A method for enriching osteogenic active peptides from collagen based on affinity adsorption

Abstract

The invention discloses a method for enriching bone collagen osteogenic active peptides based on affinity adsorption, including: installing a hydroxyapatite pre-packed column in an AKTA pure protein purification system, and enriching the bone collagen peptide solution by affinity adsorption through a hydroxyapatite column-AKTA pure protein purification system, wherein, firstly, eluting with mobile phase a as an eluting solution, collecting samples when an absorption peak occurs, and obtaining a bone collagen osteogenic active peptide sample F1, wherein the mobile phase a is a potassium chloride solution. The present invention screens out polypeptides affinity-adsorbed with a hydroxyapatite column matrix, obtains bone collagen peptides with strong ability to bind hydroxyapatite, measures the calcium binding capacity of the bone collagen peptide components obtained by enrichment and separation, and its effect on osteoblast proliferation, and provides technical support for the enrichment and preparation of affinity-adsorbed bone collagen osteogenic active peptides, which is of great significance to improving product effectiveness.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptide processing, and more specifically, to a method for enriching bone collagen osteogenic active peptides based on affinity adsorption. Background Art

[0002] China is the world's largest meat producer and consumer, with up to 17 million tons of bone byproducts produced each year from livestock and poultry slaughtering and processing. Livestock and poultry bones are rich in protein, mainly collagen (accounting for 35% to 40% of the total protein content). Collagen peptides prepared from livestock and poultry bones have the characteristics of wide sources, natural safety, and high acceptability, and are widely used in the development of functional bone-based foods. Previous studies have shown that supplementing collagen peptides can increase the regularity and firmness of the body's collagen fiber network, promote the orderly deposition of calcium salts, and increase bone density and bone strength. At the same time, collagen peptides are also a natural carrier protein that transports calcium into the intestinal wall. Peptide calcium chelates, as a new type of calcium supplement, have the characteristics of fast transport speed, low energy consumption, and difficulty in saturation. They can effectively improve the body's calcium absorption rate and biological efficacy, and are excellent nutritional supplements. With the accelerated aging of my country's population, bone health has become an important public health issue. Using collagen peptides to develop functional bone-derived foods that strengthen bones and promote dietary nutritional intervention is of great significance to promoting the bone health of Chinese residents.

[0003] The collagen peptide products currently sold on the market are usually peptide products with a relative molecular mass of less than 10,000 made from livestock and poultry bones through proteolysis or fermentation. This type of collagen hydrolysate is usually a mixture of different peptide molecules. In addition to the effective components that can effectively bind to hydroxyapatite (the main component of human bones), there are also free amino acids, peptides of different molecular weights, peptides composed of different amino acid sequences, collagen that has not been enzymatically hydrolyzed, and reaction aids. Therefore, it is of great significance to separate and enrich collagen peptide components with high affinity for hydroxyapatite from the product mixture system. However, most of these target peptide components exist in a low-abundance form and are easily affected by high-abundance proteins during the enrichment process. How to effectively extract low-abundance target peptide components from high-concentration protein mixtures is a huge challenge. A variety of enrichment and separation methods have been developed for endogenous functional peptides and exogenous functional peptides, such as electrophoresis technology, membrane separation technology, chromatography technology, etc. These technologies have the disadvantages of small scope of application, poor specificity, and high cost. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In view of the above problems, the present invention provides a method for enriching osteogenic active peptides based on affinity adsorption, which can effectively enrich osteogenic active components in collagen peptide samples and enhance the effectiveness of osteogenic active collagen peptide products.

[0006] Hydroxyapatite is one of the most characteristic mixed mode fillers at present. It has both ion exchange and metal affinity, and has special separation capabilities that other fillers do not have. Hydroxyapatite has the advantages of easy separation from the product, no product contamination, high affinity adsorption specificity, and good concentration effect. Therefore, it is of great significance to develop the technology of hydroxyapatite affinity adsorption enrichment of bone collagen osteogenic active peptides.

[0007] To this end, the present invention provides a method for enriching collagen osteogenic active peptides based on affinity adsorption, comprising the following steps:

[0008] The hydroxyapatite pre-packed column is installed in the AKTA pure protein purification system, and the collagen peptide solution is enriched by affinity adsorption through the hydroxyapatite column-AKTA pure protein purification system, wherein the mobile phase a is first used as the elution solution for elution, and the sample is collected when the absorption peak appears to obtain the collagen osteogenic active peptide sample F1, wherein the mobile phase a is a potassium chloride solution. More preferably, the mobile phase a is a 1 mol / L potassium chloride solution.

[0009] Preferably, the method for enriching bone collagen osteogenic active peptides based on affinity adsorption further comprises the following steps:

[0010] After collecting the collagen osteogenic active peptide sample F1, the elution solution is replaced with mobile phase b for elution, and the sample is collected again according to the absorption peak to obtain the collagen osteogenic active peptide sample F2, wherein the mobile phase b is a potassium phosphate solution. More preferably, the mobile phase b is a 100 mmol / L potassium phosphate solution with a pH value of 8.0.

[0011] Preferably, in the method for enriching bone collagen osteogenic active peptides based on affinity adsorption, the AKTA pure system is adjusted in the AKTApure protein purification system for automatic loading, the flow rate is 1.5 mL / min, and after complete loading, it is rinsed with a balanced solution, the flow rate is adjusted to 1.0 mL / min, and after the ultraviolet absorption is stable, it is eluted with the mobile phase a, and the balanced solution is a potassium phosphate solution with a pH value of 7.0. More preferably, the balanced solution is a 5 mmol / L potassium phosphate solution with a pH value of 7.0.

[0012] Preferably, in the method for enriching bone collagen osteogenic active peptides based on affinity adsorption, the bone collagen peptide is dissolved in a potassium phosphate buffer solution, and then filtered to obtain the bone collagen peptide solution. More preferably, the bone collagen peptide is dissolved in a 5mmol / L potassium phosphate buffer solution, and after adjusting the pH of the solution to 7.0 with a pH regulator, a bone collagen peptide solution with a concentration of 70mg / mL is prepared. Most preferably, the pH regulator is 0.1mol / L potassium hydroxide.

[0013] Preferably, in the method for enriching bone collagen osteogenic active peptides based on affinity adsorption, after elution, the collected bone collagen osteogenic active peptide samples are desalted and freeze-dried to obtain sample F1 or sample F2 respectively.

[0014] Preferably, the method for enriching bone collagen osteogenic active peptides based on affinity adsorption further comprises the following steps:

[0015] After the mobile phase a, mobile phase b and balanced solution are prepared, bubbles in the solution are removed before use. More preferably, after the balanced solution, mobile phase a and mobile phase b are prepared, bubbles in the solution are removed under an ultrasonic environment, the ultrasonic frequency is 40KHz, and the treatment time is 60min.

[0016] Preferably, in the method for enriching bone collagen osteogenic active peptides based on affinity adsorption, the bone collagen peptide is dissolved in a 5mmol / L potassium phosphate buffer solution, and the solution pH is adjusted to 7.0 with 0.1mol / L potassium hydroxide to prepare a solution with a concentration of 70mg / mL. The prepared solution is filtered using a 0.22μm pore size microporous filter membrane, and the filtrate is collected for sample loading. The hydroxyapatite pre-packed column is connected to the AKTA pure protein purification system, the purification system flow rate is maintained at 1.5mL / min, and the hydroxyapatite column is balanced with 5CV, pH=7.0, and a potassium phosphate solution with a concentration of 5mmol / L, and the absorption peak is detected at an ultraviolet absorption wavelength of 240nm, and the sample is loaded after the ultraviolet absorption is stable. The sample was injected into the Superloop 10mL loading cup with a syringe, and the AKTA pure system was adjusted for automatic loading. The loading volume was 10mL and the flow rate was 1.5mL / min. After the sample was completely loaded, it was rinsed with a balanced solution, and the flow rate was adjusted to 1.0mL / min. After the ultraviolet absorption was stable, 30mL of mobile phase a (1mol / L potassium chloride) was used for elution, and the ultraviolet absorption spectrum was observed. When the absorption peak appeared, the sample was collected, and the sample F1 was obtained by freeze-drying after desalting. After the ultraviolet absorption was stable, the elution solution was replaced with mobile phase b (pH 8.0, 100mmol / L potassium phosphate) for elution, and the sample was collected according to the peak. After desalting, freeze-dried to obtain F2. The calcium retention rate of the obtained two component samples was determined, and based on this, the component with a higher calcium binding rate was screened out to verify the activity related to osteoblast proliferation, differentiation, and mineralization. More preferably, the calcium binding capacity determination method is the EDTA titration method. More preferably, the osteoblast activity determination method is the CCK-8 method.

[0017] The bone collagen osteogenic active peptide comprises the following peptide segments: the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, which are GDFYR, GERGF, GDFYRA, FDGDFYR, IDGRPGP, LPQPPQE, AGADGARG, EGGPQGPR, GPKGEPGPA, GEPGPPGPAG, GDAGPAGPPGPP, GDAGPPGPAGPA, GEVGPPGPPGPA, respectively.

[0018] The bone collagen osteogenic active peptide is used in promoting osteoblast proliferation activity, promoting osteoblast differentiation and promoting osteoblast mineral deposition.

[0019] The application of the bone collagen osteogenic active peptide in developing functional bone-derived foods and / or dietary nutritional intervention for strengthening bones, restoring bones or strengthening bones.

[0020] The present invention has at least the following beneficial effects:

[0021] The present invention screens out polypeptides that are affinity-adsorbed to a hydroxyapatite column matrix, obtains collagen peptides with a strong ability to bind to hydroxyapatite, measures the calcium binding capacity of the collagen peptide components obtained by enrichment and separation, and their effects on osteoblast proliferation, thereby providing technical support for the enrichment and preparation of affinity-adsorbed collagen osteogenic active peptides, and is of great significance for improving the effectiveness of the product.

[0022] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a chromatogram of collagen peptide purified by hydroxyapatite column in one embodiment of the present invention.

[0024] Figure 2 This is a graph showing the calcium retention rate of collagen peptide before and after purification in one of the embodiments of the present invention.

[0025] Figure 3 This is a graph showing the molecular weight ratio of collagen peptides and their calcium chelates before and after enrichment in one of the embodiments of the present invention.

[0026] Figure 4 This is a graph showing the changes in the amino acid composition of the collagen peptide and its calcium chelate before and after enrichment in one of the embodiments of the present invention.

[0027] Figure 5 This is a Fourier transform infrared spectrum of the enriched collagen peptide and its calcium chelate in one embodiment of the present invention.

[0028] Figure 6 This is a fluorescence spectrum of the reaction between the enriched collagen peptide and calcium chloride in one of the embodiments of the present invention.

[0029] Figure 7 This is a diagram showing the effect of collagen peptides on osteoblast proliferation before and after enrichment in one of the embodiments of the present invention.

[0030] Figure 8 This is a diagram showing the effect of collagen peptides on osteoblast differentiation before and after enrichment in one of the embodiments of the present invention.

[0031] Fig. 9 This is a quantitative result diagram of the relative enzyme activity of alkaline phosphatase (ALP) treated with collagen peptides before and after enrichment in one of the embodiments of the present invention.

[0032] Fig.10This is a diagram showing the results of Alizarin red staining of osteoblasts using different samples in one embodiment of the present invention.

[0033] Fig.11 This is a quantitative analysis diagram of the Alizarin Red staining results in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0035] <Experimental example>

[0036] The specific test process is as follows:

[0037] 1. Materials and methods

[0038] 1.1 Materials and reagents

[0039] Hydroxyapatite prepacked column, Tosoh Bioscience; HiTrapdesalting desalting column, Cytiva; potassium hydroxide, Shanghai En Chemical Technology Co., Ltd.; tripotassium phosphate, Shanghai En Chemical Technology Co., Ltd.; potassium chloride, Shanghai En Chemical Technology Co., Ltd.; disodium ethylenediaminetetraacetic acid, Shanghai En Chemical Technology Co., Ltd.; calcium chloride, Beijing Solebold Technology Co., Ltd.; sodium chloride, Beijing Solebold Technology Co., Ltd.; calcium indicator, Beijing Solebold Technology Co., Ltd.; CCK-8 cell proliferation kit, Beijing Solebold Technology Co., Ltd.; MC3T3-E1 Subclone24, Wuhan Pronocell Life Science Co., Ltd.; osteoblast culture medium, Wuhan Pronocell Life Science Co., Ltd.; osteoblast mineralized nodule staining kit (Alizarin Red S method), Beyotime Biotechnology.

[0040] 1.2 Instruments and Equipment

[0041] AKTA pure protein purification system, Cytiva, vacuum freeze dryer, high-speed centrifuge, HPLC1260-II system, Agilent Technologies Inc, Fourier transform infrared spectrometer, Thermo Scientific, fluorescence spectrophotometer F-4600, Hitachi Scientific Instruments Co., Ltd.

[0042] 1.3. Affinity adsorption-based separation and enrichment of collagen peptides

[0043] Accurately weigh 7.0g of bovine collagen peptide sample, add a small amount of 5mmol / L pH7.0 potassium phosphate buffer solution and fully dissolve it, adjust the pH to 7.0 and then dilute to 100mL to obtain a 70.0mg / mL collagen peptide solution. Filter the prepared solution using a 0.22μm pore size microporous filter membrane, collect the filtrate and prepare for loading. Connect the hydroxyapatite column to the AKTA pure protein purification system, use a potassium phosphate buffer solution with a pH value of 7.0 and a concentration of 5mmol / L at a flow rate of 1.5mL / min for column balance, detect the absorption peak at an ultraviolet absorption wavelength of 240nm, and balance until the ultraviolet absorption value is stable. When loading, use a sample cup to automatically load 10mL of the prepared sample solution through the system, and adjust the flow rate to 1mL / min. After loading, rinse with 5mmol / L potassium phosphate buffer solution until the UV absorption value is stable, then elute with 1mol / L KCl solution, the elution volume is 30mL, the flow rate is 1.5mL / min, collect samples according to peaks, desalt and freeze-dry to obtain F1. Then elute with 30ml, 100mmol / L potassium phosphate buffer solution (pH=8.0), collect components according to peaks, desalt and freeze-dry to obtain F2.

[0044] 1.4 Preparation of collagen peptide chelated calcium and determination of calcium binding capacity

[0045] Accurately weigh 10g of the collagen peptide samples (F1, F2) obtained by enrichment and separation and 10g of CaCl2 and place them in a 100mL beaker. Add a small amount of ultrapure water to completely dissolve it, adjust the pH to 7.0 and then make the volume to 50mL. React in a 60℃ constant temperature oscillating water bath for 60min to obtain a reaction solution. Add 9 times the volume of anhydrous ethanol to precipitate the protein, centrifuge at 8000r / min for 10min, separate the supernatant and the precipitate, and dry the precipitate in a 40℃ oven to obtain the collagen peptide calcium chelate, and measure its calcium binding capacity.

[0046] 1.5. Determination and calculation of calcium binding capacity

[0047] The calculation method of calcium binding capacity is as shown in the formula. When calculating the chelation rate, if the sample contains trace amounts of calcium, the calcium content in the sample must be considered before calculation.

[0048]

[0049] Where: M0: represents the total calcium content in the reaction solution (mg);

[0050] M1: represents the calcium content (mg) of chelated calcium in the same volume of reaction solution.

[0051] Note: The reaction solution is the same as the one mentioned in the preparation of collagen peptide calcium chelate in 1.4

[0052] 1.5.2. Determination of total calcium content

[0053] Use a pipette to take 0.1mL of the reaction solution to a conical flask, and measure 50mL of distilled water into a measuring cylinder. Then, add 2-3mL of triethanolamine, 0.8-1mL of potassium hydroxide solution (pH 12-13, 1.25mol / L), and an appropriate amount of calcium indicator. Gently shake well, and immediately titrate with EDTA solution (diluted 10 times, Ca = 0.0456mg / mL). The end point is when the solution changes from purple-red to pure blue, and record the EDTA volume V0. The total calcium content M0 (g) can be calculated according to formula 3-4.

[0054]

[0055] 1.5.3 Determination of calcium content

[0056] Use a pipette to take 1 mL of the reaction solution into a 50 mL centrifuge tube, add 20 mL of ethanol, centrifuge at 8000 r / min for 10 min, remove the supernatant, dilute to 20 mL with distilled water, and take 2 mL of this solution. Follow the same procedure as in step 1.5.2 (EDTA titration), and record the EDTA volume V1. Calculate the chelated calcium content M1 (g) according to formula 3-5.

[0057]

[0058] Table 1. Determination of calcium retention rate of samples

[0059]

[0060] 1.6 Characterization of the physicochemical properties of collagen peptides

[0061] 1.6.1 Determination of molecular weight distribution

[0062] The molecular weight distribution of collagen peptides and their calcium chelates was determined by high performance liquid chromatography. The molecular weight distribution of collagen peptides was determined using an Agilent HPLC1260-II system (Agilent Technologies Inc., California, USA). TSK gel G2000 SWXL column (7.8×300 mm, TOSOH, Tokyo, Japan); column temperature: 40°C; mobile phase: A is a 45% (v / v) acetonitrile solution mixed with 0.1% trifluoroacetic acid; isocratic elution; flow rate: 0.5 mL / min; injection volume: 10 μL, and the response value was measured at a wavelength of 214 nm. Gly-Sar (146 Da), Gly-Gly-Tyr-Arg (451 Da), Bacitracin (1422 Da), Aprotinin (6511 Da) and Cytochrome C (12327 Da) were used as standards to establish a standard curve between retention time (X) and logarithm of molecular weight (Y) (Y = -0.1622X + 6.1169, R 2 =0.989).

[0063] 1.6.2. Amino Acid Composition Determination

[0064] Weigh 100 mg of sample into a hydrolysis tube, add 8 mL of 6M HCl, fill with nitrogen for 3 minutes, and place at 110°C for hydrolysis for 22 hours. After the reaction is completed, add 4.8 mL of 10M NaOH, and add water to make the volume 25 mL. After mixing evenly, filter with double-layer filter paper, take the filtrate and centrifuge at 10000r / min for 10 minutes, collect the supernatant. Use HPLC to analyze the amino acid composition of the supernatant.

[0065] 1.6.3 Fourier Transform Infrared Spectroscopy (FTIR)

[0066] Weigh 1 mg of completely dried sample powder, mix it with 100 mg of KBr, grind and press it into tablets under infrared light, and then scan it on the machine. The measurement parameters of the instrument are set as follows: resolution is 4 cm -1 ; Scan times: 32; Scan range: 4000~400cm -1 The results were expressed as transmittance, and the data were analyzed using OMINIC 8.2 software (Thermo Fisher Scientific Inc.).

[0067] 1.6.4. Endogenous fluorescence spectroscopy

[0068] The collagen peptide powder was prepared into a solution with a protein concentration of 0.1 mg / mL, and the metal ion solution was slowly added and stabilized for 10 minutes to make the final concentration reach the fluorescence quenching phenomenon. The fluorescence intensity was detected at an excitation wavelength of 280 nm and an emission wavelength of 290-500 nm.

[0069] 1.6.5. Mass spectrometry sequencing

[0070] F2 was analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was a Q-Exactive plus mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nano-LC1200. A total of 3 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient of 60 min. The column flow was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 2% of phase B, increased to 35% in 47 minutes with a nonlinear gradient, increased to 100% in 1 minute, and maintained for 12 minutes.

[0071] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z): 200-1800; resolution: 70,000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s.

[0072] Table 2. F2 component peptide sequencing results

[0073]

[0074] 1.7. Analysis of osteogenic activity of collagen peptide samples before and after enrichment

[0075] 1.7.1 Determination of osteoblast proliferation rate

[0076] Add 100 μL of cell suspension (1×10 4). The culture plate was cultured in a 37°C, 5% CO2 incubator for 24 hours. After the cells adhered to the wall, they were cultured in serum-free medium for 24 hours to synchronize the cells. The upper culture medium was discarded and 100 μL of complete culture medium or complete culture medium containing different test samples was added, and cultured at 37°C for 24 hours. After removing the upper culture medium, 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added, and the culture plate was incubated in the incubator for 4 hours, and its absorbance was measured at a wavelength of 450 nm.

[0077] 1.7.2 Promote osteoblast differentiation

[0078] MC3T3-E1 cells were cultured at 8x10 4 Cells were seeded at a density of 1.54 cells / mL in a 24-well plate and cultured for 24 hours. The culture medium in each well was replaced with 500 μL differentiation medium. The differentiation medium contained 10% fetal bovine serum (a-MEM), 5 mmol LB-glycerophosphate, 1% penicillin and streptomycin (w / v), 50 μg / mL ascorbic acid and 10 mol / L dexamethasone. At the same time, different concentrations of pre- and post-enrichment collagen peptides were added to the cells. Three groups were repeated in parallel for osteogenic differentiation detection. Untreated cells were used as blank controls.

[0079] After 3 days of osteoblast differentiation, the expression of ALP was detected using an alkaline phosphatase (ALP) staining kit. The cells were fixed with 200 μL of 95% ethanol at 4°C for 15 min to complete the staining of ALP. The cells were then washed three times with PBS solution, and then the ALP staining reagent was added according to the manufacturer's instructions, and the mixture was incubated for 30 min. An inverted optical microscope was used to take pictures, and ALP was quantified using ImageJ software.

[0080] 1.7.3 Characterization of osteoblast mineralization activity

[0081] Add 1 mL of cell suspension (5 × 10 4 ), culture the culture plate in a 37°C, 5% CO2 incubator for 24 hours, and after the cells adhere to the wall, culture them in serum-free medium for 24 hours to synchronize the cells, then discard the medium and add 1 mL of complete medium or complete medium containing different test samples to the culture plate, culture for 21 days, and replace the medium every two days. After the culture is completed, rinse the cells to be stained with PBS 1-3 times, add 95% ethanol to fix for 30 minutes, rinse with PBS 1-2 times after fixation, and then stain with 1 mL of Alizarin Red S staining solution for 30 minutes, wash off the excess dye with distilled water, and observe under a microscope. To quantify the bound dye, treat with 10% cetylpyridinium chloride (CPC) in the dark for 1 hour and read at 570nm with an enzyme reader.

[0082] 1.8. Results of collagen peptide enrichment and separation and calcium retention rate determination

[0083] 1.8.1 Enrichment and separation of collagen peptides

[0084] The results of enriching and separating collagen peptides using hydroxyapatite columns are as follows Figure 1 As shown, the hydroxyapatite column is washed with a balanced solution and after the UV absorption peak at 240 nm is stabilized, it is eluted with mobile phase a. When the first UV absorption peak appears, the component is collected and marked as F1. After the UV absorption is stable, the system is adjusted to select mobile phase b for elution. After the second peak appears, the sample is collected and marked as F2. The collected samples are desalted, freeze-dried and stored at 4°C for subsequent experimental analysis.

[0085] 1.8.2 Determination of calcium binding capacity of different components

[0086] The results of the EDTA titration method for determining the calcium retention rate of different components are as follows: Figure 2 As shown in the figure, compared with the samples before enrichment and separation, the calcium retention rate of the treated samples was significantly increased. The calcium binding capacity of the F1 and F2 components increased from (36.09±1.13)μg / mg of the untreated samples to (39.54±3.49)μg / mg and (42.06±0.92)μg / mg, respectively. The calcium retention rate increased significantly, and the calcium retention rate of the F2 component was the highest. The F2 component was selected for research in subsequent experiments.

[0087] 1.9. Structural characterization of collagen peptides before and after enrichment and separation

[0088] 1.9.1 Changes in the molecular weight distribution of collagen peptides before and after enrichment and separation

[0089] Depend on Figure 3It can be seen that the molecular weight distribution of CPs and F2 before and after enrichment and separation is significantly different. The molecular weight of the F2 component is mainly concentrated below 5000Da, and the components with a molecular weight of less than 1000Da account for 74.5% of the total, which is significantly higher than 44.26% of CPs. After calcium chelation, the molecular weight distribution of CPs and F2 components showed an overall upward trend, among which the proportion of peptides with a molecular weight of CPs less than 1000Da decreased from 44.26% to 34.91%, and the proportion of peptides with a molecular weight between 1000Da and 2000Da decreased from 29.27% ​​to 28.2%. The proportion of components with molecular weight greater than 2000Da increased, the proportion of peptides with molecular weight between 2000Da and 3000Da increased from 13.45% to 16.59%, the proportion of components with molecular weight between 3000Da and 5000Da increased from 9.9% to 14.73%, and the proportion of components greater than 5000Da increased from 3.12% to 5.57%; the proportion of components with molecular weight less than 1000Da in F2 decreased from 74.5% to 63.08%, the proportion of components with molecular weight between 1000-2000Da decreased from 11.89% to 9.976%, and the proportion of components with molecular weight distribution above 3000Da increased to a certain extent. The results show that calcium chelation reaction will significantly increase the molecular weight of collagen peptides.

[0090] 1.9.2 Changes in Amino Acid Composition of Collagen Peptides Before and After Enrichment and Separation

[0091] The changes in amino acid composition before and after enrichment and separation are as follows Figure 4 As shown in the figure, the overall change of amino acid composition before and after separation was not significant, and there was no significant difference. However, after calcium chelation, the amino acid composition of F2 and its calcium chelate changed. The amino acid content of Asp and Glu increased significantly after calcium chelation, which may be due to the chelation reaction between Asp and Glu and calcium due to their lone pair electrons. Consistent with previous studies, the hydroxyl groups of Asp and Glu are the main chelation sites of peptide-calcium chelates.

[0092] 1.9.3 Changes in Fourier transform infrared spectra of collagen peptides before and after enrichment and separation

[0093] FTIR spectroscopy was used to explore the chemical structure changes of the samples. The characteristic changes of FTIR absorption peaks can be used to reflect the interaction between metal ions and organic ligand groups in peptides. Figure 5 As shown in Figure 2, the FTIR spectrum changes after the calcium binding reaction. The wave number moves from 3307.12 to 3300.09 cm -1 , which may be due to the binding of -NH2 to calcium ions, indicating that NH helps to form chelates due to dipole field effects or inductive effects. 1700-1500cm -1 The vibrational spectral region in the range corresponds to amide I (1600-1700 cm -1) and amide II (1580-1510cm -1 ) stretching vibration, these two vibration bands correspond to the stretching vibration of C=O and CH NH bonds respectively. In the FTIR spectrum, after calcium treatment, F2 at 1654.87cm -1 The absorption peak of F2 at 1537.21 cm -1 The absorption peak at 1546.86 cm -1 ), which suggests that CH and NH groups may bind to calcium ions in the binding reaction. In addition, 1430-1370cm -1 The spectral region within the range represents the stretching vibration of the carboxylic acid group. In the FTIR spectrum, the band shifts to higher wavenumbers, which indicates that the carboxylic acid group may be bound to calcium ions.

[0094] 1.9.4 Changes in fluorescence spectra of collagen peptides before and after enrichment and separation

[0095] In order to study the interaction between small molecule ligands and peptides, different concentrations of metal ions were added to the peptide solution to obtain fluorescence spectra such as Figure 6 As shown in the figure, with the addition of metal ions, the fluorescence intensity decreases continuously, which indicates that the metal ions interact with the peptide and produce fluorescence quenching. As the concentration of the added drop increases, the decrease rate decreases continuously, which may be because as the binding reaction proceeds, the exogenously added metal ions are in a saturated state and slow down the decrease of fluorescence.

[0096] 1.10 Study on osteogenic activity of collagen peptides before and after enrichment and separation

[0097] 1.10.1 Effect on osteoblast proliferation

[0098] The proliferation effect of the samples on MC3T3-E1 cells was determined by CCK-8 method. Figure 7 As shown. The proliferation effects of CPs and F2 at concentrations of 5, 1, 0.5, and 0.1 mg / mL on osteoblasts were measured respectively. It can be seen that compared with CPs, the F2 component has a stronger promoting effect on the proliferation of osteoblasts. Under the above four concentrations, the osteoblast proliferation rate is higher than that of cells treated with the CPs group. And when the F2 concentration is 1 mg / mL, the proliferation activity of osteoblasts is the strongest, which is (197±11.3)%. Therefore, 1 mg / mL was selected as the subsequent experimental concentration to carry out experiments on promoting osteoblast differentiation and mineralization.

[0099] 1.10.2 Osteoblast differentiation

[0100] Alkaline phosphatase (ALP) is a product of the early differentiation of MC3T3-E1 cells and an important indicator for determining its osteogenic activity. The increase in ALP activity indicates the promotion of osteoblast differentiation. ALP was histochemically stained to determine the effects of CP and F2 on the osteogenic formation of MC3T3-E1 cells. Figure 8 Compared with (C), CP( Figure 8 Middle (A)) and F2 ( Figure 8 The number of ALP-positive cells in MC3T3-E1 cells treated with (B) increased significantly and the blue-purple color became darker, indicating that collagen peptide can significantly increase the ALP activity of osteoblasts and promote osteoblast differentiation ( Figure 8 ). In addition, the number of ALP-positive cells after F2 treatment was significantly higher than that of CPs, indicating that F2 has a stronger activity in promoting cell differentiation, e.g. Fig. 9 , the quantitative results of ALP relative enzyme activity were consistent.

[0101] 1.10.3 Osteoblast mineralization

[0102] Effects of CPs, CPs-Ca, and CaCl2 on osteoblast mineralization Fig.10 As shown, Fig.10 (A) is CPs, Fig.10 (B) is F2, Fig.10 (C) is the blank control. It can be seen from the figure that F2 has the best effect on promoting osteoblast mineral deposition, and Alizarin red staining shows a large area of ​​blocky red precipitation, followed by CPs and the blank control. The quantitative analysis of the bound dye is shown in the following figure. Fig.11 As shown in the figure, it can be seen that F2 binds the most dye, indicating that it has the best effect in promoting osteoblast mineral deposition, which is consistent with the results of microscopic observation.

Claims

1. A composition of collagen osteogenic active peptides, characterized in that: The following peptides Composition: Amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

2. Use of the composition of collagen osteogenic active peptides as claimed in claim 1 in promoting osteoblast proliferation activity, promoting osteoblast differentiation and promoting osteoblast mineral deposition, wherein the use is for non-therapeutic purposes.

3. Use of the composition of the osteogenic active collagen peptides as claimed in claim 1 in developing functional bone-derived foods and dietary nutritional interventions for strengthening bones, nourishing bones or strengthening bones.

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