using a solid-phase synthesis method. These peptides were then compared with the collagen protein hydrolysate obtained in Example 1 to detect osteoblast proliferation activity. The results are shown in Table 3.

[0040] The method for detecting osteoblast proliferation activity is as follows: Log-phase MC3T3-E1 osteoblasts were collected, digested, resuspended, and counted, and then analyzed at a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density equal to the number of wells in 96-well plates and cultured until approximately 90% confluence was achieved, at which point they were divided into three groups: a control group, a model group, and a treatment group. The control group was cultured in complete medium for 36 hours; the model group was treated with complete medium containing 50 μmol / L dexamethasone for 36 hours; and the treatment group was treated with complete medium containing 50 μmol / L dexamethasone and the sample for 36 hours. After cell culture, cell viability was determined using the CKK assay, and each experiment was performed six times.

[0041] The cell culture method is as follows: Mouse osteogenic progenitor cells MC3T3-E1 were selected as model cells. The cells were seeded in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and cultured in a constant temperature incubator at 37°C and 5% CO2. When the cells grew to the logarithmic growth phase, they were passaged or used for subsequent experiments. Before the experiment, the cells were acclimatized to serum-free medium for 2-4 hours to eliminate serum interference.

[0042] Table 3. Effects of Example 1 and Synthetic Peptides on Osteoblast Proliferation Rate

[0043] As shown in Table 3, the cell proliferation rate in the model group was only 61.2%, significantly lower than that in the control group (100%), indicating that dexamethasone treatment successfully inhibited osteoblast proliferation activity, and the model construction was effective. The proliferation rate in Example 1 group was 85.4%, significantly higher than that in the model group, indicating that the overall collagen protein hydrolysate had a certain proliferative effect, but its effect was still lower than that of the control group. Further comparison of the effects of each single short peptide revealed that GPAGPLGPV, FLPEPPQEE, and SFLPEPPQE all significantly enhanced osteoblast proliferation, with proliferation rates reaching 95.1%, 93.6%, and 94.5%, respectively, all close to the control group level and significantly better than that of Example 1 group, indicating that specific sequence peptides obtained through isolation, purification, and screening have stronger biological activity than mixed peptides. Among them, GPAGPLGPV showed the best proliferative effect, suggesting that it may be a key functional peptide for bone promotion. The above results indicate that small molecule specific short peptides have higher activity in promoting osteoblast proliferation, which may be related to their simple structure, easy uptake by cells, and easier interaction with cell surface receptors.

[0044] Example 6: Evaluation of osteogenic differentiation function The alkaline phosphatase (ALP) activity of the four short peptides GPAGPLGPV, LGVPLPL, FLPEPPQEE, and SFLPEPPQE synthesized in solid phase, as well as the collagen protein hydrolysate obtained in Example 1, was measured. The results are shown in Table 4.

[0045] The method for measuring alkaline phosphatase (ALP) activity is as follows: Logarithmic growth phase MC3T3-E1 osteoblasts were used at 5×10 4 cells / cm 2 Cells were seeded at high density into six-well plates and cultured in complete culture medium. When the cells reached approximately 90% confluence, they were divided into three groups: a control group, a model group, and a treatment group. The control group was cultured in differentiation medium for 7 days; the model group was cultured in dexamethasone medium for 7 days; and the treatment group was cultured in medium containing the sample for 7 days. After culture, the culture medium in the wells was discarded, and the cells were washed three times with PBS. The plates were placed on ice, and 200 μL of IRIPA cell lysis buffer was added to each well. The cells were gently scraped off and transferred to centrifuge tubes. After centrifugation for 20 min (4℃, 10000×g), the supernatant was collected, and ALP activity was measured using an ALP assay kit. Six replicates were performed for each group.

[0046] Table 4. Effects of Example 1 and the synthesized peptide on alkaline phosphatase activity

[0047] Table 4 shows that the ALP activity in the blank group was 65.4 U / mL, while that in the model group was only 31.5 U / mL, a significant decrease, indicating that dexamethasone treatment significantly inhibited osteoblast differentiation. Compared with the model group, the ALP activity in the Example 1 group increased to 51.3 U / mL, indicating that the collagenase hydrolysate as a whole has a certain osteogenic differentiation-promoting effect, but it has not yet returned to normal levels. Further comparison of the effects of each single peptide revealed that GPAGPLGPV, LGVPLPL, FLPEPPQEE, and SFLPEPPQE all significantly increased ALP activity, reaching 63.5 U / mL, 67.2 U / mL, 62.9 U / mL, and 69.9 U / mL, respectively, all close to or even exceeding the levels of the blank group. Among them, the SFLPEPPQE group had the highest ALP activity, followed by LGVPLPL, indicating that these two peptides have a stronger effect in promoting osteoblast differentiation. Compared with Example 1, each single short peptide treatment group showed a better promoting effect, indicating that the specific sequence peptides obtained by isolation and purification have higher biological activity than the mixed peptides.

[0048] The above detailed description is a specific description of feasible embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. An active peptide that promotes bone growth, characterized in that, The active peptide contains the amino acid sequence GPAGPLGPV as shown in SEQ ID NO: 1, the amino acid sequence LGVPLPL as shown in SEQ ID NO: 2, the amino acid sequence FLPEPPQEE as shown in SEQ ID NO: 3, and the amino acid sequence SFLPEPPQE as shown in SEQ ID NO:

4.

2. The bioactive peptide for promoting bone growth according to claim 1, characterized in that, The active peptide is derived from the enzymatic hydrolysis product of animal bone collagen. The average molecular weight of the enzymatic hydrolysis product is 500-1000 Da, and the mass percentage of the component with a molecular weight less than 1000 Da is greater than 60%.

3. A method for preparing an active peptide for promoting bone growth as described in claim 1 or 2, characterized in that, Includes the following steps: Animal bones are subjected to acid treatment and alkali treatment in sequence, and then extracted, filtered, ion exchanged and dried to obtain bone collagen. The collagen protein was enzymatically hydrolyzed under the action of protease to obtain collagen protein hydrolysate; The collagen protein hydrolysate was separated and purified to obtain the active peptide.

4. A method for preparing an active peptide that promotes bone growth according to claim 3, characterized in that, The protease is selected from one or more of alkaline protease, trypsin, and papain, and the enzymatic hydrolysis conditions are: pH 7.0-9.0, temperature 45-60℃, and hydrolysis time 2-4h.

5. The method for preparing an active peptide that promotes bone growth according to claim 3, characterized in that, The method for enzymatic hydrolysis of the collagen protein under the action of proteases is as follows: The collagen protein was added to deionized water at a material-to-liquid ratio of 1:20 (g / mL), the pH was adjusted to 8.0, alkaline protease was added at 2% of the substrate mass, and the mixture was enzymatically hydrolyzed at 50°C for 3 hours. Then adjust the pH to 7.5, add trypsin at 1% of the substrate mass, and continue enzymatic hydrolysis for 2 hours; After enzymatic hydrolysis, the system is heated to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, it is centrifuged, and the supernatant is collected to obtain the collagen protein hydrolysate.

6. The method for preparing an active peptide that promotes bone growth according to claim 3, characterized in that, The separation and purification includes ultrafiltration membrane fractionation and chromatographic separation. The ultrafiltration membrane has a molecular weight cutoff of 3 kDa, and the chromatographic separation includes gel chromatography and reversed-phase high-performance liquid chromatography.

7. The method for preparing an active peptide that promotes bone growth according to claim 6, characterized in that, The method for separating and purifying the collagen protein hydrolysate is as follows: The collagen protein hydrolysate was fractionated by ultrafiltration membrane, and the permeate with a molecular weight of less than 3kDa was collected. The permeate was then freeze-dried to obtain crude peptide powder. Crude peptide powder was dissolved in deionized water and separated by Sephadex G-15 gel chromatography with distilled water as the mobile phase and a flow rate of 0.5 mL / min. The components corresponding to each elution peak at a wavelength of 220 nm were collected. Each component was initially screened for activity, and the components with activity higher than the set value were further separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic column was a C18 column, and the mobile phase was 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B). Gradient elution was used, and single peak components were collected and lyophilized for storage.

8. The method for preparing an active peptide that promotes bone growth according to claim 3, characterized in that, The method for sequentially treating animal bones with acid and then with alkali is as follows: The dried bone granules are placed in food-grade hydrochloric acid with a mass concentration of 3%-6%, and the material-to-liquid ratio is 1:8-10 (g / mL). After soaking at a temperature of 10-15℃ for 3-7 days, the granules are washed with water until the pH reaches 3.5-4.

5. Then immerse it in lime water with a mass concentration of 1.5%-4.0%, with a material-to-liquid ratio of 1:6-8 (g / mL). Soak it at a temperature of 8-12℃ for 25-45 days, and then wash it with water until the pH reaches 6.0-6.

5.

9. The method for preparing an active peptide that promotes bone growth according to claim 3, characterized in that, The method for obtaining collagen protein through sequential extraction, filtration, ion exchange, and drying is as follows: After alkali treatment, bone particles are added to deionized water at a material-to-liquid ratio of 1:1.5-2 (g / mL), with pH controlled at 4.5-5.

5. Extraction is carried out by distillation at 55-90℃ under normal pressure to obtain a dilute gel solution with a mass concentration of 4%-5%. The diluted filtrate was then filtered through cotton cake. The resulting clear and transparent filtrate was then desalted sequentially through a strong acid cation exchange column and a strong base anion exchange column. The filtrate temperature was 30-40℃, the flow rate was 1.5-3.0 BV / h, and the final ash content was controlled to be ≤0.5%, conductivity ≤50μS / cm, and pH 4.8-5.

2. After vacuum concentration and drying, collagen is obtained.

10. The use of an active peptide as described in claim 1 or 2 in the preparation of a product for promoting bone growth, said product being a functional food, a medical food, or a nutritional supplement.

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