Collagen peptide for promoting osteoblast proliferation and application thereof

Abstract

The invention discloses collagen peptide for promoting osteoblast proliferation and application thereof, and belongs to the technical field of biological medicine. The amino acid sequence of the pigskin collagen peptide is as shown in SEQ ID NO. 3. The pigskin collagen peptide and calcium ions are chelated to serve as a core material, liposome nanoparticles can be formed through liposome embedding and biopolymer coating, and the bioavailability of calcium can be effectively improved. The absolute value of the Zeta potential of the embedded particles is increased, and the particle size and the PDI are obviously reduced, which indicates that the stability and the dispersity of the system are enhanced. Stability experiments verify that the nano-particles prepared by multilayer embedding of lecithin, chitosan and sodium alginate have optimal comprehensive performance, and have the dual advantages of promoting osteoblast proliferation and improving the digestion and absorption capacity of the body.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a collagen peptide for promoting proliferation of osteoblasts and application thereof. BACKGROUND

[0002] With the continuous growth of pork consumption, the output of pigskin, a byproduct of slaughter, has increased significantly, but its economic value is low. At present, it is mainly used in the leather industry, and only a small part is used for high-value utilization. Pigskin is rich in collagen and is a potential high-quality resource for extracting bioactive peptides. However, traditional collagen extraction methods have problems such as high cost and low efficiency, which limits their industrial application in the fields of food, medicine, etc. Therefore, how to efficiently extract and utilize pigskin collagen to improve its added value has become a problem to be solved.

[0003] Calcium is an essential mineral for the human body, and calcium deficiency can lead to osteoporosis and other diseases. Currently commonly used calcium supplements such as calcium carbonate are easily formed into insoluble salts with phytic acid, oxalic acid, etc. in the intestinal tract, and have low bioavailability. Although vitamin D can promote calcium absorption, excessive use has safety hazards. In recent years, it has been found that food-derived collagen peptides can form chelates with calcium ions to improve the solubility and absorption rate of calcium, but the existing peptide-calcium chelates have poor stability in the body environment and are easily affected by pH, enzymes, etc. and inactivated, which limits their actual application effect.

[0004] In addition, the existing screening method of bioactive peptides relies on traditional enzymatic hydrolysis and in vitro experiments, which is time-consuming, high-cost and low-efficiency. Although some studies have tried to use liposome embedding technology to improve the stability of active ingredients, ordinary liposomes have problems such as unstable membrane structure, easy oxidation, and rapid release in the gastrointestinal environment. Therefore, it is urgent to develop an efficient and stable peptide-calcium delivery system to improve the bioavailability of calcium and expand the high-value application of pigskin collagen. SUMMARY

[0005] The purpose of the present application is to provide a collagen peptide for promoting proliferation of osteoblasts and application thereof to solve the problems existing in the prior art. The present application obtains a high-activity peptide segment FYRA with potential to promote proliferation of osteoblasts through computer-aided design combined with virtual screening, chelates the peptide segment with calcium ions, and further constructs a liposome nanoparticle, which can effectively improve the bioavailability of calcium and has the dual advantages of promoting proliferation of osteoblasts and improving the body's digestive and absorptive capacity.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a collagen peptide for promoting proliferation of osteoblasts, and the amino acid sequence of the pigskin collagen peptide is shown in SEQ ID NO. 3.

[0008] The application also provides application of the pig collagen peptide in any one of the following (1)-(3):

[0009] (1) preparation of a product for promoting osteoblast proliferation;

[0010] (2) preparation of a calcium supplement product;

[0011] (3) promotion of calcium absorption.

[0012] Further, the product comprises a peptide-calcium chelate and a liposome nanoparticle comprising the peptide-calcium chelate.

[0013] The application also provides a collagen peptide-calcium chelate liposome nanoparticle, which comprises, from inside to outside, a core material, a liposome shell layer and a biopolymer coating layer.

[0014] The core material is a chelate formed by a collagen peptide with an amino acid sequence as shown in SEQ ID NO. 3 and calcium ions.

[0015] Further, the raw material of the liposome shell layer comprises cholesterol, phospholipid and surfactant; the mass ratio of the phospholipid, the cholesterol and the surfactant is 6:1:1; and the phospholipid is soybean phospholipid or lecithin.

[0016] Further, the raw material of the biopolymer coating layer comprises sodium alginate and / or chitosan.

[0017] Further, the preparation method of the chelate comprises the following steps:

[0018] The collagen peptide and a calcium source are dissolved in a solvent at a mass ratio of (2-4):1, the pH is adjusted to 8-10, chelation is carried out at 50-70℃ for 40-60min, then ethanol is added for precipitation, centrifugation is carried out, the precipitate is collected and dried.

[0019] Further, the mass ratio of the collagen peptide and the calcium source is 3:1.

[0020] Further, the pH is 9.

[0021] Further, the chelation temperature is 62℃ and the time is 52min.

[0022] The application discloses the following technical effects:

[0023] The application obtains a high-activity peptide segment FYRA with osteoblast proliferation potential by computer-aided design combined with virtual screening from mass spectrometry identification results. The peptide segment exhibits excellent biological activity, water solubility and ADMET properties, is non-toxic and can stably bind to CB2 and EPCR receptors, and molecular dynamics simulation further confirms the binding stability, thereby significantly reducing the workload and research and development cost of traditional screening.

[0024] Taking FYRA as a calcium ion carrier, the application further optimizes the preparation process of the porcine collagen peptide-calcium chelate. Under the optimal process conditions (pH 9, time 52 min, temperature 62℃, mass ratio 3:1), the peptide-calcium chelate with excellent chelation rate can be obtained. By constructing liposome nanoparticles, the application effectively improves the bioavailability of calcium. The absolute value of Zeta potential of the embedded particles increases, and the particle size and PDI significantly decrease, indicating that the stability and dispersibility of the system are enhanced. It is verified by the stability experiment that the nanoparticles prepared by multi-layer embedding of lecithin, chitosan and sodium alginate have the optimal comprehensive performance, and have the dual advantages of promoting osteoblast proliferation and improving the digestive and absorptive capacity of the body. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0026] Figure 1 Peptide sequence molecular weight distribution of pepsin enzyme hydrolysate (A) and trypsin enzyme hydrolysate (B);

[0027] Figure 2 LC-MS / MS score of bioactive peptide;

[0028] Figure 3 Molecular docking binding energy;

[0029] Figure 4 LC-MS / MS molecular weight of bioactive peptide;

[0030] Figure 5Figure 1 is a molecular dynamics result chart; wherein, A: root mean square deviation (RMSD); B: radius of gyration (Rg); C: solvent accessible surface area (SASA); D: the number of hydrogen bonds between small molecules and target proteins; E: root mean square fluctuation (RMSF) of FYRA-CB2 complex system; F: root mean square fluctuation (RMSF) of FYRA-EPCR complex system;

[0031] Figure 6 Figure 2 is a peptide-calcium chelate nanoparticle embedding effect; wherein, A: zeta potential result; B: particle size result; C: PDI; in the figure, FYRA-Ca is a calcium-peptide chelate; FYRA-Ca-L is a lecithin prepared liposome; FYRA-Ca-L-C is a chitosan-coated lecithin liposome; FYRA-Ca-L-C-SA is a sodium alginate-chitosan-coated lecithin liposome; FYRA-Ca-SL is a soy lecithin prepared liposome; FYRA-Ca-SL-C is a chitosan-coated soy lecithin liposome; FYRA-Ca-SL-C-SA is a sodium alginate-chitosan-coated soy lecithin liposome, and different letters in the figure indicate significant differences between groups;

[0032] Figure 7 Figure 3 is a nanoliposome pH stability analysis result; wherein, A: potential; B: particle size; C: PDI;

[0033] Figure 8 Figure 4 is a nanoliposome ion stability analysis result; wherein, A: potential; B: particle size; C: PDI;

[0034] Figure 9 Figure 5 is a nanoliposome in vitro simulated gastric digestion stability analysis result; wherein, A: potential; B: particle size; C: PDI;

[0035] Figure 10 Figure 6 is a nanoliposome in vitro simulated intestinal stability analysis result; wherein, A: potential; B: particle size; C: PDI;

[0036] Figure 11 Figure 7 is a nanoliposome storage stability analysis result at 4°C; wherein, A: potential; B: particle size; C: PDI;

[0037] Figure 12 Figure 8 is a nanoliposome storage stability analysis result at 25°C; wherein, A: potential; B: particle size; C: PDI;

[0038] Figure 13 Figure 9 is a nanoliposome release rate under different pH conditions;

[0039] Figure 14 Release rate of the nano-liposome under different ion conditions;

[0040] Figure 15 Release rate of the nano-liposome under different simulated digestion time;

[0041] Figure 16 Release rate of the nano-liposome under different storage time at 4°C;

[0042] Figure 17 Release rate of the nano-liposome under different storage time at 25°C. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation thereof.

[0044] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits are also included.

[0045] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0046] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0047] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0048] Example 1 Virtual screening and identification of porcine collagen peptide FYRA

[0049] 1. Experimental methods

[0050] (1) Preparation of pigskin collagen

[0051] Fresh pigskin was purchased from the market, and the hair and subcutaneous fat were removed. After washing, the pigskin was freeze-dried and ground into powder. The pigskin powder was weighed and added to 0.4 mol / L acetic acid solution at a solid-liquid ratio of 1:50 (g / mL). The mixture was ultrasonically treated for 60 min, and then extracted at room temperature for 24 h. After extraction, the supernatant was collected by centrifugation, and the precipitate was collected by salting out at 4°C. The pigskin collagen was obtained.

[0052] (2) Enzymatic hydrolysis of pigskin collagen peptides

[0053] The extracted pigskin collagen was subjected to enzymatic hydrolysis with pepsin and trypsin, respectively.

[0054] The pepsin hydrolysis conditions were as follows: pH 3.0, enzyme addition amount 5% (w / w), and reaction at 30°C for 5 h under magnetic stirring.

[0055] The trypsin hydrolysis conditions were as follows: pH 7.5, enzyme addition amount 3% (w / w), and reaction at 40°C for 5 h under magnetic stirring.

[0056] After the enzymatic hydrolysis, the enzyme was inactivated by heating the hydrolysis solution in a water bath at 90°C for 10 min. After cooling, the supernatant was collected by centrifugation to obtain the pigskin collagen peptide hydrolysate.

[0057] (3) Virtual screening and identification of peptide fragments

[0058] The two hydrolysates were entrusted to Beijing Baitai Paker Biological Technology Co., Ltd. for peptide sequence analysis and identification by LC-MS / MS. The identified peptide sequences were subjected to virtual screening according to the following steps:

[0059] ① The online tool Peptide Ranker (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pages / peptideranker.php) was used to predict the potential biological activity of the peptide fragments, and the peptide fragments with an activity score greater than 0.5 were selected.

[0060] ② The Innovagen "Peptide property calculator" (http: / / www.innovagen.com / proteomics-tools) was used to predict the solubility of the peptide fragments, and polypeptides with good water solubility were selected.

[0061] ③ADMET SAR (http: / / lmmd.ecust.edu.cn / admetsar1 / predict / ) was used to predict the ADMET properties, mainly including human intestinal aabsorption (HIA), Caco-2 permeability, blood-brain barrier penetration (BBB), and acute oral toxicity. ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to predict the toxicity, and the peptide segment with positive HIA and no toxicity was screened out.

[0062] ④The peptide segment with a score greater than 100 was screened out in combination with the mass spectrometry identification score.

[0063] ⑤The three-dimensional structures of the receptor proteins CB2 and EPCR related to osteoblast proliferation were downloaded from the PDB database (http: / / www.rcsb.org / ). The AutoDock Vina software was used to perform molecular docking of the screened peptide segment and the receptor, and the binding energy was calculated. Finally, in combination with the molecular weight size (preferably a peptide segment less than 1000 Da) and the docking binding energy, the polypeptide with the best binding effect was selected as the target peptide segment.

[0064] (4) Molecular dynamics simulation verification

[0065] Gromacs 2023 software was used to perform 100 ns molecular dynamics simulation on the complex formed by the polypeptide and the receptors CB2 and EPCR. The protein was parameterized by the CHARMM 36 force field, and the ligand topology structure was constructed by the GAFF2 force field. The periodic boundary condition was used to place the protein-ligand complex in a cubic box. The TIP3P water model was used to fill water molecules into the box. The particle mesh Ewald (PME) and Verlet algorithms were used to handle electrostatic interactions, respectively. Then, 100,000 steps of isothermal-isochoric ensemble equilibration and isothermal-isobaric ensemble equilibration were performed, with a coupling constant of 0.1 ps and a duration of 100 ps simulation. The cutoff value of 1.0 nm was used to calculate van der Waals and Coulomb interactions. Finally, the system was subjected to molecular dynamics simulation under constant temperature (300 K) and constant pressure (1 bar) using Gromacs 2023, with a total simulation time of 100 ns.

[0066] 2、Experimental results

[0067] (1) Mass spectrometry identification results

[0068] The prepared pigskin collagen peptides were identified by LC-MS / MS analysis. The results showed that 180 peptide sequences were identified in the pepsin enzyme solution, and 340 peptide sequences were identified in the trypsin enzyme solution. The peptide segments with a molecular weight less than 1000 Da accounted for 33.3% and 31.8% in the pepsin enzyme solution and the trypsin enzyme solution, respectively; the peptide segments with a molecular weight of 1000-2000 Da accounted for 42.2% and 49.4%, respectively; the peptide segments with a molecular weight of 2000-3000 Da accounted for 7.2% and 15.6%, respectively; and the peptide segments with a molecular weight greater than 3000 Da accounted for 17.25% and 3.2%, respectively. Figure 1 .

[0069] (2) Virtual screening results

[0070] First, the online platform was used for screening in combination with biological activity, water solubility, ADME properties, and toxicity. The PeptideRanker score was between 0 and 1, and it was generally considered that a score greater than 0.5 indicated potential biological activity, and the higher the score, the stronger the biological activity. The results, as shown in Tables 1 and 2, indicated that nine collagen peptides in the pepsin enzyme solution had good biological activity, water solubility, ADME properties, and no toxicity, and twelve collagen peptides in the trypsin enzyme solution had good biological activity, water solubility, ADME properties, and no toxicity. Then, in combination with the mass spectrometry analysis results, the peptides with a score greater than 100 were further screened. The results, as shown in Table 3, indicated that one peptide in the pepsin enzyme solution had a score less than 100, and three peptides in the trypsin enzyme solution had a score less than 100, so these four peptides were excluded, and further molecular docking was performed. Figure 2

[0071] Table 1 Virtual screening results of some peptide segments in the pepsin enzyme solution

[0072]

[0073] Table 2 Virtual screening results of some peptide segments in the trypsin enzyme solution

[0074]

[0075] The screened peptides were docked with CB2 and EPCR, respectively, and the results are shown in Table 4. It can be seen that the peptides with better binding energy are longer. Previous studies have shown that small molecule peptides are more easily digested and absorbed by the human body, so we further screened them by molecular weight. The molecular weight distribution is shown in Table 5. Figure 3 Figure 4 ​​As shown, there are 6 peptides with a molecular weight less than 1000 Da. Based on the molecular docking results, FYRA showed the best binding effect among these 6 peptides. Therefore, FYRA was selected for further research. Further analysis of the molecular docking results of peptide FYRA revealed that EPCR (PDB ID: 1LQV) and FYRA are mainly connected by 5 hydrogen bonds, with a binding energy of -8.7 kcal / mol and a bond distance between 2.1 and 3.4 Å. The main binding site residues involved in the hydrogen bond interaction are THR-65, THR-168, GLN-57, and GLN-171. CB2 (PDB ID: 6PT0) is mainly connected to FYRA through 9 hydrogen bonds with a binding energy of -10.5 kcal / mol and a bond distance between 1.8 and 3.1 Å. The main binding site residues involved in the hydrogen bond interaction are LEU-192, LEU-318, PHE-278, SER-277, SER-215, ASP-322, and THR-321.

[0076] (3) Molecular dynamics simulation results

[0077] Molecular dynamics simulations can provide information such as the free energy and conformational changes of protein-ligand binding.

[0078] Root mean square deviation (RMSD) is a good indicator of conformational stability of proteins and ligands, and also measures the degree of deviation of atomic positions from their initial positions. The smaller the deviation, the better the conformational stability. Therefore, RMSD was used to evaluate the balance of the simulation system. Figure 5 As shown in Figure A, the FYRA-CB2 complex system reached equilibrium after 45 ns, eventually fluctuating around 4.8 Å. The FYRA-EPCR complex system reached equilibrium after 10 ns, eventually fluctuating around 1.1 Å. The FYRA-EPCR complex system exhibited a lower RMSD value. Therefore, the EPCR small molecule showed high stability when binding to the FYRA target protein.

[0079] Further analysis revealed slight fluctuations in the radius of gyration (Rg) and solvent-accessible surface area (SASA) of the FYRA-CB2 and FYRA-EPCR complex system during movement. This indicates that the binding of small molecules caused a conformational change in the target protein. Figure 5 (BC).

[0080] Hydrogen bonds play a crucial role in the binding of ligands to proteins. The number of hydrogen bonds between small molecules and target proteins during kinetic processes is as follows: Figure 5As shown in D of the figure, the number of hydrogen bonds between FYRA-CB2 complex system is from 0 to 7, in most cases, the complex has about 3 hydrogen bonds. The number of hydrogen bonds between FYRA-EPCR complex system is from 0 to 8, in most cases, the complex has about 6 hydrogen bonds, which indicates that FYRA-CB2 and FYRA-EPCR complex system have good hydrogen bond interaction.

[0081] Root mean square fluctuation (RMSF) can represent the flexibility of amino acid residues in the protein. As shown in E-F of the figure, the RMSF value of FYRA-CB2 and FYRA-EPCR complex system is relatively low (mostly below 3 Å), so its flexibility is low and the stability is high. Figure 5

[0082] In summary, FYRA-CB2 and FYRA-EPCR complex system are stable. Among them, the RMSD value of FYRA-EPCR complex system is low, and the complex has good hydrogen bond interaction. Therefore, the EPCR small molecule binds well with the FYRA target protein.

[0083] Example 2 Optimization of chelation process of pigskin collagen peptide FYRA and calcium ions

[0084] 1. Experimental method

[0085] (1) Determination of chelation rate

[0086] EDTA titration method was used to determine the chelation rate. FYRA-Ca chelate was dissolved in deionized water at 0.4 g / mL, 1 mL of triethanolamine (200 g / L) was added as a masking agent, the pH was adjusted to 12.0 with 1 mol / L NaOH solution, two drops of phenothalin T (5 g / L) were added as an indicator, and the solution was titrated with EDTA standard solution until the solution changed from purple red to blue. The volume of EDTA consumed (V) was recorded. At the same time, a blank control (V0) was made. The chelation rate calculation formula is as follows:

[0087] ;

[0088] In the formula: C--EDTA standard solution concentration mol / L;

[0089] V--consumed EDTA standard solution volume L;

[0090] V0--blank control consumed EDTA standard solution volume L;

[0091] M--molar mass of calcium g / moL;

[0092] ​m - FYRA-Ca chelate mass g.

[0093] (2) Single factor and response surface optimization

[0094] With synthetic purified FYRA peptide (purity 95%) and anhydrous calcium chloride as raw materials, the ratio of material to liquid was fixed at 25 mg / mL, and the chelation rate was used as an index. The effects of four single factors, including chelation temperature (30℃, 40℃, 50℃, 60℃, 70℃), chelation time (30 min, 40 min, 50 min, 60 min, 70 min), pH (6, 7, 8, 9, 10) and peptide-calcium mass ratio (1:1, 2:1, 3:1, 4:1, 5:1), were investigated in turn. After chelation for a certain time, 9 times the volume of anhydrous ethanol was added and allowed to stand for 1 h. The precipitate was collected by centrifugation and freeze-dried. The chelation rate was then determined.

[0095] On the basis of single factor experiments, a Box-Behnken response surface experiment with four factors and three levels was designed. The influencing factors and levels are shown in Table 3. The experimental results were analyzed by regression analysis using Design Expert software.

[0096] Table 3 Factors and levels of response surface experiment

[0097]

[0098] 2、Experimental results

[0099] (1) Single factor experiment results

[0100] The results of single factor experiments showed that each process parameter had a significant effect on the chelation rate of peptide-calcium. The chelation rate increased with increasing pH and reached a peak of 64.7% at pH 9. With the extension of chelation time, the chelation rate first increased and then decreased, reaching a maximum of 68.47% at 50 min. The temperature experiment showed that the chelation rate was best at 60℃, reaching 69.54%. When the mass ratio of peptide to calcium was 3:1, the chelation rate reached a maximum of 67.91%. In general, when the pH was 8, 9, 10; the chelation time was 40, 50, 60 min; the chelation temperature was 50, 60, 70℃; and the mass ratio of peptide to calcium was 2:1, 3:1, 4:1, the chelation rate was higher. Therefore, these four levels were selected for the response surface experiment.

[0101] (2) Response surface experiment results

[0102] On the basis of single factor experiments, a Box-Behnken response surface experiment with four factors and three levels was designed. The influencing factors and levels are shown in Table 3. The experimental results were analyzed by regression analysis using Design Expert software.

[0103] Table 4 Design and results of response surface experiment for chelation of bioactive peptide FYRA and calcium ions

[0104]

[0105] Using software Design Expert, the regression equation of collagen extraction conditions (R) is obtained as follows:

[0106] R = 66.47 + 0.3383A + 0.2758B + 0.3267C + 0.1608D + 0.745AB + 0.45AC - 0.08AD + 0.7475BC - 0.095BD + 5575CD - 1.75A 2 -1.3B2-1.74C 2 -1.2D 2 According to the test design results and the quadratic multiple regression equation, the optimal chelation conditions are pH 9.152, chelation time 51.972, chelation temperature 62.093, and FYRA to calcium ion mass ratio 3.095:1. Considering the process operation convenience, the parameters are adjusted to pH 9, chelation time 52 min, chelation temperature 62℃, and FYRA to calcium ion mass ratio 3:1. The variance analysis is shown in Table 5, R 2 (0.9992), p<0.0001, the model difference is extremely significant; the p difference of the misfit term is not significant, indicating that the equation fitting is good.

[0107] Table 5 Variance analysis table

[0108]

[0109] The response surface analysis result shows that the influence degree of each factor on the chelation rate is significantly different. Among them, the pH value is the most important factor affecting the chelation rate, and its influence is significantly greater than the chelation time, chelation temperature and peptide calcium mass ratio. The influence of chelation time is only second to pH, but significantly greater than chelation temperature and peptide calcium mass ratio. The influence of chelation temperature on chelation rate is obviously greater than that of peptide calcium mass ratio. The importance of each factor is ranked as follows: pH > chelation time > chelation temperature > peptide calcium mass ratio.

[0110] According to the Box-Behnken test, the optimal enzymolysis process combination is as follows: pH 9.152, chelation time 51.972 min, chelation temperature 62.093℃, and FYRA to calcium ion mass ratio 3.095:1. Considering the process operation convenience, the parameters are adjusted to pH 9, chelation time 52 min, chelation temperature 62℃, and FYRA to calcium ion mass ratio 3:1. Under this process condition, 3 independent repeated experiments are carried out, and the chelation rate is 66.37±0.09%, which is 0.194% different from the predicted value, indicating that the model prediction result is reliable.

[0111] 3. Preparation of chelate

[0112] According to the above-mentioned optimized process, FYRA and anhydrous calcium chloride were dissolved in deionized water (25 mg / mL of feed liquid) at a mass ratio of 3:1, the pH was adjusted to 9 with NaOH solution, and the reaction was carried out at 62°C in a water bath with magnetic stirring for 52 min. After the reaction was completed, 9 times the volume of anhydrous ethanol was added, and the precipitate was collected by centrifugation at 8000 x g at 4°C for 15 min, and freeze-dried to obtain the FYRA-Ca chelate powder.

[0113] Example 3 Preparation and performance evaluation of pigskin collagen peptide chelated calcium liposome nanoparticles

[0114] 1. Experimental method

[0115] Soybean phospholipid, cholesterol / lecithin, and cholesterol were used as raw materials to prepare the first layer of liposome shell, and then high-pressure homogenization or shearing was used to form a uniform texture and nanoscale size carrying system (see Table 6). Then, sodium alginate and chitosan were used as the second layer of coating biopolymer to improve the thermodynamic properties of the nanoliposomes. The effects of six groups of wall materials on the chelate were evaluated based on the embedding rate, Zeta potential, and particle size. The physicochemical stability (pH, ionic stability), storage stability, and in vitro simulated digestion stability of the above nanoparticles were analyzed. The crude liposomes without embedding were used as a reference to determine whether the pigskin collagen peptide was effectively protected by embedding.

[0116] Table 6 Lipid nanoparticle wall material combinations

[0117]

[0118] (1) Preparation of crude liposomes

[0119] Soybean lecithin, cholesterol, and Tween-80 were dissolved in 10 mL of anhydrous ethanol solution at a mass ratio of 6:1:1, stirred at 60°C for 3 min to dissolve completely, and then rotary evaporated at 60 r / min and 60°C for 20 min to form a thin film. In a round-bottom flask, 10 mL of PBS buffer (0.01 mol; pH 7.4) containing 0.02 g of chelate was added, and the mixture was hydrated at 60°C for 1 h to obtain a crude liposome solution. The solution was ultrasonicated at 4°C for 8 min (ultrasonic frequency 20 kHz; power 500 W; interval 2 s cycle) to obtain nanoliposomes. The sample was placed in a 4°C refrigerator for 12 h to obtain a crude liposome solution.

[0120] Lecithin, cholesterol and Tween-80 were dissolved in 10 mL absolute ethanol solution at a mass ratio of 6:1:1, stirred at 60 °C water bath for 3 min to dissolve completely, and formed a film by rotary evaporation at 60 r / min and 60 °C for 20 min. In a round-bottom flask, 10 mL PBS buffer (0.01 mol; pH 7.4) containing 0.02 g chelate was added, hydrated at 60 °C for 1 h to obtain a crude liposome solution. The solution was ultrasonicated at 4 °C for 8 min (ultrasonic frequency 20 kHz; power 500 W; interval 2 s cycle) to obtain nanoliposomes. The sample was placed in a refrigerator at 4 °C for 12 h to obtain a crude liposome solution.

[0121] (2) Preparation of stock solution

[0122] Dissolve 1 g of chitosan in 100 mL of 1% acetic acid solution, stir for 12 h, water bath ultrasonic for 30 min (ultrasonic frequency 40 kHz; temperature 25 °C), centrifuge at 4000 x g for 20 min, collect the supernatant and pass through a 0.22 μm filter, and finally adjust the pH to 5.5.

[0123] Dissolve 1 g of sodium alginate in 100 mL of distilled water, stir for 12 h, water bath ultrasonic for 30 min (ultrasonic frequency 40 kHz; temperature 25 °C), centrifuge at 4000 x g for 20 min, collect the supernatant and pass through a 0.22 μm filter, and finally adjust the pH to 5.5.

[0124] (3) Preparation of nanoparticles

[0125] Dilute the chitosan stock solution (1%) with 1% acetic acid solution to a chitosan solution with a concentration of 0.6%. Take 2 mL of the chitosan solution in a beaker, and add an equal amount of liposome solution dropwise to the chitosan solution while stirring. After completion, continue stirring for 1 h to form a chitosan-core polypeptide liposome nanoparticle solution, and place the solution in a refrigerator at 4 °C for 12 h.

[0126] Dilute the sodium alginate stock solution (1%) with distilled water to a sodium alginate solution with a concentration of 0.5%. Take 2 mL of the sodium alginate solution in a beaker, and add an equal amount of liposome solution dropwise to the chitosan solution while stirring. After completion, continue stirring for 1 h to form a sodium alginate-core polypeptide liposome nanoparticle solution, and place the solution in a refrigerator at 4 °C for 12 h.

[0127] Preparation of double-layered embedded nanoparticles (FYRA-Ca-L-C-SA): Chitosan stock solution (1%) was diluted with 1% acetic acid solution to a concentration of 0.6% chitosan solution. The chitosan solution was placed in a beaker, and an equal amount of liposome solution was added dropwise to the chitosan solution while stirring magnetically. After completion, the stirring was continued for 1 h to form a chitosan-coated liposome. Subsequently, 2 mL of 0.5% sodium alginate solution was added dropwise to this system under magnetic stirring, and the stirring was continued for 1 h after the dropwise addition was completed. The final solution was allowed to stand at 4°C for 12 h to obtain a sodium alginate-chitosan- lecithin three-layer embedded nanoparticle suspension, which can be freeze-dried to obtain a solid powder.

[0128] (4) Performance evaluation index

[0129] pH stability: solutions with different pH values (pH 2.0, pH 5.0, and pH 8.0) were prepared, respectively, and then mixed with the liposome nanoparticle solution at a volume ratio of 1:1. After incubation for 30 min, the changes in size and zeta potential were recorded by DLS method.

[0130] Ion stability: the stability of the liposome system to ion pressure was studied by measuring the particle size and zeta potential (ΔP) by incubating the liposome nanoparticles in different concentrations (0-1000 mM) of sodium chloride solution at room temperature for 1 h at a volume ratio of 1:1. The ion strength of each liposome system was evaluated by the relative change rate of particle size.

[0131] Storage stability: all liposome systems were tested for storage stability in the dark for 32 days at two different temperatures (4°C and 25°C). Therefore, samples were collected every 4 days, and the changes in size and zeta potential were recorded by DLS method.

[0132] In vitro simulated digestion stability: 10 mL of crude liposomes and liposome nanoparticles were dissolved in 25 mL of ultrapure water and incubated at 37°C with shaking for 30 min. The pH was adjusted to 2.0, and the simulated gastric juice was added at a volume ratio of 1:1. The simulated gastric juice was digested at 37°C, and 5 mL of solution was taken out at 0, 30, 60, and 90 min, respectively, and the enzyme was inactivated by heating in a 100°C water bath for 5 min. After 90 min of simulated gastric digestion, the pH was adjusted to 7, and the simulated intestinal juice was added at a volume ratio of 1:1. The simulated intestinal juice was continued to be digested at 37°C, and 5 mL of sample was taken out at 0, 30, 60, 90, and 150 min, respectively. After heating at 100°C for 10 min to inactivate the enzyme, the samples were stored in a -80°C freezer for subsequent experiments.

[0133] Calcium ion release rate: EDTA titration method was used to determine the calcium ion release rate. The solution treated by stability test was diluted with PBS solution at a certain concentration, 1 mL triethanolamine (200 g / L) masking agent was added, then 1 mol / L NaOH solution was used to adjust the pH to 12.0, two drops of indigo black T (5 g / L) indicator was added, and EDTA standard solution was used for titration. The solution changed from purple red to blue as the end point of titration.

[0134] 2. Experimental results

[0135] (1) Peptide chelated calcium nanoparticle embedding effect

[0136] Liposomes were prepared respectively with soybean phospholipid, cholesterol and lecithin, cholesterol as raw materials, chitosan as the first layer of packaging material to prepare the liposome shell, and then high pressure homogenization or shearing was used to form a uniform texture, size in nanometer level of the delivery system. Then, sodium alginate was used as the second layer of coating biopolymer to improve the thermodynamic properties of the nanoliposome. With embedding rate, Zeta potential, particle size as evaluation index, the influence of six groups of wall materials on the liposome nanoparticles was analyzed. The embedding effect is shown in Figure 6 Compared with the chelate, the absolute value of the zeta potential of the six groups of samples after embedding increased, indicating that the stability of the liposome dispersion system was enhanced compared with before embedding. The particle size and PDI after embedding showed a significant decreasing trend, indicating that the dispersibility of the liposome was better.

[0137] (2) pH stability

[0138] The evaluation of ζ potential, particle size, PDI performance at different pH values Figure 7 shows that at pH 5 and 8, the absolute value of the potential of each group of samples is larger, relatively stable, and the particle size and dispersibility are also better. While at pH 2.0, the particle size and ζ potential have undergone mutation, and the stability and dispersibility are relatively worse. This may be due to the lower or neutral ζ potential of phospholipid in acidic medium, leading to swelling of the nanoparticles, thereby increasing their size. It can be seen from Figure 13 When the pH is 8, the release rate of the chelate is significantly reduced, which may be because in alkaline conditions, the amino group tends to deprotonate, the repulsive force is weakened, leading to particle coagulation, thereby inhibiting the release of the contents. Among them, FYRA-Ca-L-C-SA has the best stability and dispersibility, and the release rate is also relatively lower, with better pH stability.

[0139] (3) Ionic stability

[0140] The ionic stability of the liposome system was evaluated under the gradient change of sodium chloride concentration (0-1000 mM). The relative change rate of the particle size and ζ potential of the liposome is shown in Figure 8The zeta potential of all liposome systems decreased with increasing sodium chloride concentration, while the particle size and PDI increased. This change can be attributed to the electrostatic shielding of the counterions, which reduces the repulsive forces between the particles, thus facilitating their aggregation. The release rate of the peptide-calcium chelate increased with increasing sodium chloride concentration, and then decreased. At low ionic concentrations, the thicker electric double layer formed by the surface charge of the liposomes (high absolute zeta potential) inhibits liposome aggregation or membrane fusion, and the structure is stable, with the contents tightly packed. When the ionic concentration in the solution increases, the counterions (such as Na + 、Cl - ) neutralize the surface charge of the liposomes, compressing the thickness of the electric double layer (reducing the absolute zeta potential), and weakening the electrostatic repulsion. The liposomes easily aggregate or lose membrane stability, resulting in the leakage of the contents. In an extremely high salt environment, the liposomes can completely disintegrate (such as membrane dissolution or micelle formation), and the contents are released instantaneously. However, if the experimental conditions do not reach the critical point, the membrane may be too tightly packed due to excessive aggregation. Figure 14 .

[0141] (4) In vitro simulated digestion stability

[0142] The enhanced physicochemical stability of single and double coated liposomes can stimulate the sustained release behavior of these carriers. Therefore, we verified this feature by in vitro simulated digestion. During the simulated gastric digestion process, the potential of the pigskin collagen peptide FYRA chelated calcium-loaded nanoliposomes was about -18 mV. However, during the simulated intestinal digestion process ( Figure 9 ), the potential of the pigskin collagen peptide FYRA chelated calcium nanoliposomes was close to -40 mV. During intestinal digestion ( Figure 10 ), the potential value can be attributed to the presence of different anions in the intestinal fluid particles (such as bile salts) or to the hydrolysis products of phospholipids in the liposomes, such as free fatty acids, in trypsin. A higher absolute potential value (≥ 30 mV) indicates that the liposomes are more stable in terms of their physicochemical properties. During intestinal digestion, the high stability of the nanoliposomes protects the egg white peptide chelated calcium from the damage of biological enzymes and the alkaline environment, avoids the formation of more calcium ions into insoluble precipitates, and allows more calcium to be absorbed by the intestinal tract. The release of the chelate is as follows Figure 15As shown, the release rate gradually increased as the digestion process proceeded. When entering the simulated intestinal digestion, the release rate sharply increased, and this burst release tendency was due to the conformational change of the phospholipid bilayer caused by the protonation of the liposome surface at acidic pH. This release burst can also be caused by bile salts and pancreatin, which rupture and hydrolyze the liposome bilayer. Due to the deposition of biopolymers on the surface of nanoliposomes, electrostatic bridges were formed, resulting in decreased liposome membrane fluidity and permeability, thus enhancing its stability and drug release control ability. Among all the liposome systems, FYRA-Ca-L-C-SA had the highest absolute value of zeta potential and the best stability. FYRA-Ca-SL-C-SA had smaller particle size and PDI and the best dispersibility. However, FYRA-Ca-L-C-SA had a higher release rate in the intestine and a higher bioavailability of the peptide segment FYRA-calcium chelate. Overall, FYRA-Ca-L-C-SA is more meaningful for research.

[0143] (5) Storage stability

[0144] The effect of long-term storage on all liposome systems was studied by monitoring the changes in zeta potential, particle size, PDI, and peptide-calcium chelate release rate over 32 days at two different temperatures (4°C and 25°C). At a temperature of 4°C, the changes in the potential, particle size, and PDI parameters of all carrier systems were relatively small. In contrast, the absolute value of the zeta potential of FYRA-Ca-L-C-SA showed a clear upward trend, and the dispersibility also became better with the storage time ( Figure 11 ). However, at room temperature (25°C), the particle sizes of FYRA-Ca-L-C-SA and FYRA-Ca-SL liposomes increased significantly ( Figure 12 ), and this phenomenon was due to the weakening of the repulsive force between particles, leading to particle aggregation. The release rate of all liposome carriers increased with the extension of the storage time, and the liposomes prepared from soy lecithin had a higher release tendency ( Figure 17 ). It may be because the aggregation and hydrolysis of nanoparticles prepared from soy lecithin led to the leakage of the chelate by destroying the phospholipid bilayer. Therefore, it can be concluded that the liposomes prepared from lecithin have better storage stability, and among them, FYRA-Ca-L-C-SA has the best stability.

[0145] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A collagen peptide for promoting proliferation of osteoblasts, characterized by, The amino acid sequence of the collagen peptide is shown as SEQ ID NO.

3.

2. The collagen peptide of claim 1 is used in any one of (1)-(3) as follows: (1) to prepare a product for promoting proliferation of osteoblasts; (2) to prepare a calcium supplement product; (3) to promote calcium absorption.

3. Use according to claim 2, characterized in that, The product comprises a peptide-calcium chelate and a liposome nanoparticle containing the peptide-calcium chelate.

4. A collagen peptide chelated calcium liposome nanoparticle, characterized by, It comprises, in order from inside to outside, a core material, a liposome shell layer and a biopolymer coating layer. The core material is a chelate formed by a collagen peptide with an amino acid sequence shown as SEQ ID NO. 3 and calcium ions.

5. The collagen peptide-chelated calcium liposome nanoparticle according to claim 4, characterized by, The raw materials of the liposome shell layer comprise cholesterol, phospholipids and a surfactant; the mass ratio of the phospholipids, the cholesterol and the surfactant is 6:1:1; the phospholipids are soybean phospholipids or lecithin.

6. The collagen peptide-chelated calcium liposome nanoparticle according to claim 4, characterized by, The raw materials of the biopolymer coating layer comprise sodium alginate and / or chitosan.

7. The collagen peptide-chelated calcium liposome nanoparticle according to claim 4, characterized by, The preparation method of the chelate comprises the following steps: The collagen peptide and a calcium source are dissolved in a solvent at a mass ratio of (2-4):1, the pH is adjusted to 8-10, chelation is carried out at 50-70℃ for 40-60 min, then ethanol is added for precipitation, centrifugation is carried out, the precipitate is collected and dried.

8. The collagen peptide-chelated calcium liposome nanoparticle according to claim 7, characterized by, The mass ratio of the porcine collagen peptide and the calcium source is 3:

1.

9. The collagen peptide-chelated calcium liposome nanoparticle according to claim 7, characterized by, The pH is 9.

10. The collagen peptide-chelated calcium liposome nanoparticle according to claim 7, characterized by, The chelation temperature is 62℃ and the time is 52 min.

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