A chitosan oligosaccharide peptide calcium chelate, its preparation method and application

Abstract

The present invention discloses a chitosan oligosaccharide peptide calcium chelate, its preparation method and application. In the present invention, polypeptides with potential calcium absorption promoting activity in walnut protein peptides are screened by molecular docking and glycosylated modification is carried out to prepare peptide calcium complexes. Based on structural characterization, in vitro stability experiments, Caco-2 cell monolayer model, and MC3T3-E1 cell induced differentiation model, the binding effect, stability and calcium absorption promoting mechanism of glycosylated walnut peptide chelates are explored. The results show that COS-AERGVLYR-Ca has good thermal stability, gastrointestinal stability, anti-phosphate crystallization inhibition ability and better calcium transport ability.

Description

Technical Field

[0001] The present invention relates to the field of peptide-calcium chelates, and particularly to a chitosan oligosaccharide-peptide calcium chelate, a preparation method thereof, and an application thereof. Background Art

[0002] At present, three types of calcium supplements, namely inorganic calcium salts, amino acid calcium, and organic acid calcium salts, have been developed in the market to prevent and resist calcium deficiency. However, since these calcium supplements are prone to form calcium phosphate precipitates under the slightly alkaline conditions of the intestine, they are likely to cause irritation to the intestine and are not suitable for long-term use. Peptide chelated calcium, as the fourth-generation new calcium supplement, can overcome the limitations brought by low bioavailability at low concentrations and biotoxicity at high concentrations. However, after some polypeptides are chelated with calcium, their stability is poor and their bioavailability is low. The purpose of the present invention is to develop a chitosan oligosaccharide-peptide calcium chelate for preparing a new calcium supplement to improve the calcium loading efficiency and promote the calcium absorption effect. Summary of the Invention

[0003] The purpose of the present invention is to screen out polypeptides with potential for promoting calcium absorption through molecular docking and calcium-binding ability and prepare their chelates, and to explore the binding mechanism between calcium and glycosylated peptides and their calcium-holding ability after chelation by using various structural characterizations and stability experiments. A Caco-2 cell monolayer model and an MC3T3-E1 cell induced differentiation model are established to explore the calcium absorption promotion and osteogenic ability under the synergistic action of glycosylated peptides and calcium.

[0004] To solve the above technical problems, the technical solution of the present invention is as follows: A peptide-calcium chelate, and the structural formula of the peptide-calcium chelate is as follows: AERGVLYR-Ca.

[0005] The present invention also provides a chitosan oligosaccharide-peptide calcium chelate, in which the polypeptide is glycosylated with chitosan oligosaccharide COS and then chelated with calcium ions to prepare a chitosan oligosaccharide-peptide calcium chelate, and the structural formula of the chitosan oligosaccharide-peptide calcium chelate is as follows: chitosan oligosaccharide COS-AERGVLYR-Ca.

[0006] The present invention also provides a preparation method of the peptide-calcium chelate or the chitosan oligosaccharide-peptide calcium chelate. The process of obtaining the glycosylated peptide COS-AERGVLYR by using COS modification is as follows:

[0007] Mix the polypeptide AERGVLYR and chitosan oligosaccharide COS in water, adjust the pH of the reaction system to 6.5-7.5, react in a water bath at 85-95 °C for 2-5 h, quickly cool to room temperature to end the reaction, and then place it at 2-5 °C overnight to make it fully hydrated. Dialyze with a 500-1000 Da dialysis bag at 18-25 °C for 20-30 h, centrifuge at 2500-3500 r / min for 8-15 min, take the supernatant and freeze-dry it to obtain the freeze-dried glycosylated peptide COS-AERGVLYR;

[0008] The mass ratio of the polypeptide AERGVLYR to chitosan oligosaccharide COS is 0.8 - 1.0:1.0 - 1.2, and their total concentration in water is 40 - 60 mg / mL.

[0009] Furthermore, the freeze-dried glycosylated peptide COS-AERGVLYR or polypeptide AERGVLYR is dissolved in deionized water at a concentration of 10 - 20 mg / mL, and CaCl2 is added so that the mass ratio of the glycosylated peptide COS-AERGVLYR or polypeptide AERGVLYR to CaCl2 is 1:3 - 6, and the pH is adjusted to 6.5 - 7.5. The reaction is carried out in a water bath at 50 - 65 °C for chelation for 40 - 80 min. After the chelation reaction is completed, anhydrous ethanol with a volume 8 - 10 times that of the reaction solution is added to the reaction system. Finally, the mixture is centrifuged at 2 - 5 °C at 6000 - 11000×g for 8 - 12 min, the precipitate is collected, and freeze-dried to obtain the glycosylated peptide calcium COS-AERGVLYR-Ca chelate or AERGVLYR-Ca chelate.

[0010] The present invention also provides the application of the peptide calcium chelate or chitosan oligosaccharide peptide calcium chelate in the preparation of a biological calcium supplement.

[0011] Through molecular docking and calcium binding ability determination, the present invention clarifies that AERGVLYR is the peptide sequence with the best affinity for TRPV6 protein and Cav1.3 protein, and hydrophobic interaction and hydrogen bond are its main interaction forces. The carbonyl oxygen of leucine, the carboxyl oxygen of glutamic acid, and the carbonyl oxygen of valine may be the calcium ion binding sites. The calcium binding ability of AERGVLYR to calcium ions is 66.08 μg / mg, and the calcium binding ability of the glycosylated COS-AERGVLYR to calcium ions is increased to 78.29 μg / mg.

[0012] The present invention clarifies the structural changes of the peptide calcium chelate formed by COS-AERGVLYR and calcium ions. The results show that after binding with calcium ions, the peptide structure of COS-AERGVLYR folds, the crystallinity increases, and it is a smooth block structure. Calcium ions may bind to the peptide through the carboxyl oxygen atom and amino nitrogen atom of the peptide segment.

[0013] Clarify the stability and calcium retention ability of COS-AERGVLYR-Ca. COS-AERGVLYR-Ca has good thermal stability, gastrointestinal stability, and the ability to inhibit phosphate crystallization.

[0014] To investigate the effects of COS-AERGVLYR in combination with calcium on calcium transport in the Caco-2 cell monolayer model and osteogenic activity of MC3T3-E1 cells. COS-AERGVLYR + CaCl2 can resist the inhibitory effect of the nutritional factor phytic acid and exhibits better calcium transport ability compared with CaCl2 and AERGVLYR + CaCl2. The calcium channels TRPV6 and Cav1.3 regulate the promotion of calcium transport in the Caco-2 cell monolayer model by COS-AERGVLYR. The combination of COS-AERGVLYR and calcium can promote the proliferation, differentiation, and mineralization of MC3T3-E1 cells.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The COS-AERGVLYR-Ca provided by the present invention has good thermal stability, gastrointestinal stability, and ability to resist phosphate crystallization inhibition. COS-AERGVLYR + CaCl2 can resist the inhibitory effect of the nutritional factor phytic acid and exhibits better calcium transport ability compared with CaCl2 and AERGVLYR + CaCl2. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Technical roadmap of the present invention;

[0017] Figure 2 Total ion current chromatogram;

[0018] Figure 3 Molecular weight distribution of walnut peptides with MW < 3000 Da;

[0019] Figure 4 Visualization results of molecular docking of AERGVLYR with TRPV6 and Cav1.3;

[0020] Figure 5 Prediction of calcium binding sites of AERGVLYR;

[0021] Figure 6 Fourier transform infrared spectra of AERGVLYR, AERGVLYR-Ca, COS-AERGVLYR, and COS-AERGVLYR-Ca;

[0022] Figure 7 Ultraviolet-visible absorption spectra of AERGVLYR, AERGVLYR-Ca, COS-AERGVLYR, and COS-AERGVLYR-Ca;

[0023] Figure 8 Fluorescence spectra of chelation of different concentrations of CaCl2 with AERGVLYR and COS-AERGVLYR;

[0024] Figure 9 X-ray diffraction patterns of AERGVLYR, AERGVLYR-Ca, COS-AERGVLYR, and COS-AERGVLYR-Ca;

[0025] Figure 10 Scanning electron microscope images;

[0026] Figure 11 TG-DSC of different samples: A: AERGVLYR, B: AERGVLYR-Ca, C: COS-AERGVLYR, D: COS-AERGVLYR-Ca;

[0027] Figure 12 Thermal stability of AERGVLYR-Ca and COS-AERGVLYR-Ca. Different letters indicate significant differences between different groups ( P <0.05);

[0028] Figure 13 Gastrointestinal simulated digestion of AERGVLYR-Ca and COS-AERGVLYR-Ca;

[0029] Figure 14 Results of the phosphoric acid crystallization inhibition experiment;

[0030] Figure 15 Effects of different concentrations of samples on the viability of Caco-2 cells. Different letters indicate significant differences between different groups ( P <0.05);

[0031] Figure 16 Changes in TEER values during the Caco-2 cell modeling process;

[0032] Figure 17 Calcium transport of AERGVLYR and COS-AERGVLYR acting on the Caco-2 cell monolayer model at different time periods. Different letters indicate significant differences between different groups ( P <0.05);

[0033] Figure 18 Effects of different concentrations of Cav1.3 calcium channel inhibitors on calcium absorption. Different letters indicate significant differences between different groups ( P <0.05);

[0034] Figure 19 Effects of different concentrations of TRPV6 calcium channel inhibitors on calcium absorption. Different letters indicate significant differences between different groups ( P <0.05 );

[0035] Figure 20 Under the inhibition of phytic acid, the effects of AERGVLYR and COS-AERGVLYR on calcium transport in the Caco-2 cell monolayer model; different letters indicate significant differences between different groups ( P <0.05);

[0036] Figure 21 The calcium influx of COS-AERGVLYR and AERGVLYR acting on cells;

[0037] Figure 22 The gene expression of Caco-2 cells; different letters indicate significant differences between different groups ( P <0.05);

[0038] Figure 23 The proliferation effects of samples with different concentrations on MC3T3-E1 cells; different letters indicate significant differences between different groups (P<0.05);

[0039] Figure 24 The effects of AERGVLYR, COS-AERGVLYR, AERGVLYR+CaCl2, and COS-AERGVLYR+CaCl2 on the ALP activity of MC3T3-E1 cells. Different letters indicate significant differences between different groups ( P <0.05);

[0040] Figure 25 The effects of AERGVLYR, COS-AERGVLYR, AERGVLYR+CaCl2, and COS-AERGVLYR+CaCl2 on the mineralization of MC3T3-E1 cells; different letters indicate significant differences between different groups ( P <0.05). Detailed implementation manners

[0041] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention is not limited to the following technical solutions. The technical roadmap of the present invention is as Figure 1 shown. The experimental data in the following examples are all expressed as the mean values ± standard deviation (Mean values ± SD) of 3 independent experiments. One-way ANOVA is performed using SPSS 21.0 statistical software, and charts are drawn using ORIGIN 2024. Different marked letters indicate significant differences between data ( P <0.05).

[0042] Example 1 Screening of bioactive peptides

[0043] Extraction of walnut meal protein

[0044] Defatted walnut powder → mix with water at a solid-liquid ratio of 1:20 (w / v) → adjust the solution pH to 11.0 → stir in a 55℃ water bath for 1.5 h → cool → centrifuge (4℃, 3500 rpm, 10 min) → take the supernatant and adjust the pH to 4.5 → let stand for 1 h → centrifuge (4℃, 3500 rpm, 10 min) → wash the precipitate with distilled water → adjust the pH to 7.0 → dialysis desalination → vacuum freeze drying → walnut protein isolate → refrigerate at 4℃ for later use.

[0045] Walnut protein hydrolyzate

[0046] The freeze-dried walnut protein isolate was dissolved in ultrapure water and mixed thoroughly to obtain a solution with a substrate concentration of 2%. 2% (enzyme mass / substrate mass) of alkaline protease was added, and the pH of the mixed solution was adjusted to 9.0 with 1.00 mol / L NaOH, and hydrolyzed at 55°C for 3 h. After the hydrolysis was completed, the mixture was heated in boiling water for 10 min to inactivate the enzyme. After the enzymatic hydrolyzate was cooled to room temperature, it was centrifuged at 4000 rpm for 20 min, and the supernatant was collected and freeze-dried for later use.

[0047] Isolation of walnut peptides

[0048] Ultrafiltration: Walnut peptides were separated using an ultrafiltration tube with a molecular weight cutoff of 3 kDa and the lower fraction of the ultrafiltration tube was collected and freeze-dried for later use. Peptides with high calcium binding capacity were screened out through Trince-SDS-PAGE gel electrophoresis verification, liquid phase secondary mass spectrometry LC-MS / MS identification and molecular docking.

[0049] Figure 2 The total ion current chromatogram of walnut mixed peptides. The LC-MS / MS method was used to detect the molecular weight of walnut mixed peptides to identify the amino acid sequence. The mass spectrometry test raw file was searched by the search engine MaxQuant 1.6.14 to retrieve the corresponding database, and 496 peptides were obtained. The length of these peptides ranged from 8 to 24 amino acids. Figure 3 As shown, there are 25 peptides with a molecular weight of less than 1000 Da, accounting for 5.04% of the species and 4.08% of the content; there are 419 peptides with a molecular weight of 1000-2000 Da, accounting for 84.48% of the species and 89.65% of the content; there are 52 peptides with a molecular weight of 2000-3000 Da, accounting for 10.48% of the species and 6.27% of the content.

[0050] In the previous experiment, the same walnut peptide sample was divided into different molecular weights by ultrafiltration. The results of the calcium chelation experiment showed that the walnut peptide with MW < 1000 Da had the strongest calcium chelation ability. Therefore, 25 walnut polypeptides with a molecular weight < 1000 Da were selected for molecular docking with the calcium ion channel proteins TRPV6 and Cav1.3. At the same time, in order not to miss other walnut peptide sequences with good calcium-promoting ability, multiple polypeptides were selected from walnut peptides with a molecular weight of 1000 - 3000 Da through scoring, signal intensity, and amino acid sequence screening for molecular docking, and walnut peptide sequences with good affinity and high credibility were selected from them.

[0051] The score represents the credibility of the peptide segment. The relative abundance can represent the detected ion signal intensity in mass spectrometry. The higher the score and the higher the abundance, the higher the credibility and content of the peptide segment in the component. Therefore, through three conditions: the top 100 in terms of score, the top 40 in terms of signal intensity, and the top 10 in terms of the sum of the numbers of Asp, Glu, and Ser, 10 polypeptides with a molecular weight of 1000 - 3000 Da were screened in sequence for molecular docking.

[0052] Active transport (i.e., transcellular transport) and passive transport (i.e., paracellular transport) are two ways of intestinal calcium absorption. TRPV6 calcium channel (the transient receptor potential cation of the vanilloid subfamily V member 6) and Cav1.3 (calcium channel, voltage-dependent, L-type, alpha 1D subunit) are two typical active transport methods. Therefore, TRPV6 protein and Cav1.3 protein were selected for docking with polypeptides. The lower the binding energy, the stronger the affinity between the receptor and the ligand. According to the docking results in Table 1, peptide sequences with a signal intensity of 0 in the table were excluded. LPSFSNAPR is a polypeptide with a relatively high binding energy among those with MW < 1000 Da. The docking scores of LPSFSNAPR with TRPV6 protein and Cav1.3 protein were -8.6 kcal / mol and -8.9 kcal / mol respectively, with a total of -17.5 kcal / mol; the scores of VVQGRGLHGA, VIAFPAGVAH, NSFNLPIL, and AERGVLYR were similar, but AERGVLYR had a higher score among them, which means higher credibility. The docking scores of AERGVLYR with TRPV6 protein and Cav1.3 protein were -7.9 kcal / mol and -8.1 kcal / mol respectively (compared with VIAFPAGVAH, AERGVLYR had similar binding energies with TRPV6 protein and Cav1.3 protein and stronger regulatory potential), with a total of -15.9 kcal / mol. In summary, two polypeptides, LPSFSNAPR and AERGVLYR, were selected for synthesis.

[0053] Table 1 Molecular Docking Data Table

[0054]

[0055] Synthesis Verification of AERGVLYR and LPSFSNAPR

[0056] The AERGVLYR and LPSFSNAPR polypeptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and verified by mass spectrometry and HPLC liquid chromatography. From the HPLC chromatogram of AERGVLYR, it can be seen that the peak area is the largest at a retention time of 9.594 min, the peak area is 12413907, the peak area percentage is 97.329, the peak height is 886292, and the peak height percentage reaches 98.246%; from the HPLC chromatogram of LPSFSNAPR, it can be seen that the peak area is the largest when the retention time is 10.170 min, the peak area is 15242131, the peak area percentage is 96.286, the peak height is 1212635, and the peak height percentage reaches 95.112%.

[0057] The molecular weights of the synthetic peptides were determined by electrospray ionization (ESI) mass spectrometry. The theoretical molecular weight of AERGVLYR is 962.5298 Da, [M + 3 H] 3+ type, [M + 2 H] 2+ type, [M + 1 H] 1+ type molecules, and the actual molecular weight is 962.85 Da; the theoretical molecular weight of LPSFSNAPR is 987.5138 Da, belonging to [M + 2 H] 2+ type, [M + 1 H] 1+ type molecules, and the actual molecular weight is 988.10 Da.

[0058] Example 2 Preparation of Peptide-Calcium Chelates

[0059] COS and the pure peptide were dissolved in deionized water with a total concentration of 50 mg / mL, and the mass ratio of COS to the pure peptide was 1:0.8. Then, the pH value of the solution was adjusted to 7 and heated in a water bath at 90 °C for 2.5 h. Subsequently, the solution was cooled to room temperature and then placed at 4 °C overnight for complete hydration. To separate the mixture, it was placed in a dialysis bag with a molecular weight of 500 - 1000 Da and dialyzed at 20 °C for 24 h, centrifuged at 2500 - 3500 r / min for 8 - 15 min, and the supernatant was freeze-dried to obtain freeze-dried glycosylated peptide;

[0060] Preparation of Peptide-Calcium Chelates and Chitosan Oligosaccharide Peptide-Calcium Chelates

[0061] According to the previous research method in the laboratory, the polypeptide or glycosylated peptide was dissolved in deionized water at a concentration of 10 - 20 mg / mL, the mass ratio of the peptide to Ca was 1:5, the total concentration after mixing the peptide and calcium was 60 mg / mL, and the pH value was set to 7.4. The chelation reaction was carried out at 60 °C for 1 h, and 9 times the volume of anhydrous ethanol of the solution was used to separate the chelate, centrifuged at 10733×g at 4 °C for 10 min, and the precipitate was collected and freeze-dried for subsequent use.

[0062] Determination of calcium binding capacity

[0063] After dissolving the polypeptide in deionized water to 1.0 mg / mL, it was mixed with 5 mM / L CaCl2, the pH was adjusted to 8.0, and it was stirred at 37 °C for 2 h. Subsequently, it was reacted at 37 °C for 1 h under the condition of mixing with 0.2 mol / L sodium phosphate buffer (pH 8.0). After the reaction mixture was centrifuged to remove the insoluble calcium phosphate salt, it was further operated at room temperature. After freeze-drying the remaining supernatant, the calcium content was determined by o-cresolphthalein complexone colorimetry. All experiments were carried out in triplicate, and the obtained results were expressed as mean ± standard deviation (SD).

[0064] As shown in Table 2, the calcium binding capacities of the three selected peptide segments were determined. The calcium binding capacity of AERGVLYR was 66.08 ± 6.18 μg / mg, and the calcium binding capacity of LPSFSNAPR was 65.99 ± 3.49 μg / mg. After glycosylation modification, the calcium binding capacities were increased to 78.29 ± 4.37 μg / mg and 72.84 ± 1.21 μg / mg respectively. The results showed that glycosylation modification could enhance the calcium binding capacity of walnut polypeptides.

[0065] Table 2 Calcium binding capacity

[0066]

[0067] Result analysis of molecular docking and prediction of calcium binding sites of AERGVLYR

[0068] As Figure 4 The visualization results of molecular docking of peptide AERGVLYR with proteins TRPV6 and Cav1.3 are shown. The docking result of AERGVLYR with protein TRPV6 is shown in the figure. Arg in AERGVLYR formed hydrogen bonds with Glu (B:605) and Leu (B:608) of TRPV6, and Gly formed a hydrogen bond with Lys (B:607) of TRPV6. The exposed Glu carboxyl side chain and Leu side chain carboxyl oxygen may provide binding sites for calcium ions; from the docking site, it can be seen that hydrogen bonds and hydrophobic interactions play important roles in the binding of AERGVLYR to TRPV6 and Cav1.3. Hydrophobic force belongs to weak intermolecular force, and hydrogen bond force is stronger than hydrophobic force. Therefore, the combination of AERGVLYR and Cav1.3 has stronger affinity.

[0069] From Figure 5As can be seen, the carbonyl oxygen of leucine, the carboxyl oxygen of glutamic acid, and the carbonyl oxygen of valine in AERGVLYR may form binding sites with calcium ions. In addition, the molecular docking results show that hydrogen bonds are mainly formed at the C-terminal and N-terminal of the peptide. Acidic amino acids have a high calcium-binding ability, and the carboxyl oxygen of the exposed Glu side chain after molecular docking may chelate calcium ions. In the calcium-peptide chelate, the oxygen and nitrogen atoms of the polypeptide mainly act as electron donors, and the metal ions mainly act as electron acceptors to form coordination bonds. Treating the calcium-peptide chelate with dissociating agents at different action sites, it was found that the chelate disintegrated when the hydrophobic interaction and hydrogen bond were disrupted. This result confirms that the formation and stability of the chelate depend on the existence of hydrogen bonds and hydrophobic interactions.

[0070] Example 3 Structural Characterization of COS-AERGVLYR-Ca

[0071] Based on the analysis of the structural characteristics of COS-AERGVLYR-Ca by spectroscopy, chromatography, etc., the measurement methods used in this example are all existing methods.

[0072] Fourier Transform Infrared Spectroscopy (FTIR)

[0073] The significant change in the position of the absorption peak in the FTIR spectrum indicates that the organic ligand groups in the peptide interact with calcium ions. The amide I band (1700 - 1600 cm -1 ) is mainly caused by the vibration of C=O. As Figure 6 shown, after chelating with calcium ions, the corresponding peak vibration of AERGVLYR shifts from 1640.72 cm -1 to 1649.03 cm -1 , and the absorption peak of COS-AERGVLYR shifts from 1641.52 cm -1 to 1648.6 cm -1 . The amide II band (1600 - 1500 cm -1 ) corresponds to the C-N bending vibration and the N-H bending vibration. As shown in the figure, after glycosylation modification, the absorption peak of AERGVLYR redshifts from 1545.81 cm -1 to 1559.82 cm -1 , and the absorption peak of AERGVLYR-Ca redshifts from 1552.29 cm -1 to 1553.25 cm -1 . This may be due to the introduction of COS into AERGVLYR and AERGVLYR-Ca, causing the C=O stretching vibration. After chelating with calcium ions, AERGVLYR changes from 1438.73 cm -1 , 1203.63 cm -1 , 1139.44 cm -1Red-shifted to 1444.66 cm respectively -1 、1205.84 cm -1 、1141.66 cm -1 , COS-AERGVLYR shifted from 1407.58 cm -1 、1204.89 cm -1 、1062.57 cm -1 Red-shifted to 1441.58 cm respectively -1 、1205.29 cm -1 、1141.44 cm -1 , which may be caused by the N-H bending vibration and C-N stretching vibration after the peptide segment binds to calcium ions. These results indicate that calcium ions may bind to the peptide through the carboxyl oxygen atom and amino nitrogen atom of the peptide.

[0074] Ultraviolet-visible absorption spectrum

[0075] Chelates formed by metal ions and organic ligands may cause the appearance of shifted, disappeared or new absorption peaks in the ultraviolet-visible absorption spectrum. As Figure 7 shown, AERGVLYR, AERGVLYR-Ca, COS-AERGVLYR, COS-AERGVLYR-Ca show multiple absorption peaks in the range of 200 - 275 nm, among which significant absorption peaks appear at 230 nm, 248 nm, 260 nm, and 274 nm, which are attributed to the electronic transitions of carbonyl, carboxyl and amide groups. The absorption intensities of glycosylated COS-AERGVLYR-Ca are higher than those of AERGVLYR and AERGVLYR-Ca respectively. From the perspective of calcium chelation, the absorption intensities of COS-AERGVLYR and AERGVLYR show different degrees of hypochromic effects after complexing with calcium ions respectively. The carbonyl or amino group in the amide bond forms a coordination complex with calcium ions. These spectral changes indicate that COS-AERGVLYR forms a COS-AERGVLY-Ca complex by binding to calcium ions. This phenomenon may be due to the changes in the spatial structure and electronic transitions of the chelate caused by the binding reaction with calcium ions. The results of ultraviolet spectrophotometry show that glycosylation modification and calcium ions have an impact on the structure of AERGVLYR, and finally COS-AERGVLYR-Ca is formed.

[0076] Fluorescence spectrum

[0077] Fluorescence spectroscopy can be used to study the interaction between proteins and small molecules. Aromatic amino acids such as phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr) can act as chromophores to produce endogenous fluorescence at specific excitation wavelengths. Therefore, changes in the molecular structure of peptides can be inferred from the changes in these fluorescent amino acid residues. As Figure 8 shown, COS-AERGVLYR and AERGVLYR contain tyrosine, and the fluorescence intensity decreases with the increase in calcium ion concentration. The decrease in intensity may be due to the fluorescence quenching of peptides containing aromatic amino acids after binding to calcium ions. This may be caused by the reduced exposure of fluorescent amino acid residues in the solvent due to the folding of the peptide structure caused by chelation, which changes the structure of AERGVLYR and COS-AERGVLYR. This may be the reason for the decrease in fluorescence intensity. In addition, compared with AERGVLYR, the fluorescence intensity of COS-AERGVLYR is relatively low, which may be due to glycosylation further reducing the exposure of tyrosine and decreasing the fluorescence intensity. The experimental results show that the COS-AERGVLYR-Ca complex formed by the reaction of CaCl2 with COS-AERGVLYR changes the structure of the functional binding site of COS-AERGVLYR, and the coordination bond is the coordination bond connecting calcium ions and the OSPH binding site.

[0078] XRD X-ray diffraction

[0079] As Figure 9 shown, at 31.72°, 45.44°, and 56.42°, several sharp and narrow diffraction peaks appear in COS-AREGVLYR-Ca. At 31.66°, 45.46°, and 56.38°, several sharp and narrow diffraction peaks also appear in AREGVLYR-Ca. No obvious diffraction peaks appear in COS-AREGVLYR and AREGVLYR. This result indicates that the presence of calcium increases the crystallinity of COS-AREGVLYR and AREGVLYR.

[0080] Scanning electron microscopy

[0081] The microstructures of AERGVLYR, COS-AERGVLYR, AERGVLYR-Ca, and COS-AERGVLYR-Ca are as Figure 10As shown. The AERGVLYR is evenly distributed and has a loose strip structure. The surface of COS-AERGVLYR is smooth and shows a flaky structure. The surface of AERGVLYR-Ca is rough and has relatively large voids in its internal structure, while COS-AERGVLYR-Ca presents a smooth block. Research shows that the addition of calcium ions will interfere with or damage the protein structure. This change in microstructure may be due to the fact that calcium ions bind to the peptide through coordination bonds, thus partially destroying the original structure of COS-AERGVLYR. SEM images show that during the modification and chelation processes, due to the interaction between the peptide, chitosan oligosaccharide, and calcium ions, the initial structure of the peptide has changed.

[0082] Example 4 Stability Analysis of COS-AERGVLYR-Ca

[0083] TG-DS detection method: The thermal properties of AERGVLYR, AERGVLYR-Ca, COS-AERGVLYR, and COS-AERGVLYR-Ca copolymer were tested using a TG-DSC simultaneous thermal analyzer. During the test, we weighed each sample powder (5 mg) and placed them in an aluminum pan. Then the samples were heated from room temperature to 900 °C at a rate of 10 °C per minute, and simultaneously purged with liquid nitrogen (50 mL / min) to maintain a stable experimental environment.

[0084] Thermal stability detection method: AERGVLYR-Ca and COS-AERGVLYR-Ca were dissolved in deionized water to a final concentration of 1 mg / mL, and were kept in a water bath (20 °C, 40 °C, 60 °C, 80 °C, 100 °C) for 2 h, centrifuged at 4000 rpm for 10 min, and the calcium release amount in the supernatant was measured.

[0085] Gastrointestinal stability detection method: Prepared 1 mg / mL pepsin solution and 1 mg / mL trypsin solution were used to simulate gastric juice and intestinal juice. First, the sample was dissolved in deionized water (concentration of 1 mg / mL), the pH value of the solution was adjusted to 2.0, then 1 mg / mL pepsin solution was added, and magnetic stirring was carried out at 37 °C for 2 h with an enzyme / chelator mass ratio of 1:50, and the enzyme was inactivated in boiling water for 5 min. Subsequently, the pH value of the solution was adjusted to 7.00, 1 mg / mL trypsin solution was added to make the enzyme / sample mass ratio 1:25, and the reaction was carried out at 37 °C for 2 h and then the enzyme was inactivated in boiling water for 5 min again. During the whole process, the digestive fluid was taken every 30 min, centrifuged at 8000 g for 10 min, and the calcium content was measured.

[0086] Phosphoric acid crystallization inhibition detection method: Add 10 mg of glycosylated walnut peptide, walnut peptide, and distilled water into 2 mL of 0.8 mol / L CaCl2 respectively, and react in a 37°C water bath for 5 min. Then, transfer the mixture into 100 mL of NaH2PO4 (0.008 mol / L), and immediately adjust the pH to 7.4. Use a pH meter to monitor the pH value of the system in real time, record it every 2 min until it stabilizes.

[0087] TG-DSC: In this example, thermogravimetry-differential scanning calorimetry (TG-DSC) was used to analyze the thermal stability of AERGVLYR, COS-AERGVLYR, AERGVLYR-Ca, and COS-AERGVLYR-Ca. During the thermal decomposition of compounds, changes in heat and mass occur. Thermogravimetry-differential scanning calorimetry (TG-DSC) can be used to predict changes in physical and chemical properties such as evaporation, sublimation, and redox of substances. The endothermic peak is mainly caused by the initial evaporation of free water and bound water absorbed by the sample, followed by pyrolysis, including the destruction of amide bonds at different positions (such as deamination, decarboxylation, and depolymerization), and finally thermal decomposition. The first stage is mainly the loss of free water and bound water in the sample, resulting in mass loss. The second stage is the thermal degradation of the sample, and the third stage of degradation is mainly the carbonization of organic matter. Therefore, AERGVLYR, COS-AERGVLYR, AERGVLYR-Ca, and COS-AERGVLYR-Ca all showed mass loss in this stage. As Figure 11As shown in the figure, the thermal decomposition process of AERGVLYR can be divided into three stages. Two endothermic peaks appear at 244 °C and 283.3 °C, and obvious weight loss occurs on the TG curve, with a weight loss rate of 87.7644%. This may be due to the breaking of the C-N bond in AERGVLYR at high temperature. After glycosylation modification, the thermal stability of COS-AERGVLYR is slightly improved, with a weight loss rate of 86.4593%, and endothermic peaks appear at 250.1 °C and 281.7 °C. Compared with AERGVLYR, the thermal stability of AERGVLYR-Ca is significantly improved. The thermal decomposition can be divided into four stages. An obvious endothermic peak appears at 129.3 °C, while there is no obvious mass loss on the corresponding TG curve. This is because the adsorbed water in AERGVLYR-Ca volatilizes during heating. There are three weak endothermic peaks at 379.9 °C, 472.9 °C, and 598.2 °C, with a weight loss rate of 61.1289%. The endothermic peaks and the accompanying mass loss indicate that the N-Ca and COO-Ca ionic bonds break during heating. These differences show that AERGVLYR-Ca has stronger thermal stability than AERGVLYR. COS-AERGVLYR-Ca has endothermic peaks at 131.3 °C, 367.3 °C, 408.1 °C, 468.6 °C, and 600.8 °C, with a weight loss rate of 55.7884%. Generally speaking, after glycosylation modification and calcium chelation, the structure of the complex changes, requiring stronger energy to break chemical bonds, having a more stable structure and stronger stability.

[0088] Thermal stability: Thermal processing plays an important role in food industrial production. Therefore, thermal stability is crucial for the calcium delivery system. As Figure 12 , the calcium retention rates of COS-AERGVLYR-Ca and AERGVLYR-Ca were studied at different temperatures (20, 40, 60, 80, and 100 °C), and it was found that as the temperature increased, the calcium retention rate gradually decreased, both remaining above 80%. After heating at 100 °C for 30 min, the calcium retention rate of COS-AERGVLYR-Ca was 89.83%, and the calcium retention rate of AERGVLYR-Ca was 88.06%. This shows that the Ca 2+ delivery system has good stability. The research results show that COS may have a protective effect on AERGVLYR-Ca at high temperatures.

[0089] Gastrointestinal stability: The nutrients in food can be effectively absorbed only after being digested, absorbed, and utilized through the gastrointestinal tract. To study the digestion process of the Ca 2+ delivery system, a simulated human digestion experiment was carried out. Figure 13Shows the digestive process of the AERGVLYR-Ca and COS-AERGVLYR-Ca delivery systems. During the simulated gastrointestinal digestion process, the final calcium release rate of AERGVLYR-Ca reached 68.63%, and the calcium release rate of COS-AERGVLYR-Ca reached 61.71%, indicating that COS-AERGVLYR-Ca exhibited a better sustained-release effect. Therefore, COS-AERGVLYR-Ca can help calcium enter the intestine in the form of soluble chelates, thereby improving the bioavailability of calcium. The AERGVLYR of AERGVLYR-Ca is directly exposed to the gastrointestinal environment, making AERGVLYR more easily degraded by pepsin, resulting in rapid calcium release. In contrast, COS forms a protective layer for AERGVLYR-Ca in the COS-AERGVLYR-Ca delivery system, thus preventing the destruction of enzymes in the gastrointestinal environment, enabling calcium to be released slowly. The change in pH during gastrointestinal digestion affects the release of calcium ions, while pepsin has little effect on the stability of calcium ions. In addition, the reduced solubility of minerals under neutral intestinal conditions is a major cause of low bioavailability. However, mineral ions chelated with peptides before digestion can be protected and are beneficial to their solubility, thus promoting the absorption of mineral ions. Therefore, COS-AERGVLYR-Ca can effectively promote calcium absorption due to its high stability and calcium retention rate in the form of chelates after digestion. The experimental results show that COS-AERGVLYR-Ca can maintain stability under the action of digestive enzymes and the acid-base environment of the human gastrointestinal tract, preventing the dissociation of calcium ions in the intestine and further improving the bioavailability of COS-AERGVLYR-Ca.

[0090] Phosphate crystallization inhibition ability: The inhibitory effect on calcium phosphate crystallization can be estimated as the decrease in the pH value of the system. During the reaction, H atoms are ionized by H2PO -4 / HPO2 -4 resulting in a decrease in pH value. As crystallization nucleation and aggregation become the main trend, the pH value drops rapidly, which is defined as the crystallization stage. As Figure 14As shown in the figure, the pH value of the control group decreased significantly. After adding COS-AERGVLYR, AERGVLYR, and COS respectively, the downward trend of the system pH value slowed down to varying degrees, indicating that the crystallization of calcium phosphate was inhibited, and AERGVLYR, COS-AERGVLYR, and COS could effectively bind calcium ions. The pH of the control group stabilized at 10 min, and the pH stabilized between 6.6 - 6.7. The pH of AERGVLYR stabilized at 6.71 after 15 min, the pH of COS-AERGVLYR stabilized between 6.7 - 6.8 after 20 min, and the pH of COS stabilized between 6.8 - 6.9 at 20 min. The pH decrease process of COS was slower, and the degree of pH change was smaller. This may be because the molecular structure of COS monomers contains amino / acetamido, secondary hydroxyl, and primary hydroxyl groups at C-2, C-3, and C-6 respectively. While strengthening the hydrogen bond interaction between COS monomers, it also enables COS monomers to have the ability to chelate metal ions, thus enabling COS to form a competitive effect with phosphate ions in the solution. COS binds a part of calcium ions, and the dissociation of H2PO -4 / HPO2 -4 is inhibited, and the range of pH decrease is reduced. Compared with AERGVLYR and the control group, the pH decrease process of COS-AERGVLYR is slower. This may be because the polypeptide has the ability to chelate calcium ions, and glycosylated AERGVLYR further improves the calcium ion chelating ability. In summary, COS-AERGVLYR has a good inhibitory effect on the formation of calcium phosphate precipitation. This excellent calcium-holding ability enables COS-AERGVLYR to play an important role in the absorption and utilization of calcium ions in the intestine.

[0091] Example 5 Caco-2 cell calcium absorption study

[0092] The MTT cell toxicity test method is as follows: The MTT assay was used to calculate the toxicity of the drug to Caco-2 cells. It was divided into 4 groups: ① glycosylated walnut peptide; ② glycosylated walnut peptide + 2 mM CaCl2; ③ walnut peptide; ④ walnut peptide + 2 mM CaCl2.

[0093] The cell viability was calculated according to the following formula:

[0094]

[0095] where As: absorbance value of the experimental group; Ac: absorbance value of the control group; Ab: absorbance value of the blank group.

[0096] Such as Figure 15As shown in the figure, the MTT method was used to determine the cytotoxic effects of samples at concentrations of 0, 50, 100, 200, 400, 800, and 1000 mg / mL on Caco-2 cells. The results showed that AERGVLYR had no cytotoxic effect on Caco-2 cells at 50 μg / mL, and the cell viability decreased significantly at 100, 200, 400, and 800 mg / mL. The cell viabilities were 91.22%, 78.9%, 82.27%, 72.31%, and 72.31% respectively (P<0.05). AERGVLYR-Ca had no cytotoxic effect on Caco-2 cells at 50 and 100 μg / mL. The cell viability decreased to 96.53% and 93.64% at 200 and 400 μg / mL respectively, but there was no significant difference. The cell viability decreased significantly at 800 and 1000 μg / mL, being 81.89% and 83.44% respectively ( P <0.05). COS-AERGVLYR had no cytotoxic effect on Caco-2 cells at 50, 100, 200, and 400 μg / mL. The cell viability reached the maximum value of 112.85% at 200 μg / mL, and cytotoxic effects occurred at 800 and 1000 μg / mL. The cell viabilities decreased significantly to 88.93% and 89.19% respectively ( P <0.05). COS-AERGVLYR-Ca had no cytotoxic effect on Caco-2 cells at 50, 100, 200, and 400 μg / mL. At 800 and 1000 μg / mL, the cell viabilities decreased to 80.58% and 83.98% respectively ( P <0.05). Therefore, a concentration of 400 μg / mL was selected for subsequent experiments.

[0097] Establishment of Caco-2 cell monolayer model

[0098] The establishment of the Caco-2 cell monolayer model played an important role in the study of the absorption and utilization rate of calcium. The transepithelial electrical resistance ( Transepithelial electrical resistance, TEER ) can reflect the permeability and integrity of the cell monolayer. Caco-2 cells were inoculated on TransWell culture plates, and the transepithelial electrical resistance value of the cell monolayer was measured every 2 days. From the TEER value results in Figure 16 , it can be seen that the TEER value of Caco-2 cells increased slowly within the first 6 days of modeling. This may be because Caco-2 cells need some time to adhere, proliferate, and divide after being inoculated on TransWell TM culture plates and have not reached confluence. The TEER value of Caco-2 cells began to increase rapidly from the 8th day and reached 482 Ω·cm -2 on the 16th day, and then tended to a relatively stable state, reaching 508 Ω·cm on the 21st day.-2 According to the literature report, when the TEER value is higher than 300 Ω·cm -2 it indicates that Caco-2 cells have formed a dense monolayer membrane structure, which can be used for subsequent transport experiments. From the above research results, it can be concluded that Caco-2 cells have reached a high degree of polar differentiation and successfully established a cell monolayer model, whose structure is similar to the human intestinal epithelial absorption system, indicating that this cell model can be used as an effective tool for studying intestinal absorption.

[0099] Study on Calcium Transport in Caco-2 Cells

[0100] Based on the cytotoxicity experiment, appropriate drug concentrations were selected to explore the effect on calcium transport. The concentration of CaCl2 used was 5 mM / L, and the peptide concentration was determined by MTT cytotoxicity test. After incubation for 21 days, the culture medium was discarded, and the monolayer cells were immediately washed twice with Hanks balanced salt solution (HBSS, without calcium and magnesium). 0.5 mL was added to the AP side and 1.5 mL HBSS was added to the BL side. After incubation in the incubator for 20 min, the HBSS was aspirated. 0.5 mL of drugs in different groups was added to the AP side, and 1.5 mL HBSS was added to the BL side. At different time points (30, 60, 120, 180 min), 1 mL HBSS was collected from the basolateral side to measure the calcium content, and at the same time, 1 mL of fresh HBSS buffer was added to the basolateral side to maintain a constant volume.

[0101] The effect of COS-AERGVLYR on calcium transport in Caco-2 cell monolayers is shown in Figure 17 Compared with the CaCl2 group, the AERGVLYR+CaCl2 treatment group and the COS-AERGVLYR+CaCl2 treatment group showed significant calcium transport activity at the 30-180 min time points ( P <0.05). Compared with the AERGVLYR treatment group, after the action of COS-AERGGVLYR for 30 min, the intracellular calcium transport amount increased significantly, with an increase amplitude of 27%. The calcium transport amounts at 30, 60, 120, and 180 min were 45.13±0.12, 58.04±1.89, 64.77±0.74, and 66.62 ± 1.10 μg / mL, respectively, which were 1.63, 1.44, 1.77, and 1.10 times that of the control group, and 1.27, 1.05, 1.01, and 1.03 times that of AERGVLYR. Calcium absorption increased in a time-dependent manner. This result indicates that COS-AERGVLYR can promote calcium transport in Caco-2 cell monolayers.

[0102] Cav1.3 Calcium Ion Channel

[0103] AsFigure 18 , Caco-2 cell monolayers were treated with different concentrations of nimodipine (0, 10, 15, 20 μM). Compared with the blank group, after adding the inhibitor nimodipine (Nimodipine), calcium transport in both the AERGVLYR+CaCl2 and COS-AERGVLYR+CaCl2 groups was inhibited. After introducing 10, 15, and 20 μM nimodipine, calcium transport in AERGVLYR+CaCl2 decreased significantly from 49.26 μg to 22.12, 14.2, and 15.9 μg respectively ( p <0.05). In the COS-AERGVLYR+CaCl2 group, the calcium transport in CaCo-2 cell monolayers treated with 10 μM nimodipine decreased significantly from 52.14 μg to 28 μg, and the calcium transport in cell monolayers treated with 15 and 20 μM nimodipine decreased significantly, reaching 12.70 and 11.80 μg respectively ( p <0.05). The above experimental results indicate that the calcium absorption promoted by COS-AERGVLYR-Ca may be achieved through the L-type calcium channel Cav1.3.

[0104] TRPV6 calcium channel

[0105] After 2-APB interacts with the lipid-protein of TRPV6, the hydrophobic residues in TRPV6 tightly bind to form hydrophobic clusters. During this process, there are accompanied by the elimination of hydrogen bond forces and salt bridges between the bond positions in TRPV6 and the rearrangement of the structure, thus promoting the closure of the channel. Therefore, cell monolayers were treated with 6.25, 12.5, and 25 μM 2-APB respectively, and the experimental results are as Figure 19 shown, and the calcium transport amount decreased significantly with the increase in the inhibitor concentration ( P <0.05). Compared with the calcium transport amount in the cell monolayers without drug addition, the calcium transport amounts in the cell monolayers treated with 6.25, 12.5, and 25 μM 2-APB decreased significantly. For COS-AERGVLYR +CaCl2, they reached 17.19, 13.38, and 7.63 μg respectively, and for AERGVLYR+ CaCl2, they reached 19.6, 9.45, and 7 μg respectively ( P <0.05). The above results indicate that COS-AERGVLYR may promote calcium transport through the TRPV6 calcium channel.

[0106] Calcium transport inhibited by phytic acid (PA)

[0107] Six groups were set up in this experiment: ① CaCl2; ② PA + CaCl2; ③ Glycosylated walnut peptide + CaCl2; ④ Glycosylated walnut peptide + CaCl2 + PA; ⑤ Walnut peptide + CaCl2; ⑥ Walnut peptide + CaCl2 + PA. Among them, the concentrations of glycosylated walnut peptide and walnut peptide were determined by MTT toxicity test, and the concentrations of both CaCl2 and phytic acid were 5 mmol / L. 1.5 mL was collected from the BL side every 30 min within 2 h after adding the drugs, and 1.5 mL of HBSS was supplemented to maintain a constant volume. The calcium transport amount was detected using a calcium content kit.

[0108] During gastrointestinal digestion, various factors may reduce the bioavailability of calcium. For example, calcium ions may form insoluble precipitates with phytic acid or oxalic acid in chelates, or generate Ca(OH)2, thus affecting the absorption and utilization of calcium. As Figure 20 shown, COS-AERGGVLYR can improve calcium transport in the Caco-2 cell monolayer model. Calcium passes through the intestinal wall in a soluble form. After introducing PA (phytic acid) into the CaCl2 treatment group, AERGVLYR treatment group, and COS-AERGVLYR treatment group, calcium transport was significantly inhibited ( P <0.05). It is worth noting that in the calcium transport system with the introduction of PA, the calcium transport amount in the AERGVLYR group increased by 1.10 times compared with the CaCl2 group, but there was no significant difference. The COS-AERGVLYR group increased by 1.15 times and 1.04 times compared with CaCl2 and AERGVLYR respectively ( P <0.05). The results indicate that the presence of COS-AERGVLYR can resist the inhibitory effects of nutritional factors.

[0109] Calcium imaging

[0110] Caco-2 cells were seeded in 3-cm culture dishes, and drug culture media of different groups were prepared using 1640 medium. Among them, CaCl2 was 5 mM / L, and the concentrations of glycosylated walnut peptide and walnut peptide were determined by MTT toxicity test. After culturing the cells for 24 h, the culture medium was aspirated and the cells were rinsed 3 times with HBSS. Then, a working solution of Fluo-4 AM diluted to 4 μM with HBSS was added, 200 μL for each dish, and incubated at 37 °C in the dark for 30 min; the cells were washed 2 - 3 times with HBSS and incubated for another 10 min in the dark, and the fluorescence images of the cells were observed using an inverted fluorescence microscope.

[0111] Figure 21The calcium uptake effects of AERGVLYR+CaCl2, COS-AERGVLYR+CaCl2, and CaCl2 were shown in the Caco-2 cell monolayer model. Compared with the CaCl2 group, after introducing AERGVLYR in calcium transport, the fluorescence intensity increased, and the fluorescence intensity after adding COS-AERGVLYR was higher than that of the AERGVLYR group. This result indicated that the addition of COS-AERGVLYR+CaCl2 significantly improved calcium uptake in the Caco-2 cell monolayer model. In summary, COS-AERGVLYR effectively promoted calcium uptake in Caco-2 cells.

[0112] Q-PCR analysis

[0113] (1)Total RNA extraction

[0114] Caco-2 cells were seeded in 6-cm culture dishes. After culturing with drugs of different groups for 24 h, the total RNA of Caco-2 cells was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit, and the operation was carried out on an ice plate throughout the extraction process. The extracted RNA was detected for its concentration and purity using Nanodrop 2000. The extracted RNA was stored in a -80 °C refrigerator.

[0115] (2)Reverse transcription

[0116] Adjust the RNA concentration of the sample to 500 ng, and configure the reverse transcription system according to the existing method.

[0117] Carry out reverse transcription according to the program parameters set in Table 4.

[0118] (3)PCR quantification

[0119] Synthesize primers, dilute the primers to 0.4 μM, dilute the DNA template to 100 ng, configure the PCR reaction system according to Table 6, pipette 25 μL of each sample into a 96-well PCR plate, and then use a 300-μL multichannel pipette to dispense 10 μL of each sample into a 384-well plate for PCR reaction.

[0120] Previously, the glycosylated walnut peptide COS-AERGVLYR and Ca were explored using a calcium channel inhibitor 2+Calcium transport pathways when acting on Caco-2 monolayers together, and the results show that TRPV6 calcium channels and Cav1.3 calcium channels may be involved in calcium transport in Caco-2 monolayers. To further verify this conclusion, Q-PCR was used to verify the expression of genes related to TRPV6 calcium channels (TRPV6, Calbindin D9k, PMCA1b) and the expression of Cav1.3 gene in Caco-2 cells under the action of different drugs. Figure 22 A shows the expression of TRPV6 gene in Caco-2 cells. Compared with the control group, the gene expression level of TRPV6 increased in each treatment group. Among them, the expression level of COS-AERGVLYR+CaCl2 was the highest, increasing significantly by 44%, followed by AERGVLYR+CaCl2, increasing by 42%. Figure 22 B and Figure 22 C respectively show the expression of Calbindin D9k and PMCA1b genes in Caco-2 cells. Compared with the control group, COS-AERGVLYR+CaCl2 significantly increased the expression of Calbindin D9k and PMCA1b genes, increasing by 52.14% and 37% respectively. Compared with AERGVLYR+CaCl2, the expression levels of TRPV6 and PMCA1b genes of COS-AERGVLYR+CaCl2 were slightly higher, but there was no significant difference ( P <0.05). The above research results show that COS-AERGVLYR promotes the expression of TRPV6, Calbindin D9k, and PMCA1b in Caco-2 cells. Figure 22 D shows the expression of Cav1.3 gene in Caco-2 cells. Compared with the control group, CaCl2, AERGVLYR, COS-AERGVLYR, AERGVLYR+CaCl2, and COS-AERGVLYR+CaCl2 significantly increased the expression of Cav1.3 in Caco-2 cells ( p <0.05), which were 1.17, 1.16, 1.22, 1.34, and 1.37 times that of the control group respectively. In summary, COS-AERGVLYR regulates calcium transport in the Caco-2 cell monolayer model by promoting the expression of TRPV6, Calbindin D9k, PMCA1b, and Cav1.3 genes.

[0121] Investigation of osteogenic effect

[0122] CCK8 cell proliferation assay

[0123] MC3T3-E1 cells were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37 °C and 5% CO2. MC3T3-E1 cells in the logarithmic growth phase were seeded in a 96-well plate at a concentration of 8×10 3 cells / well, 100 µL per well, and PBS was added to the edge wells. After culturing for 24 h, the old medium was discarded, and complete culture medium with different concentrations of the sample (0, 50, 100, 200, 400 μg / mL), where CaCl2 was 5 mM / L, was added and cultured for 24 h. There were 6 replicates for each concentration. 10 μL of CCK8 solution was added to each well. After culturing in an incubator at 37 °C and 5% CO2 for 1 h, the absorbance value of each well was measured at 450 nm using a microplate reader.

[0124]

[0125] The proliferation activity of MC3T3-E1 cells was as Figure 23 shown. The proliferation rate of MC3T3-E1 cells with AERGVLYR showed concentration dependence and reached 123.63±1.14% at 400 μg / mL. The proliferation rate of AERGVLYR+CaCl2 on MC3T3-E1 showed a trend of first increasing and then decreasing, and reached the maximum value of 125.14±3.52% at 100 μg / mL. Similarly, the proliferation rate of MC3T3-E1 cells with COS-AERGVLYR also showed a trend of first increasing and then decreasing, and reached the highest proliferation rate of 124.25±6.55% at 200 μg / mL. It is worth noting that the proliferation ability of MC3T3-E1 cells with COS-AERGVLYR+CaCl2 showed concentration dependence, and the proliferation rate reached the highest at 400 μg / mL, which was 140.84±6.56%, significantly higher than that of AERGVLYR+CaCl2 and COS-AERGVLYR groups at the same concentration ( P <0.05), indicating that glycosylation modification can promote the proliferation of peptide calcium chelate on MC3T3-E1 cells to a certain extent.

[0126] Alkaline phosphatase (ALP) assay

[0127] MC3T3-E1 cells in the logarithmic growth phase were seeded at 5×10 4Cells were inoculated into a 24-well plate at a concentration of cells per well. After culturing for 24 h, the medium was changed to an induction differentiation medium containing samples of different groups to culture MC3T3-E1 cells. Three replicate wells were set for each group. Among them, the concentration of CaCl2 was 5 mM / L, and the concentrations of glycosylated walnut peptides and walnut peptides were determined by the CCK8 cell proliferation experiment. The induction differentiation culture medium was α-MEM medium containing 10 mM sodium glycerophosphate, 50 μg / mL ascorbic acid, 10% fetal bovine serum, and 1% penicillin-streptomycin. The medium was changed every 3-4 days for 7 days of induction differentiation. After the culture was completed, the supernatant was collected and placed in a centrifuge tube, centrifuged at 1500 rpm / min for 10 min, and the supernatant was stored at -20°C for later use. Samples were added to the enzyme-linked immunosorbent assay (ELISA) plate according to the kit instructions, and the absorbance values of each well were measured at a wavelength of 520 nm using an enzyme-linked immunosorbent assay reader.

[0128]

[0129] Among them, the concentration of the phenol standard was 0.02 mg / mL.

[0130] ALP is an exogenous enzyme of osteoblasts, and its expression activity is a significant feature of osteoblast differentiation. As Figure 24 shown, the ALP activities of MC3T3-E1 cells treated with COS-AERGVLYR+CaCl2 and AERGVLYR+CaCl2 were significantly higher than those of the control group ( P <0.05). Among them, the ALP activity of COS-AERGVLYR+CaCl2 was the highest. This may be because COS-AERGVLYR+CaCl2 contains more hydroxyl groups after glycosylation and can interact better with the surface receptors of MC3T3-E1 cells (the "external-internal signal" on MC3T3-E1 cells). More hydroxyl groups in COS-AERGVLYR+CaCl2 enhanced cell interaction and triggered signal transduction, thus stimulating cell differentiation through a receptor-mediated mechanism.

[0131] Mineralization staining of MC3T3-E1 cells

[0132] MC3T3-E1 cells were seeded at 5×10 4Cells were seeded at a density of [cells / per well] into 24-well plates. After culturing for 24 h, the old culture medium was discarded and replaced with induction differentiation culture media of different groups, where CaCl2 was 5 mM / L, and the concentrations of glycosylated walnut peptides and walnut peptides were determined by the CCK8 cell proliferation assay. They were cultured for 14 d and 21 d respectively. The induction differentiation culture medium was changed every 3 - 4 d in the first week, and then half of the culture medium was changed every 2 d. After the culture was completed, an alizarin red staining experiment was carried out: the old culture medium was discarded, and the cells were gently washed twice with pre-cooled PBS; fixed with 4% paraformaldehyde for 30 min, the fixing solution was discarded, and the cells were washed three times with ultrapure water; alizarin red staining solution was added to each well, and stained at room temperature for 5 min. The staining could be ended when obvious orange-red calcium nodule aggregations were observed under the microscope. If the effect was not obvious enough, the staining time could be appropriately extended; the dye was aspirated, and the cells were washed three times with ultrapure water. The calcium nodules were observed under a low-power microscope field (×10), and representative photos were taken. Subsequently, 2% cetylpyridinium chloride was used to semi-quantify the calcium nodules induced for 21 d, and the absorbance value measured at 562 nm by an enzyme-linked immunosorbent assay was used to quantify the mineralization degree of MC3T3-E1 cells.

[0133] Mineralization is the final stage of osteoblast differentiation and can directly indicate the degree of bone formation. After the calcium ions in the mineralized nodules bind to alizarin red S, they show a dark red color, and its absorbance is related to the amount of calcium deposition. As Figure 25 shown, compared with the Control group, the mineralization degree of all treatment groups increased. At 14 days, the mineralization degree of MC3T3-E1 cells was not very obvious. At 21 days, compared with the control group and AERGVLYR, COS-AERGVLYR increased the mineralization activity of MC3T3-E1 cells. Among the groups with calcium ions introduced, the calcium nodules produced by MC3T3-E1 mineralization were more than those in the groups without calcium ions introduced. Among them, the content of calcium nodules produced by COS-AERGVLYR+CaCl2 was the highest, which was 1.07±0.01 times, 1.11±0.02 times, 1.5±0.01 times, 1.76±0.01 times, and 2.57±0.06 times that of the AERGVLYR+CaCl2, CaCl2, COS-AERGVLYR, AERGVLYR, and Control groups respectively ( P <0.05). These results indicate that the synergistic effect of COS-AERGVLYR and CaCl2 can improve the mineralization ability of MC3T3-E1 cells.

Claims

1. A peptide-calcium chelate, characterized in that, The structural formula of the peptide-calcium chelate is as follows: AERGVLYR-Ca.

2. A chitosan oligosaccharide peptide calcium chelate, characterized in that, The polypeptide is glycosylated with chitosan oligosaccharide COS and then chelated with calcium ions to prepare a chitosan oligosaccharide peptide-calcium chelate. The structural formula of the chitosan oligosaccharide peptide-calcium chelate is as follows: COS-AERGVLYR-Ca.

3. The preparation method of the peptide calcium chelate according to claim 1, characterized in that, Dissolve the polypeptide AERGVLYR in deionized water at a concentration of 10 - 20 mg / mL, add CaCl2 so that the mass ratio of polypeptide AERGVLYR to CaCl2 is 1:3 - 6, and adjust the pH to 6.5 - 7.

5. React in a water bath at 50 - 65 °C for chelation for 40 - 80 min. After the chelation reaction is completed, add anhydrous ethanol with a volume 8 - 10 times that of the reaction solution to the reaction system. Finally, centrifuge the mixture at 2 - 5 °C at 6000 - 11000×g for 8 - 12 min, collect the precipitate, and freeze-dry to obtain the peptide-calcium chelate AERGVLYR-Ca.

4. The preparation method of the chitosan oligosaccharide peptide calcium chelate according to claim 2, characterized in that, Mix the polypeptide AERGVLYR and chitosan oligosaccharide COS in water, adjust the pH of the reaction system to 6.5 - 7.5, react in a water bath at 85 - 95 °C for 2 - 5 h, quickly cool to room temperature to end the reaction, and then place it at 2 - 5 °C overnight to allow it to fully hydrate. Dialyze with a dialysis bag of 500 - 1000 Da at 18 - 25 °C for 20 - 30 h, centrifuge at 2500 - 3500 r / min for 8 - 15 min, take the supernatant and freeze-dry to obtain the freeze-dried glycosylated peptide COS-AERGVLYR. Chelate the freeze-dried glycosylated peptide COS-AERGVLYR with calcium chloride to obtain the chitosan oligosaccharide peptide-calcium chelate. The mass ratio of the polypeptide AERGVLYR to chitosan oligosaccharide COS is 0.8 - 1.0:1.0 - 1.2, and their total concentration in water is 40 - 60 mg / mL.

5. The preparation method of the chitosan oligosaccharide peptide calcium chelate according to claim 4, wherein, Dissolve the freeze-dried glycosylated peptide COS-AERGVLYR in deionized water at a concentration of 10 - 20 mg / mL, add CaCl2 so that the mass ratio of glycosylated peptide COS-AERGVLYR to CaCl2 is 1:3 - 6, and adjust the pH to 6.5 - 7.

5. React in a water bath at 50 - 65 °C for chelation for 40 - 80 min. After the chelation reaction is completed, add anhydrous ethanol with a volume 8 - 10 times that of the reaction solution to the reaction system. Finally, centrifuge the mixture at 2 - 5 °C at 6000 - 11000×g for 8 - 12 min, collect the precipitate, and freeze-dry to obtain the chitosan oligosaccharide peptide-calcium chelate COS-AERGVLYR-Ca.

6. Use of the peptide-calcium chelate according to claim 1 or the chitosan oligosaccharide peptide-calcium chelate according to claim 2 in the preparation of a biological calcium supplement.

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