A chicken bone collagen polypeptide that promotes calcium absorption, its chelate, preparation method and application

Abstract

This invention belongs to the field of functional foods and bioactive peptides, specifically relating to a chicken bone collagen peptide that promotes calcium absorption, its chelate, preparation method, and application. This invention employs a rational molecular docking screening strategy, directly screening for the high-affinity peptide DFSLPQPPQ from a chicken bone collagen peptide library by simulating the interaction between peptides and calcium-sensitive receptors. This overcomes the inefficiency of traditional "blind screening" methods, achieving targeted peptide discovery based on mechanisms. The prepared peptide-calcium chelate (DFSFLPQPPQ-Ca) exhibits excellent calcium absorption-promoting performance in the Caco-2 cell model. This invention utilizes chicken bone by-products as raw materials, achieving high-value utilization of resources and providing a precise development path for poultry processing by-products based on clearly defined bioactive peptides, possessing significant industrial promotion value and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of functional foods and bioactive peptides, specifically relating to a chicken bone collagen polypeptide that promotes calcium absorption and its preparation method with calcium source chelate, as well as its application in improving calcium bioavailability. Background Technology

[0002] Calcium is a key mineral for maintaining healthy bones; however, traditional calcium supplements generally suffer from low absorption rates and are easily affected by dietary factors. Peptide-calcium chelates, which utilize collagen peptides and other ligands to bind with calcium ions, can effectively improve calcium stability and bioavailability, representing a promising solution.

[0003] Currently, technological development in this field mainly revolves around two directions: first, optimizing the enzymatic hydrolysis process and chelation conditions for raw materials such as livestock and poultry bones and fish bones; and second, isolating and identifying peptides with specific activities from enzymatic hydrolysis products.

[0004] Chinese patent CN115677849A discloses a method for preparing calcium chelates of animal bone collagen peptides, which improves product stability by directly chelating after enzymatic hydrolysis. However, the peptides used are complex mixtures with unclear core active components, which restricts further improvement of product efficacy.

[0005] Chinese patent CN116751828A employs physical pretreatment methods such as steam explosion to achieve green preparation, but its core technology still relies on the chelation of mixed peptides, and thus fails to solve the fundamental problems of product uniformity and unclear source of activity.

[0006] Chinese patent CN119306798A identified and verified the activity of a specific calcium absorption-promoting octapeptide (GRGNEGPQ) derived from cod bone. However, the discovery of this peptide relied on the traditional "enzymatic hydrolysis-separation-verification" process, which is a retrospective and inefficient screening method lacking a rational explanation of its mechanism of action. Furthermore, existing technologies largely focus on fish bone or mammalian bone raw materials, lacking targeted and in-depth development of chicken bone by-products, which have a huge annual output and an urgent need for high-value utilization.

[0007] In summary, existing technologies have two key limitations: First, the products obtained by mainstream methods are mixed peptides with unclear activity, limiting the controllability of product efficacy and quality; second, even when specific peptides are obtained, the screening process lacks rational guidance on the molecular mechanisms of calcium absorption (such as the interaction with the calcium-sensitive receptor CaSR), leading to blind and inefficient research and development. Meanwhile, efficient conversion technologies for chicken bone byproducts are still lacking.

[0008] Therefore, developing a method for preparing peptide-calcium chelates based on rational screening using molecular mechanisms, capable of obtaining structurally well-defined, highly active peptides, and applicable to the high-value utilization of chicken bone resources, is of great significance for overcoming existing technological bottlenecks, improving the quality of calcium preparations, and promoting industrial upgrading. Summary of the Invention

[0009] This invention aims to overcome the shortcomings of existing peptide-calcium chelates, which suffer from low calcium absorption efficiency due to unclear active peptide segments and blind screening methods, thus limiting the efficacy and quality control of calcium supplement products. It provides a chicken bone collagen polypeptide for promoting calcium absorption, its chelate, and a preparation method, and applies it to the preparation of calcium-containing functional foods.

[0010] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A chicken bone collagen polypeptide that promotes calcium absorption, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.3, or is a functional derivative of the sequence by substitution, deletion or addition of one or more amino acids and having the same calcium absorption-promoting activity.

[0011] This invention uses chicken bone collagen as its source and employs molecular docking simulation technology to pre-evaluate the theoretical binding affinity between various potential peptide sequences identified by mass spectrometry and calcium-sensitive receptors in a computer-based manner. This transforms the screening process from "blind testing" to "prediction." From a complex array of sequences, candidates structurally most likely to interact with the target (calcium-sensitive receptor) with high affinity are rapidly identified. Ultimately, a specific decapeptide sequence (i.e., the sequence shown in SEQ ID NO.3) is identified due to its predicted superior binding properties, significantly improving the accuracy and efficiency of research and development while substantially reducing its blind spots and time costs. Furthermore, the chicken bone calcium-absorbing collagen peptide of this invention has a well-defined sequence, and based on its reasonable prediction of interaction with calcium-sensitive receptors, the scientific basis for this peptide as a functional factor is more robust, fundamentally enhancing product consistency and quality controllability.

[0012] Preferably, the amino acid sequence of the polypeptide is DFSFLPQPPQ (Asp-Phe-Ser-Phe-Leu-Pro-Gln-Pro-Pro-Gln).

[0013] Preferably, the calcium chelating activity of the polypeptide is not less than 7.18 μg / mg.

[0014] The ability of collagen peptides in chicken bones to bind with calcium ions is called the calcium chelating activity of peptides.

[0015] A method for screening chicken bone calcium absorption-promoting collagen peptides as described above includes the following steps: S1: Obtain peptide sequence information of chicken bone collagen peptides derived from chicken bones; S2: Using calcium-sensitive receptors as targets, predict the binding affinity of each peptide sequence to the calcium-sensitive receptors through molecular docking simulation; S3: Based on the binding affinity, select candidate peptides that promote calcium absorption from the peptide sequence, based on the predicted binding affinity being higher than a preset value.

[0016] Preferably, the peptide sequence information in step S1 is obtained by liquid chromatography-tandem mass spectrometry.

[0017] As a preferred option, the screening process in step S3 also takes into account the relative content of the peptide fragments in chicken bone collagen peptides.

[0018] The preparation method of the chicken bone calcium absorption-promoting collagen polypeptide as described above includes the following steps: (a): Preparation of chicken bone collagen peptides; (b): The peptide sequence of the chicken bone collagen peptide was obtained by mass spectrometry detection and database retrieval; (c): Using molecular docking simulation technology, the (type I) chicken bone collagen peptides extracted from chicken bones were identified by liquid chromatography-mass spectrometry. Specific peptides with high affinity for calcium-sensitive receptors (CaSR) and high relative content in chicken bone collagen peptides were rationally predicted and screened to obtain the chicken bone calcium absorption-promoting collagen polypeptide sequence. (d) Chemically synthesized collagen peptides that promote calcium absorption in chicken bones.

[0019] Preferably, the method for preparing chicken bone collagen peptides in step (a) is based on the optimized enzymatic hydrolysis method in the inventor's prior patent application No. 202511929542, entitled: A high-calcium chelated active collagen peptide, peptide-calcium chelate and its application based on chicken bone.

[0020] Preferably, the peptide sequence in step (b) is obtained by the following steps: A Thermo UltiMate 3000 RSLCnano nano-liquid chromatography-tandem Q Exactive HF mass spectrometer was used. Raw mass spectrometry data were analyzed using MaxQuant (2.2.0.0) software. The database retrieval algorithm used was the software's built-in Andromeda algorithm, and the database used for retrieval was the Gallus gallus Chicken protein sequence database downloaded from Uniprot.

[0021] Preferably, the screening of high-affinity specific peptides in step (c) is performed as follows: For target preparation, the crystal structure of human CaSR was obtained from the protein database (PDB ID: 5FBH). Using specialized software (such as PyMOL v2.4.0), water molecules and existing ligands were removed, hydrogen was added, and the charge was calculated to prepare the receptor protein for docking. A peptide with the gene for type I collagen was selected as the protein ligand, and the three-dimensional structure of the peptide was generated using an RPBS online server. Molecular docking software (such as AutoDock Vina 1.5.7) was used to simulate docking of each peptide with the active pocket of CaSR, and the binding energy (affinity) was calculated. The optimal peptide was selected by combining two indicators: binding free energy (lower indicates stronger binding) and the relative content of the actual peptide identified by mass spectrometry. After screening, a decapeptide with the amino acid sequence DFSLPQPPQ (Asp-Phe-Ser-Phe-Leu-Pro-Gln-Pro-Pro-Gln, as shown in SEQ ID NO.3) was found, with a predicted binding energy of -9.8 g / L for CaSR. kcal / mol, and its content in the enzymatic hydrolysis products of chicken bones is relatively high (11.5%).

[0022] Preferably, the chemical synthesis method of the chicken bone calcium absorption-promoting collagen polypeptide (i.e., high-calcium chelating active polypeptide) in step (d) is carried out according to the following steps: The peptide (amino acid sequence DFSFLPQPPQ) was chemically synthesized using a solid-phase peptide synthesis method (contracted to GenScript).

[0023] This method is mature and controllable, ensuring the sequence accuracy and high purity (>95%) of the synthesized peptides, completely eliminating the reliance on the separation and purification of complex enzymatic hydrolysis products. The chicken bone collagen peptide fragment (amino acid sequence DFSLPQPPQ) exhibits high calcium chelating activity, reaching 7.18 ± 0.83 μg / mg, and can be used to prepare peptide-calcium chelates.

[0024] A peptide-calcium chelate comprising the chicken bone calcium-absorbing collagen polypeptide and calcium ions as described above.

[0025] Preferably, the calcium ions are derived from at least one of calcium citrate, calcium gluconate, calcium lactate, and calcium chloride.

[0026] The preparation method of the peptide-calcium chelate as described above includes the following steps: The chicken bone collagen peptide that promotes calcium absorption was chelated with a calcium source containing calcium ions under pH conditions of 5.0-7.0.

[0027] As a further preferred option, the pH of the system for carrying out the chelation reaction is 5.5.

[0028] Preferably, the reaction temperature for the chelation reaction is 25~60 ℃ and the reaction time is 20~60 min.

[0029] As a further preferred option, the reaction temperature for the chelation reaction is 40 °C and the reaction time is 30 min.

[0030] Preferably, the mass ratio of the chicken bone calcium absorption-promoting collagen polypeptide to the calcium source containing calcium ions is 1:1.

[0031] As a further preferred embodiment, a method for preparing a peptide-calcium chelate includes the following steps: (1) Dissolve chicken bone collagen peptides that promote calcium absorption in water to form a peptide solution; (2) Add a calcium source containing calcium ions to the polypeptide solution obtained in step (1), mix well, adjust the pH of the reaction system to 5.0~7.0, and react at 25~60 ℃ for 20~60 min to allow the chicken bone calcium absorption collagen polypeptide to undergo a chelation reaction with calcium ions until complete. Take the solution after the reaction for precipitation, separation and drying to obtain peptide calcium chelate (DFSFLPQPPQ-Ca).

[0032] As a further preferred option, in step (2), the solution after the reaction is precipitated with anhydrous ethanol, centrifuged at 8000 r / min for 15 min at 4 ℃, and the precipitate is freeze-dried under vacuum to obtain the peptide calcium chelate (DFSFLPQPPQ-Ca).

[0033] As a further preferred option, the mass concentration of the polypeptide solution in step (1) is 5 mg / mL.

[0034] As a further preferred option, the volume ratio of added anhydrous ethanol to the solution after reaction is 6:1.

[0035] As a further preferred option, the vacuum freeze drying temperature is -60 ℃, the drying time is 48 h, and the pressure is 10 Pa.

[0036] The application of a chicken bone calcium-absorbing collagen polypeptide or a peptide-calcium chelate as described above in the preparation of functional foods.

[0037] Preferably, when combined with pharmaceutically or food-grade excipients, the resulting functional food is prepared in any of the following dosage forms: tablets, capsules, powders, or oral liquids.

[0038] Preferably, the functional foods include calcium supplements, high-calcium foods, and foods for special medical purposes.

[0039] A functional food prepared from the peptide-calcium chelate as described above.

[0040] Therefore, the present invention has the following beneficial effects: (1) The rational molecular docking screening strategy adopted in this invention is significantly innovative. By simulating the interaction between peptides and calcium-sensitive receptors (CaSR), the high-affinity peptide DFSLPQPPQ is directly screened from the chicken bone collagen peptide library. Its predicted binding energy is -9.8 kcal / mol, and its content in the enzymatic hydrolysis product is as high as 11.5%. This method overcomes the inefficiency of the traditional "blind screening" mode and realizes the discovery of targeted peptides based on mechanisms. (2) The chemically synthesized active peptide DFSLPQPPQ provided by the present invention has a purity of >95% and a calcium chelation activity of 7.18±0.83 μg / mg, which is about 64.7% higher than that of some peptides in the prior art (such as the cod bone peptide GRGNEGPQ in patent CN119306798A, which has a purity of 4.36 μg / mg), showing a stronger calcium ion binding ability; (3) The peptide calcium chelate (DFSFLPQPPQ-Ca) prepared in this invention showed excellent calcium absorption-enhancing performance in the Caco-2 cell model. The calcium bioavailability was significantly higher than that of the calcium chloride control group after 120 minutes of transport. In particular, the calcium bioavailability of the group bound to calcium gluconate and calcium citrate was significantly higher than that of the calcium chloride control group alone (P<0.05), which verified its potential to enhance calcium absorption through the CaSR pathway. (4) This invention uses chicken bone by-products as raw materials, realizing the high-value utilization of resources and providing a precise development path for poultry processing by-products based on clearly defined active peptides, which has significant industrial promotion value and economic benefits. Attached Figure Description

[0041] Figure 1 The images show the total ion chromatogram of chicken bone collagen peptides and the secondary mass spectrometry of chicken bone calcium absorption-promoting collagen polypeptides DFSLPQPPQ; among them, Figure 1 In the figure, 'a' represents the total ion chromatogram of chicken bone collagen peptides. Figure 1 b in the image represents the secondary mass spectrum of the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ.

[0042] Figure 2 The structural diagram of the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ and its molecular docking visualization with CaSR are shown; among them, Figure 2 In the diagram, 'a' represents the structure of the chicken bone collagen polypeptide DFSFLPQPPQ that promotes calcium absorption. Figure 2 In the figure, b is a visualization of the molecular docking between the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ and CaSR.

[0043] Figure 3This is a diagram showing the growth morphology of a Caco-2 cell monolayer membrane structure.

[0044] Figure 4 The graph shows the changes in transmembrane resistance of Caco-2 cells.

[0045] Figure 5 This is a graph showing the calcium bioavailability of peptide-calcium chelates in Caco-2 cells.

[0046] Figure 6 Physicochemical properties of chicken bone calcium absorption-promoting collagen peptide DFSFLPQPPQ and its calcium chelate DFSFLPQPPQ-Ca are shown in the figure; Figure 6 In the image, 'a' represents the UV-Vis full spectrum scan of DFSFLPQPPQ and DFSFLPQPPQ-Ca. Figure 6 In the diagram, b represents the circular dichroism chromatograms of DFSLPQPPQ and DFSLPQPPQ-Ca; Figure 6 In the figure, c represents the Fourier transform infrared spectrum of DFSFLPQPPQ and DFSFLPQPPQ-Ca; Figure 6 In the figure, d represents the Raman spectrum of DFSFLPQPPQ and DFSFLPQPPQ-Ca. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0048] Example 1: Preparation of high calcium affinity collagen peptides (i.e., chicken bone calcium absorption promoting collagen peptides) DFSFLPQPPQ from chicken bones 1. Preparation and peptide identification of chicken bone collagen peptides (CBCP) Chicken bone collagen peptides (CBCP) were prepared by an optimized enzymatic hydrolysis method according to the inventor's prior patent application number 202511929542, entitled "A High-Calcium Chelated Active Collagen Peptide, Peptide-Calcium Chelate, and Its Application Based on Chicken Bone." The peptide sequence was determined using a Thermo UltiMate 3000 RSLCnano nano-liquid chromatography-tandem QExactive HF mass spectrometer. Peptide samples were injected via an autosampler and bound to a C18 trapping column (75 μm * 2 cm, 3 μm particle size, 100 Å pore size, Thermo), followed by separation on an analytical column (75 μm * 25 cm, 1.9 μm particle size, 100 Å pore size). An analytical gradient was established using mobile phase A (0.1% formic acid / 3% DMSO / 97% H2O) and mobile phase B (0.1% formic acid / 3% DMSO / 97% acetonitrile (ACN)). The gradient program was: initial 7% B phase, linearly increasing to 12% B after 5 min, 15% B after 20 min, 25% B after 43 min, 47% B after 57 min, and 90% B after 57.5 min, held for 7.5 min. The flow rate was set to 300 nL / min. Mass spectrometry was performed in DDA mode. The parameters for the MS1 full scan were: resolution 60 K @200 m / z, scan range 350–1500 m / z, AGC target set to 3E6, and maximum injection time set to 30 ms. The precursor ion selection window was set to 1.4 Da, the top 20 precursor ions were selected for fragmentation, and the HCD collision energy was set to 28%. MS2 scan parameters were set as follows: resolution 15 K @ 200 m / z, AGCtarget 1E5, maximum injection time 50 ms, and dynamic exclusion time 30 seconds. Raw mass spectrometry data were analyzed using MaxQuant (2.2.0.0) software, employing the software's built-in Andromeda database retrieval algorithm. The database used for retrieval was the Gallus gallus chicken protein sequence database downloaded from Uniprot.

[0049] like Figure 1 As shown, the total ion chromatogram of CBCP and the secondary mass spectrometry of DFSFLPQPPQ were identified by liquid chromatography-mass spectrometry. Figure 1 The images show the total ion chromatogram of chicken bone collagen peptides and the secondary mass spectrometry of chicken bone calcium absorption-promoting collagen polypeptides DFSLPQPPQ; among them, Figure 1 In the figure, 'a' represents the total ion chromatogram of chicken bone collagen peptides. Figure 1 b in the image represents the secondary mass spectrum of the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ.

[0050] As shown in Table 1, a total of 16 polypeptides with type I collagen genes were identified.

[0051] Table 1: Identification of chicken bone collagen peptides and results of docking of each peptide with CaSR molecules. SEQ ID NO. Sequence Molecular Weight Peptide Content (%) Gene Affinity (kcal / mol) 1 FSFLPQPPQE 1188.5815 35.81 COL1A1 -9.3 2 FSFLPQPPQ 1059.5389 12.12 COL1A1 -8.4 3 DFSFLPQPPQ 1174.5659 11.50 COL1A1 -9.8 4 FDFSFLPQPPQE 1450.6769 8.20 COL1A1 -8.9 5 SFLPQPPQE 1041.5131 3.82 COL1A1 -8.6 6 DFSFLPQPPQEK 1431.7034 2.64 COL1A1 -9.1 7 FDFSFLPQPPQ 1321.6343 1.92 COL1A1 -9.1 8 FDFSFLPQPPQEK 1578.7718 1.06 COL1A1 -8.0 9 GFDFSFLPQPPQE 1507.6983 0.73 COL1A1 -9.4 10 DFSFLPQPP 1046.5073 0.57 COL1A1 -8.7 11 GFDFSFLPQPPQ 1378.6558 0.44 COL1A1 -9.5 12 DFSFLPQPPQE 1303.6085 0.31 COL1A1 -8.3 13 FDLSFLPQPPQE 1416.6925 0.22 COL1A1 -9.0 14 ARGPSGPQGPSG 1066.5156 0.00 COL1A1 -8.5 15 VGPTGPAGPR 907.48756 0.56 COL1A2 -9.1 16 ARGSDGSAGPTGPAGPIG 1523.7328 0.26 COL1A2 -9.1

[0052] 2. Rational screening of high calcium affinity peptides based on molecular docking Target receptor preparation: The crystal structure of the human calcium-sensitive receptor (CaSR) (PDB ID: 5FBH) was downloaded from the Protein Database (PDB). The structure was processed using the molecular visualization software PyMol (v2.4.0): water molecules, existing ligand molecules (A-TCR609 and B-TCR609), and other small ligand molecules were removed, retaining only the protein receptor itself. The processed CaSR protein was pre-processed using AutoDockTools 1.5.7 software, including hydrogenation and Gasteiger charge calculation, to generate a receptor file (.pdbqt format) suitable for molecular docking.

[0053] Ligand peptide library construction and processing: The amino acid sequence of the α1 chain of chicken type I collagen (COL1A1, UniProt accession number based on the Gallusgallus database) was used as a template. Combined with the actual peptide identification results from previous mass spectrometry analysis of enzymatic hydrolysate of chicken bone collagen, the identified peptide sequences with high abundance were selected as candidate ligand libraries. Using an online server (such as RPBS) or molecular simulation software, the most stable three-dimensional conformation of each peptide was generated based on its amino acid sequence. Similarly, AutoDockTools software was used to perform hydrogenation, charge calculation, and other processing on all candidate peptides to generate ligand files (.pdbqt format).

[0054] Molecular docking simulation and scoring: Batch molecular docking was performed using the AutoDock Vina program. The active pocket of the CaSR protein was defined as the docking search space (grid box), ensuring coverage of known ligand-binding regions. After running docking calculations, the program outputs the optimal conformation for each candidate peptide binding to the CaSR receptor and its corresponding binding free energy (Affinity, in kcal / mol). A higher absolute value of the binding free energy indicates a more stable binding and higher affinity.

[0055] As shown in Table 1, the initial screening criteria were set as binding energy < -9.0 kcal / mol and peptide content > 10%. Docking results showed that the decapeptide with the sequence Asp-Phe-Ser-Phe-Leu-Pro-Gln-Pro-Pro-Gln (abbreviated as DFSFLPQPPQ, i.e., the sequence shown in SEQ ID NO. 3) exhibited extremely high predicted affinity for CaSR, with a binding free energy of -9.8 kcal / mol. Simultaneously, mass spectrometry analysis showed that this peptide accounted for as much as 11.5% of the actual enzymatic hydrolysis products of chicken bone collagen. Considering both high predicted affinity and high natural abundance, DFSFLPQPPQ was identified as the most promising novel chicken bone-derived calcium absorption-promoting candidate peptide. Figure 2 The structural diagram of the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ and its molecular docking visualization with CaSR are shown; among them, Figure 2 In the diagram, 'a' represents the structure of the chicken bone collagen polypeptide DFSFLPQPPQ that promotes calcium absorption. Figure 2 In the figure, b is a visualization of the molecular docking between the chicken bone calcium absorption-promoting collagen polypeptide DFSFLPQPPQ and CaSR.

[0056] The 3D structure of chicken bone calcium absorption-promoting collagen peptide DFSLPQPPQ is as follows: Figure 2 As shown in Figure a. (As shown in Figure a) Figure 2 As shown in Figure b, docking conformation analysis revealed that the peptide can stably insert into the active pocket of CaSR and form multiple hydrogen bond interactions with key amino acid residues such as GLN-179, ASN-176, ARG-220, SER-247, GLU-251, and LYS-225, theoretically revealing its potential mechanism of action.

[0057] 3. Chemical synthesis and calcium chelation activity verification of chicken bone collagen peptides DFSLPQPPQ that promote calcium absorption Chemical synthesis of the peptide: The target peptide DFSLPQPPQ was synthesized by a professional biotechnology company (GenScript Biotechnology Co., Ltd.) using a standard solid-phase peptide synthesis method. Synthesis requirements: purity >95%. The resulting chicken bone calcium absorption-promoting collagen peptide lyophilized powder was stored at -20 ℃ for later use.

[0058] Calcium chelating activity assay: Accurately weigh 3 mg of the above-mentioned synthetic peptide and dissolve it in 2 mL of 5 mmol / L calcium chloride aqueous solution. Incubate at 37 °C for 20 min. Add 4 mL of sodium phosphate buffer (20 mmol / L, pH 7.4) and continue incubation at 37 °C for 30 min to form calcium phosphate precipitate. Centrifuge at 8000 rpm / min for 20 min and remove the precipitate. Take the supernatant and determine the free calcium content according to national standard GB 5009.92-2016 (atomic absorption spectrometry or EDTA titration). Use a solution containing only calcium chloride without added peptide as a blank control.

[0059] Calcium chelation activity is calculated using the following formula: ; Among them, Ca s The calcium content in the supernatant of each sample group is expressed in μg; Ca c The calcium content in the supernatant of the control group was measured in μg; m s The value is the peptide sample mass, expressed in mg.

[0060] The experimental results showed that the calcium chelating activity of the peptide DFSLPQPPQ was 7.18 ± 0.83 μg / mg. The calcium content of the supernatant in the control group (no sample) was 77.22 μg, while the calcium content of the supernatant in the experimental group (with sample) was as high as 98.76 μg. This difference (+21.54 μg) proves that the synthetic peptide (i.e., the chemically synthesized chicken bone calcium absorption-promoting collagen polypeptide DFSLPQPPQ) has a clear calcium ion binding capacity.

[0061] Example 2: Preparation of peptide-calcium chelate (DFSFLPQPPQ-Ca) 1. DFSFLPQPPQ-Ca prepared from different calcium sources Dissolve 10 mg of chicken bone calcium-absorbing collagen peptide DFSLPQPPQ in 2 mL of purified water to prepare a 5 mg / mL peptide solution. Weigh 10 mg of each of the following calcium sources (calcium chloride, calcium citrate, calcium gluconate, and calcium lactate) separately (i.e., a peptide-to-calcium mass ratio of 1:1) and add them to the peptide solution, stirring until dissolved. Adjust the pH of the mixture to 5.5 with 0.1 mol / L NaOH solution. Place the reaction system in a 36 ℃ water bath and stir continuously for 30 minutes. After the reaction is complete, add 6 volumes of pre-cooled anhydrous ethanol to the mixture and allow it to stand at 4 ℃ for 1 hour to allow the chelate to precipitate completely. Centrifuge at 8000 rpm for 15 min at 4 ℃ and collect the precipitate. After vacuum freeze-drying, the precipitate yielded a white powdery product, which is a peptide calcium chelate, and was designated as DFSFLPQPPQ-CaCl2, DFSFLPQPPQ-Calcium citrate, DFSFLPQPPQ-Calcium gluconate, and DFSFLPQPPQ-Calcium lactate, respectively.

[0062] 2. Validation of the in vitro calcium absorption-promoting function of peptide-calcium chelates The bioavailability of the peptide-calcium chelate of this invention was verified using a Caco-2 cell model. Cell model establishment: Caco-2 cells were cultured and differentiated to establish a complete intestinal epithelial monolayer cell model in Transwell chambers. The model was established when cell differentiation was complete and the transmembrane resistance was consistently above 800 Ω·cm. 2When the time is right, it indicates that the model has been successfully established and can be used for transport experiments. Dissolve an appropriate amount of peptide-calcium chelate in HBSS buffer to prepare samples with the same calcium concentration of 10 μg / mL (CaCl2, DFSLPQPPQ-CaCl2, DFSLPQPPQ-Calcium citrate, DFSLPQPPQ-Calcium gluconate, DFSLPQPPQ-Calcium lactate). Finally, filter the samples through a 0.22 μm filter membrane for sterilization and set aside. After Caco-2 cells have fully differentiated in 6-well Transwell plates, discard the culture medium. Then wash the Caco-2 monolayer cell membrane three times with preheated HBSS (Hank's balanced salt solution). For the final wash, incubate the cells in a cell culture incubator for 30 min to remove impurities from the cell membrane. Discard the HBSS. Add 2 mL of preheated peptide-calcium chelate at different concentrations to the AP side (intestinal lumen side) and 2.5 mL of HBSS to the BL side (blood side). Every 30 min over 2 h, 200 μL of sample was taken from the BL side and the same volume of HBSS was added. After all BL side sampling was completed, all liquids from both the AP and BL sides were removed. The cell surface on the AP side was rinsed twice quickly and gently with pre-chilled HBSS to remove non-specific calcium adhering to the surface. 200 μL of pre-chilled RIPA lysis buffer was added directly to the cell membrane in the Transwell chamber, and the culture plate was incubated on ice for 20 min with gentle shaking. Then, the cells were repeatedly pipetted to ensure complete detachment and lysis, and all lysis buffer was collected. The calcium bioavailability of CaCl2 and peptide calcium chelates was calculated by measuring the total calcium content, cellular calcium content (calcium retention), and extracellular calcium content (calcium transport) of the samples using the calcium assay kit microplate method (Nanjing Jiancheng Bioengineering Institute). The formula for calculating calcium bioavailability (CBA) is as follows: ; Where: A—transported calcium content, mmol / L Ca / well; B—cellular calcium content, mmol / L Ca / well; C—total calcium content of the sample, mmol / L Ca / well. Figure 3 This is a diagram showing the growth morphology of a Caco-2 cell monolayer membrane structure. Figure 4 The graph shows the changes in transmembrane resistance of Caco-2 cells. Figure 5 This is a graph showing the calcium bioavailability of peptide-calcium chelates in Caco-2 cells.

[0063] like Figure 3 and Figure 4 As shown, after 14 days of proliferation and differentiation, Caco-2 cells formed a complete membrane, and the transmembrane resistance of the cells tended to stabilize at 969 Ω / cm. 2 This verified the successful establishment of the cell monolayer model.

[0064] like Figure 5 As shown, at 120 minutes of transport, the bioavailability of the chelate groups with DFSLPQPPQ as ligand—CaCl2 group, DFSLPQPPQ-CaCl2 group, DFSLPQPPQ-Calcium citrate (DFSFLPQPPQ-CC) group, DFSLPQPPQ-Calcium gluconate (DFSFLPQPPQ-CG) group, and DFSLPQPPQ-Calcium lactate (DFSFLPQPPQ-CaLac) group—were 24.85%, 26.75%, 43.88%, 44.33%, and 22.93%, respectively. Among them, the calcium bioavailability of the groups bound to calcium gluconate and calcium citrate was significantly higher than that of the control group containing only calcium chloride (P < 0.05).

[0065] Among them, the DFSLPQPPQ-CG chelate group exhibited the highest calcium bioavailability, but its lyophilized chelate product had a strong static electricity problem, making it impossible to weigh properly. Therefore, calcium citrate, with slightly lower bioavailability, was used as the preferred calcium source. This is consistent with the optimized mixed peptide-bound calcium source in the inventor's earlier patent application No. 2025119295421, entitled: "A High-Calcium Chelated Active Collagen Peptide, Peptide-Calcium Chelate, and Its Application Based on Chicken Bone." This result not only verifies the calcium absorption-promoting function of the DFSLPQPPQ peptide, but also shows that by combining with organic calcium sources (such as calcium citrate), a synergistic effect can be produced, further improving the bioavailability of calcium.

[0066] The peptide DFSLPQPPQ (calcium chelation activity 7.18 μg / mg) screened and synthesized in this invention was compared with the cod bone calcium absorption-promoting peptide GRGNEGPQ (calcium chelation activity 4.36 μg / mg) disclosed in reference patent CN119306798A.

[0067] Under similar Caco-2 cell model and testing conditions, the DFSLPQPPQ-Ca chelate of this invention exhibits higher calcium transport efficiency and bioavailability. This demonstrates that rational molecular docking screening targeting the key calcium absorption receptor (CaSR) can yield more promising and efficient peptides than traditional random screening or activity-directed separation methods.

[0068] Example 3: Physicochemical properties of DFSFLPQPPQ and DFSFLPQPPQ-Ca 1. Preparation of DFSFLPQPPQ-Ca 50 mg of calcium citrate was dissolved in a 0.2 mol / L hydrochloric acid aqueous solution. Then, 50 mg of DFSFLPQPPQ lyophilized powder prepared according to the method in Example 1 was dissolved in the calcium solution, so that the concentration of the chicken bone calcium absorption collagen peptide solution was 5 g / L and the mass ratio of chicken bone calcium absorption collagen peptide to calcium citrate was 1:1. The pH of the solution was adjusted to 5.5 using 0.1 M NaOH aqueous solution, and the mixture was stirred at 36 °C for 30 min. After the chelation reaction was completed, 6 volumes of anhydrous ethanol were added to remove free calcium and precipitate the peptide-calcium chelate. The mixture was centrifuged at 8000 rpm for 15 min at 4 °C, the precipitate was collected, and freeze-dried under vacuum at -50 °C for 48 h to obtain 37.4 mg of peptide-calcium chelate, denoted as DFSFLPQPPQ-Ca.

[0069] 2. Ultraviolet-Visible Full Spectrum Scan The sample was dissolved in 5 mM phosphate buffer at pH 7.2–7.4 to achieve a concentration of 0.2 mg / mL. The UV-Vis spectrum of the sample was measured using a double-beam UV-Vis spectrophotometer (Purchip TU-1900) in the wavelength range of 190–400 nm. Blank correction was performed using phosphate buffer. Figure 6 Physicochemical properties of chicken bone calcium absorption-promoting collagen peptide DFSFLPQPPQ and its calcium chelate DFSFLPQPPQ-Ca are shown in the figure; Figure 6 In the image, 'a' represents the UV-Vis full spectrum scan of DFSFLPQPPQ and DFSFLPQPPQ-Ca. Figure 6 In the diagram, b represents the circular dichroism chromatograms of DFSLPQPPQ and DFSLPQPPQ-Ca; Figure 6 In the figure, c represents the Fourier transform infrared spectrum of DFSFLPQPPQ and DFSFLPQPPQ-Ca; Figure 6 In the figure, d represents the Raman spectrum of DFSFLPQPPQ and DFSFLPQPPQ-Ca.

[0070] like Figure 6 As shown in Figure a, the maximum absorption wavelength of DFSLPQPPQ is 221 nm, and that of DFSLPQPPQ-Ca is 217 nm. The blue shift observed before and after chelation may be due to the coordination of calcium ions with the carboxyl, amino, or peptide bond oxygen atoms of the polypeptide, altering the local electron cloud density; a decrease in the polarity of the aromatic ring microenvironment or an increase in conformational constraint; and changes in the folding or aggregation state of the peptide chain, affecting the exposure of the chromophore.

[0071] 3. Circular dichroism spectrum and second-order structure diagram A circular dichroism spectrometer (JASCO J-1500, Japan) was used with the liquid CD method. The test wavelength range was set to 185~260 nm. The sample was dissolved in 5 mM phosphate buffer (pH 7.2~7.4) with a target sample concentration of 0.2 mg / mL. The sample was filtered to remove bacteria and particles, and pure phosphate buffer was used as a blank control.

[0072] like Figure 6 As shown in b, the decrease in random coil from 14% to 2% indicates that calcium binding leads to a reduction in disordered structure and conformational ordering. The weakening of the 200 nm negative peak: The strong negative peak at approximately 203 nm in the CD spectrum is a typical feature of random coil. The negative value of the peptide-calcium chelate at this point decreases significantly (from -24.2 to -6.5 mdeg), strongly indicating that calcium binding greatly reduces the disordered and flexible structure of the peptide. The overall negative ellipticity decreases: Throughout the 195–220 nm range, the negative ellipticity of the peptide-calcium chelate is smaller than that of the free peptide, further supporting the conclusion that the structure tends towards order and rigidity. The increase in α-helix from 25% to 44% may indicate the formation of a more stable secondary structure after chelation.

[0073] 4. Fourier transform infrared spectroscopy Fourier transform infrared spectrometer (Thermo Fisher Scientific Nicolet iS20, USA) was used. Samples were prepared using the KBr pellet method, and the spectral range was set to 4000–400 cm⁻¹. -1 The resolution is 4 cm. -1 , scanned 32 times.

[0074] like Figure 6 As shown in c, DFSFLPQPPQ exhibits a strong peak at 1662 cm⁻¹ in amide I. -1 The absence of chelates suggests that calcium binding may directly participate in coordination, leading to a decrease in the C=O electron cloud density and a change in vibrational modes; the amide II band is 1532.96 cm⁻¹. -1 Transformed to 1576.51 cm -1 A significant redshift and enhancement suggest that NH or CN are involved in coordination, indicating an altered hydrogen bonding environment; the NH / OH stretching region (3700~3000 cm⁻¹) -1 A relatively broad peak, with a center of approximately 3325 cm. -1 The peak shape broadens and shifts to higher wavenumbers, reaching approximately 3340 cm⁻¹. -1 The altered hydrogen bond network may be related to the coordination of coordinated water molecules or the participation of NH4+ molecules, leading to an increase in the OH / NH4+ bond force constant. The carboxylate region is 1447.41 cm2. -1 Transformed to 1412.91 cm -1 This may correspond to the symmetric stretching of COO⁻, suggesting that calcium binds to the carboxyl group.

[0075] 5. Raman spectroscopy A Raman spectrometer (Thermo Scientific DXR, USA) was used, employing a 785 nm laser to avoid fluorescence interference. Confocal mode was used, covering the spectral range of 400–2000 cm⁻¹. -1 .

[0076] like Figure 6 As shown in figure d, Raman spectroscopy analysis indicates that the structure of the polypeptide DFSLPQPPQ undergoes a fundamental change after chelation with calcium ions. Most notably, the phenylalanine residues in the free peptide show a change at 1003 cm⁻¹. -1 The characteristic strong peaks at approximately 849 and 964 cm⁻¹ completely disappeared, while those at approximately 849 and 964 cm⁻¹ disappeared at the same time. -1 A new characteristic peak belonging to the Ca-O coordination bond appeared. Furthermore, the peak in the CH2 / CH3 bending vibration region split and shifted. These changes collectively confirm that calcium binding not only involves coordination with carboxyl and carbonyl groups but also profoundly affects the microenvironment of aromatic amino acids, driving the peptide to form a more ordered and rigid conformation. This provides a structural explanation for its enhanced stability and function as a highly efficient calcium carrier.

[0077] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A chicken bone collagen polypeptide that promotes calcium absorption, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.3, or it is a functional derivative of the sequence with one or more amino acid substitutions, deletions, or additions that have the same calcium absorption-promoting activity.

2. The chicken bone calcium absorption-promoting collagen polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is DFSFLPQPPQ.

3. The chicken bone calcium absorption-promoting collagen polypeptide according to claim 1 or 2, characterized in that, The calcium chelating activity of the polypeptide is not less than 7.18 μg / mg.

4. A peptide-calcium chelate, characterized in that, It contains the chicken bone calcium absorption-promoting collagen polypeptide and calcium ions as described in any one of claims 1 to 3.

5. The peptide-calcium chelate according to claim 4, characterized in that, The calcium ions are derived from at least one of calcium citrate, calcium gluconate, calcium lactate, and calcium chloride.

6. A method for preparing a peptide-calcium chelate as described in claim 4 or 5, characterized in that, Includes the following steps: The chicken bone collagen peptide that promotes calcium absorption was chelated with a calcium source containing calcium ions under pH conditions of 5.0-7.

0.

7. The method for preparing a peptide-calcium chelate according to claim 6, characterized in that, The chelation reaction was carried out at a temperature of 25–60 °C for 20–60 min.

8. The application of a chicken bone calcium absorption-promoting collagen polypeptide as described in any one of claims 1 to 2 or a peptide-calcium chelate as described in claim 4 or 5 in the preparation of functional foods.

9. The application according to claim 8, characterized in that, The functional foods include calcium supplements, high-calcium foods, and foods for special medical purposes.

10. A functional food prepared from the peptide-calcium chelate according to claim 4 or 5.

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