Calcium ion binding peptide, peptide-calcium chelate and application thereof

Abstract

The invention provides a calcium ion binding peptide, a peptide-calcium chelate and application of the peptide-calcium chelate. The calcium ion binding peptide has at least one of the sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 8. The peptide-calcium chelate is formed by chelating at least one of the sequences as shown in SEQ ID NO: 1-SEQ ID NO: 8 and Ca < 2 + >. The provided peptide-calcium chelate can be used as a calcium supplement, thereby treating calcium deficiency.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a calcium ion binding peptide, a peptide-calcium chelate and applications thereof. Background Art

[0002] Calcium is an important element of human bones and plays a vital role in maintaining bone health. In addition, calcium is also involved in regulating a variety of physiological activities, including blood coagulation, hormone secretion, and nerve conduction. However, calcium deficiency is currently very common. Statistics show that most people have serious calcium deficiency problems, with an average daily calcium intake of less than 700 mg. Calcium deficiency can lead to a variety of diseases, including osteoporosis and rickets. Calcium cannot be synthesized by the human body itself and must be supplemented through food or supplements. At present, the mainstream calcium supplements are ionized calcium (calcium carbonate and calcium citrate, etc.), but these supplements easily form insoluble complexes with phytic acid or oxalic acid in the intestine, reducing calcium absorption and causing constipation. In addition, these supplements have certain side effects, such as causing abdominal distension. Therefore, the development of calcium supplements with high bioavailability and low side effects has broad prospects. In recent years, peptide-calcium complexes have been proven to be a highly effective calcium supplement. Among these chelates, Ca 2+ The peptide is chelated and surrounded by the peptide, thus avoiding the interference of phytic acid in the intestine. In addition, the peptide can be specifically recognized by intestinal epithelial cells and absorbed by endocytosis, so it can serve as a Ca 2+ Peptide-calcium complexes have been developed using a variety of food-derived proteins, such as soy protein, casein, and collagen, which effectively enhance calcium absorption.

[0003] The key step in preparing peptide-calcium chelates is to screen for Ca 2+ Calcium-binding peptides (CBPs) with high affinity. A variety of separation techniques such as ultrafiltration, gel chromatography, and anion exchange chromatography are usually used to separate potential calcium-binding peptides, and then their amino acid sequences are identified by liquid chromatography-electrospray ionization-mass spectrometry / mass spectrometry (LC-ESI-MS / MS). However, the number of peptides identified is still large, ranging from dozens to thousands. Experimental verification of each sequence will be time-consuming and wasteful of raw materials, so new screening methods are needed. In recent years, molecular docking has been valued as a new technology that can be used to calculate the affinity of peptides with Ca 2+ Calcium-binding peptides were screened by the binding energy between calcium and calcium. In addition, the researchers also used the calcium sensing receptor (CaSR) as a target to screen calcium-binding peptides through molecular docking. CaSR can regulate calcium levels in the body. When the body is calcium deficient, CaSR is activated, thereby accelerating the intestinal uptake of calcium. Therefore, if the calcium-binding peptide can activate CaSR, the peptide-calcium chelate prepared thereby will effectively promote calcium absorption. This emerging method for screening calcium-binding peptides has broad prospects.

[0004] Hericium erinaceus (HE) is a nutritious edible mushroom. It is rich in protein (24%-27%) and is an excellent raw material for bioactive peptides. Currently, a variety of bioactive peptides have been extracted from Hericium erinaceus, including immunomodulatory peptides and antioxidant peptides. In addition, Hericium erinaceus contains high levels of acidic amino acids (E and D), which are important for the production of calcium 2+ With high affinity, Hericium erinaceus has the nutritional advantage of developing peptide-calcium chelate. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. The present invention isolates a calcium ion binding peptide from Hericium erinaceus, which can chelate with calcium ions to prepare peptide-calcium chelates, promote calcium absorption, and thus can be used as a calcium supplement to treat diseases caused by calcium deficiency. The present invention first uses ultrafiltration and anion exchange chromatography to separate and purify Hericium erinaceus hydrolysate to obtain a component with the highest calcium binding rate (CBR), and then identifies the peptide sequence by LC-ESI-MS / MS. On this basis, molecular docking is used to evaluate the affinity of these peptides with Ca 2+ / CaSR binding energy, thereby screening out polypeptides with dual functions (calcium ion binding ability and CaSR activation). Then, the screened polypeptide (WYEEGTDKA, SEQ ID NO: 1) was used to prepare a peptide-calcium chelate (WYEEGTDKA-Ca), which can promote the absorption of calcium.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides an isolated calcium ion binding peptide having at least one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8.

[0008] The second aspect of the present invention provides an isolated nucleic acid encoding the isolated calcium ion binding peptide according to the first aspect.

[0009] The third aspect of the present invention provides a construct, which comprises the isolated nucleic acid according to the second aspect.

[0010] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the construct described in the third aspect.

[0011] The fifth aspect of the present invention provides a peptide-calcium chelate, characterized in that at least one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8 and Ca 2+ Chelated.

[0012] The sixth aspect of the present invention provides a method for preparing the peptide-calcium chelate according to the fifth aspect, comprising:

[0013] Providing a calcium ion-binding peptide solution, wherein the calcium ion-binding peptide solution is obtained by dissolving the isolated calcium ion-binding peptide described in the first aspect in water;

[0014] The calcium ion binding peptide solution and Ca 2+ The reaction is carried out at a predetermined temperature. After the reaction is completed, anhydrous ethanol is used for precipitation, and the peptide-calcium chelate is obtained by centrifugation.

[0015] According to an embodiment of the present invention, the mass concentration of the calcium ion binding peptide solution is 1-5 wt %.

[0016] According to an embodiment of the present invention, the Ca 2+ It is calcium chloride, and the mass ratio of the calcium ion binding peptide to the calcium chloride is (2-3):1.

[0017] According to an embodiment of the present invention, the predetermined temperature is 45 to 60 degrees Celsius.

[0018] The seventh aspect of the present invention provides a method for promoting calcium absorption by a sample, comprising: incubating the sample with the isolated calcium ion binding peptide described in the first aspect or with the peptide-calcium chelate described in the fifth aspect;

[0019] According to an embodiment of the present invention, the sample is an in vitro biological sample.

[0020] The eighth aspect of the present invention provides a calcium absorbent, comprising the isolated calcium ion binding peptide according to the first aspect or the peptide-calcium chelate according to the fifth aspect, and an acceptable excipient.

[0021] The ninth aspect of the present invention provides use of the isolated calcium ion binding peptide of the first aspect or the peptide-calcium chelate of the fifth aspect in the preparation of a drug or an agent for enhancing calcium absorption in a subject.

[0022] The tenth aspect of the present invention provides use of the isolated calcium ion binding peptide described in the first aspect or the peptide-calcium chelate described in the fifth aspect in the preparation of a medicament for treating diseases or complications caused by calcium deficiency.

[0023] According to an embodiment of the present invention, the disease or complication caused by calcium deficiency is selected from at least one of osteoporosis, rickets, and muscle spasm.

[0024] The beneficial effects achieved by the present invention are at least:

[0025] The present invention separates a calcium ion binding peptide from Hericium erinaceus. The binding peptide has a high calcium binding capacity and can chelate with calcium ions to prepare a peptide-calcium chelate. The peptide-calcium chelate can promote the body's absorption of calcium, so it can be used as a calcium supplement to treat diseases caused by calcium deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the calcium ion binding rate results of each component of the Hericium erinaceus protein hydrolysate ultrafiltration provided by an embodiment of the present invention.

[0027] Figure 2 According to the embodiment of the present invention, the Hericium erinaceus polypeptide F 3 Figure 1. Component anion exchange chromatography elution results.

[0028] Figure 3 According to the embodiment of the present invention, the Hericium erinaceus polypeptide F 33 Calcium ion binding rate results of subfractions.

[0029] Figure 4 It is a 3D diagram and a cross-interaction diagram of the binding of the SEQ ID NO: 1 polypeptide to the calcium sensing receptor (CaSR) provided in an embodiment of the present invention.

[0030] Figure 5 The present invention provides a polypeptide shown in SEQ ID NO: 1 and a calcium-sensing receptor (CaSR) binding site and interaction force.

[0031] Figure 6 It is a scanning electron microscope image, an element energy spectrum stacking image and an element energy spectrum intensity image of the SEQ ID NO: 1 polypeptide and the peptide-calcium chelate provided according to the embodiments of the present invention.

[0032] Figure 7 It is a UV-visible spectrum scanning diagram of the polypeptide shown in SEQ ID NO: 1 and the peptide-calcium chelate provided according to the examples of the present invention.

[0033] Figure 8 It is a fluorescence spectrum diagram of the polypeptide shown in SEQ ID NO: 1 and the peptide-calcium chelate provided according to the examples of the present invention.

[0034] Fig. 9 1 is an X-ray diffraction diagram of the polypeptide shown in SEQ ID NO: 1 and the peptide-calcium chelate provided according to an embodiment of the present invention.

[0035] Fig.10 It is an infrared spectrum diagram of the polypeptide shown in SEQ ID NO: 1 and the peptide-calcium chelate provided according to the examples of the present invention.

[0036] Fig.11 1 is a diagram of simulated in vitro digestion of a peptide-calcium chelate provided according to an embodiment of the present invention.

[0037] Fig.12 These are the results of the effects of peptide-calcium chelates of different concentrations on the viability of CaCo-2 cells provided by the examples of the present invention.

[0038] Fig.13 This is the result of promoting calcium absorption of CaCo-2 cells by the peptide-calcium chelate provided in an example of the present invention. DETAILED DESCRIPTION

[0039] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0040] The term "comprise" or "comprising" means including the mentioned elements or steps, but not excluding other elements or steps. Of course, unless otherwise specified, including or comprising also covers the situation consisting of the mentioned elements or steps.

[0041] Herein, the term "isolated" means capable of being identified and separated and / or recovered from cells or cell cultures expressing a polypeptide or protein or from an artificially synthesized culture fluid. Typically, an isolated polypeptide will be prepared by at least one purification step.

[0042] The present invention provides an isolated calcium ion binding peptide having at least one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:8. According to a preferred embodiment, the isolated calcium ion binding peptide is the sequence shown in SEQ ID NO:1, the sequence shown in SEQ ID NO:2 or the sequence shown in SEQ ID NO:3. The sequence shown in SEQ ID NO:1 is: WYEEGTDKA, the sequence shown in SEQ ID NO:2 is: DTVVEPY, and the sequence shown in SEQ ID NO:3 is: AEVVKSADKEYW. The provided calcium ion binding peptides have high calcium ion binding ability and can chelate with calcium ions to prepare peptide calcium chelates as calcium supplements.

[0043] The present invention also provides an isolated nucleic acid encoding the isolated calcium ion binding peptide described in the first aspect. The nucleic acid mentioned is separable, including but not limited to DNA, RNA or cDNA, etc. The isolated nucleic acid sequence encoding the calcium ion binding peptide can be obtained by conventional methods in the art.

[0044] In order to prepare the calcium ion binding peptide mentioned above, it can be directly synthesized by artificial synthesis or by cell expression. The sequence encoding the calcium ion binding peptide can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. In order to, the present invention also provides a construct, which includes the isolated nucleic acid described above. Various methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. For example, the nucleic acid can be inserted into the multiple cloning site of the expression vector to form a construct. The construct can contain a variety of operating factors such as promoters, terminators, marker genes, etc. as needed, and these operating factors can be operably connected to the corresponding nucleic acid. The promoter is usually used to provide a signal to start transcription. The promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, phage PL and PR promoter as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening. Of course, there can also be an enhancer as needed to enhance the expression of the protein. The expression vector is not particularly limited, and may be some commercially available expression vectors, or may be an expression vector artificially modified as needed, such as a plasmid, a bacteriophage, a virus, etc. The virus may be a plant cell virus, a mammalian cell virus, etc. The construct may express antibodies or proteins in vitro, or may be transferred into cells to express the corresponding polypeptide.

[0045] The present invention also provides a host cell, which includes the construct described above. Any cell suitable for expressing a nucleic acid or a construct for polypeptide or protein can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, COS cells, Drosophila S2 or Sf9 insect cells. Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0046] The present invention further provides a peptide-calcium chelate, characterized in that any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8 and Ca 2+ Chelated.

[0047] The peptide-calcium chelate described above can be prepared by the following method, including:

[0048] Providing a calcium ion-binding peptide solution, wherein the calcium ion-binding peptide solution is obtained by dissolving the isolated calcium ion-binding peptide described in the first aspect in water;

[0049] The calcium ion binding peptide solution and Ca2+ The reaction is carried out at a predetermined temperature. After the reaction is completed, anhydrous ethanol is used for precipitation, and the peptide-calcium chelate is obtained by centrifugation.

[0050] According to a specific embodiment, the mass concentration of the calcium ion binding peptide solution is 1 to 5 wt %.

[0051] According to a specific embodiment, the Ca 2+ Derived from calcium chloride, it can be provided in the form of a calcium chloride solution. The mass ratio of the calcium ion binding peptide to the calcium chloride in the calcium chloride solution is (2-3):1.

[0052] According to a specific embodiment, the predetermined temperature is 45 to 60 degrees Celsius, and according to a preferred embodiment, it is 50 to 55 degrees Celsius.

[0053] The present invention further provides a method for promoting calcium absorption by a sample, comprising: incubating the sample with the isolated calcium ion binding peptide described in the first aspect or with the peptide-calcium chelate described in the fifth aspect;

[0054] According to the embodiment of the present invention, the sample is an in vitro biological sample, including but not limited to tissues, cells, organs, etc.

[0055] The present invention also provides a calcium absorbent, comprising the isolated calcium ion binding peptide or the peptide-calcium chelate described above, and an acceptable excipient.

[0056] The acceptable excipients mentioned are acceptable to the subject at the dose or concentration used. Acceptable excipients include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtering with a sterile filter membrane. Those skilled in the art can select different acceptable excipients as needed to prepare different dosage forms. The acceptable excipients mentioned can be pharmaceutically acceptable excipients.

[0057] The isolated calcium ion binding peptide or the peptide-calcium chelate can be used in the preparation of a drug or an agent for enhancing calcium absorption in a subject.

[0058] The calcium ion binding peptide or peptide-calcium chelate is used in the preparation of a medicament for treating diseases or complications caused by calcium deficiency.

[0059] According to a specific embodiment, the disease or complication caused by calcium deficiency is selected from at least one of osteoporosis and rickets.

[0060] The technical scheme of the present invention is described below by specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples can be obtained commercially.

[0061] Example 1

[0062] Example 1 First, Hericium erinaceus protein hydrolysate was prepared, and then calcium ion binding peptide (CBP) was separated and purified from the Hericium erinaceus protein hydrolysate.

[0063] (1) Preparation of Hericium erinaceus protein hydrolysate

[0064] Hericium erinaceus protein was extracted from Hericium erinaceus and dissolved in water to obtain Hericium erinaceus protein solution. Then, the Hericium erinaceus protein solution (mass concentration of 5%, w / w) was incubated at 50°C and pH 7.5, and the protein was hydrolyzed with papain for 2 h. The amount of protease added was 1×10 5 U / g protein. After the enzyme was inactivated in a boiling water bath, the supernatant was separated by centrifugation (4000 rpm, 15 minutes). The supernatant was then freeze-dried to obtain Hericium erinaceus protein hydrolysate.

[0065] (2) Isolation and purification of CBP from Hericium erinaceus protein hydrolysate

[0066] First, CBP was separated from Hericium erinaceus protein hydrolysate by ultrafiltration:

[0067] The above Hericium erinaceus protein hydrolysate was dissolved in deionized water to obtain a Hericium erinaceus protein hydrolysate solution (2%, w / w), and then the solution was passed through 50kD and 10kD ultrafiltration membranes in sequence, and ultrafiltration was performed using an ultrafiltration cup for 6 hours, wherein the ultrafiltration pressure was maintained at 0.2MPa. After ultrafiltration, the CBR of each component was determined, and F 1 (>50kD), F 2 (10-50 kD) and F 3 (<10kD) three components, such as Figure 1 As shown, F 3 The molecular weight of the component was the lowest, but the calcium ion binding rate (CBR) was the highest (79.21±3.37%), which was significantly higher than the other two components (p<0.05). 3 The components were then freeze-dried.

[0068] F was then further separated by anion exchange chromatography 3 component, and obtain three subcomponents:

[0069] The F with the strongest calcium ion binding ability 3 The fraction (<10 kD) was dissolved in Tris-HCl buffer (0.02 mol / L, pH 9.0) and filtered with a microporous membrane (0.45 μm). The fraction was separated by isocratic elution (2.5 mL / min) using Diamond Q anion exchange medium (Bestchrom Biosciences Ltd.). The elution buffer was Tris-HCl (0.02 mol / L, pH 9.0) containing 0, 0.25, 0.5, and 1 mol / L NaCl, respectively. After separation, three subfractions were obtained: F 31 、F 32 and F 33 ,like Figure 2 As shown in Figure 2, the CBR of the three subcomponents gradually increased, among which F 33 The CBR of the component was the highest (85.17 ± 3.70%). Figure 3 The obtained subcomponent (F 31 、F 32 、F 33 ) were dialyzed and freeze-dried.

[0070] Finally from F 33 Identification of CBP in components:

[0071] Determination of F by LC-ESI-MS / MS 33 The amino acid sequence of the subcomponents. Peptides with a molecular weight range of 200-2000Da were selected (peptides in this range have strong calcium binding ability), and a total of eight peptides that met this standard were selected: WYEEGTDKA (SEQ ID NO: 1), DTVVEPY (SEQ ID NO: 2), AEVVKSADKEYW (SEQ ID NO: 3), WYEEGTDKAKV (SEQ ID NO: 4), SADKEYWVN (SEQ ID NO: 5), SADKEYWV (SEQ ID NO: 6), GHYTEGAELVD (SEQ ID NO: 7) and EWDIWRDONDOSA (SEQ ID NO: 8). The secondary mass spectra of these peptides and their positions in the parent protein were characterized and annotated.

[0072] Example 2

[0073] Example 2 further screened calcium ion binding peptides.

[0074] In order to further screen the Ca 2+ The peptides with strong affinity were docked by molecular docking method to identify the binding sites of each peptide with Ca2 + The binding energy between Ca 2+ The structure file of Ca was obtained from PubChem database and imported into Chem3D software to obtain 2+ The peptide structure was used as a macromolecular receptor and energy minimized using Discovery Studio 2016. 2+ Both the peptide and the peptide were imported into AutoDock Tools for hydrogenation and charge calculations. Then, molecular docking was performed using AutoDockVina. The higher the absolute value of the binding energy, the more stable the binding.

[0075] The binding between the peptide and the calcium-sensing receptor (CaSR) was also evaluated by molecular docking. The structure of CaSR was obtained from the PDB database (PDB ID: 2FLU). The peptide structure was predicted using AlphaFold. The ligand (peptide) and receptor (CaSR) were imported into AutoDock Tools v1.5.7 for preprocessing, including hydrogenation, energy minimization, removal of water molecules, and charge calculation. Molecular docking was then performed using AutoDock Vina v1.1.2. Potential binding cavities were defined based on the TCR (C12H12N2O2) ligand. The interaction force between CaSR and the peptide was analyzed using PyMOL v2.5.4. The 3D and 2D graphics of the CaSR-peptide complex were obtained using the ligand interaction module of MOEv2022.02.

[0076] Lower binding energy scores indicate that the peptide binds Ca 2+ The eight peptides had a stronger affinity with Ca and formed a more stable peptide-calcium chelate. 2+ The binding energy scores between the eight peptides were all negative, indicating that these eight peptides can spontaneously bind to Ca 2+ Among them, three peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) had low binding energy scores (less than -8 kcal / mol) (see Table 1 below). Therefore, these peptides were selected for further screening.

[0077] The CaSR is responsible for maintaining calcium homeostasis in the body, and once activated, it enhances calcium absorption in the gastrointestinal tract. If CBPs can activate this receptor, they will have multiple functions: calcium binding ability and promoting calcium uptake. Therefore, using molecular docking technology, CaSR was used to further screen Hericium erinaceus-derived CBPs. Figure 4 and Figure 5It was shown that the polypeptide shown in SEQ ID NO: 1 can enter the active pocket of CaSR, and the amino acid residues (W1, E3, E4, A9 and K8) in the polypeptide shown in SEQ ID NO: 1 can interact with S272, D275, Y246, T103, V104 and A45 of CaSR through various forces. In addition, the binding energy scores between the three peptides and CaSR were -8.3, -7.1 and -7.2 kcal / mol, respectively (as shown in Table 2 below), indicating that the polypeptide shown in SEQ ID NO: 1 is a better CaSR activator. Overall, the polypeptide shown in SEQ ID NO: 1 exhibits a strong affinity for CaSR. 2+ It has a strong affinity for CaSR and was selected for the preparation of peptide calcium chelates.

[0078] Table 1Ca 2+ The binding energy between peptides

[0079] Peptides Binding energy (kcal / moL) WYEEGTDKA (SEQ ID NO: 1) -8.99 DTVVEPY (SEQ ID NO: 2) -8.55 AEVVKSADKEYW (SEQ ID NO: 3) -8.42 WYEEGTDKAKV (SEQ ID NO: 4) -6.88 SADKEYWVN (SEQ ID NO:5) -7.13 SADKEYWV (SEQ ID NO:6) -7.26 GHYTEGAELVD (SEQ ID NO:7) -6.52 EWDIWRDONDOSA (SEQ ID NO: 8) -6.22

[0080] Table 2 Binding energy between CaSR and peptides

[0081]

[0082]

[0083] Example 3

[0084] Example 3 Taking the sequence shown in SEQ ID NO: 1 as an example, a peptide-calcium chelate (expressed as WYEEGTDKA-Ca for the convenience of description) was prepared as a calcium ion binding peptide, and the calcium ion binding rate was measured, including:

[0085] The calcium ion binding peptide was dissolved in water to obtain a Hericium erinaceus calcium ion binding peptide solution (mass concentration 1%, w / w) and CaCl 2 The reaction was stirred at 50°C for 30 min. The reaction pH was 8, and the peptide was mixed with CaCl 2 The ratio (w / w) was 2: 1. After the reaction was completed, ten times the volume of anhydrous ethanol was added to precipitate the peptide calcium chelate, the chelate was separated by centrifugation, and the sample was collected after freeze-drying.

[0086] The CBR was calculated according to formula (a), and the content of chelated calcium was determined using an atomic absorption spectrophotometer.

[0087] CBR (%) = (amount of chelated calcium) / (total amount of added calcium) × 100% (a)

[0088] The experimental results show that the CBR result is 92%.

[0089] Example 4

[0090] Example 4 characterized the structure of the peptide-calcium chelate prepared in Example 3 from different perspectives.

[0091] (1) Microstructure and elemental composition analysis

[0092] The microstructure and elemental composition of the peptide-calcium chelate and the polypeptide shown in SEQ ID NO: 1 were observed using a scanning electron microscope (SEM) equipped with an energy dispersive spectrometer (EDS). The results showed that the sequence shown in SEQ ID NO: 1 was mainly composed of irregularly curled sheets ( Figure 6 (shown in A); in the presence of Ca 2+ After chelation, the irregular curled structure disappears and presents two forms: one is a more regular, smooth and compact plate-like structure, and the other is a smooth and regular spherical structure ( Figure 6 The element distribution of the two samples was analyzed using a scanning electron microscope. The sequence shown in SEQ ID NO: 1 consists of three elements (C, O and N) ( Figure 6 (B), while Ca labeled in purple was found in the peptide-calcium chelate. Most of the Ca existed in the form of nanospheres (marked with white circles), further confirming that Ca 2+ Involved in the formation of spherical particles. In addition, after chelation, the Ca signal was clearly detected in the peptide-calcium chelate ( Figure 6 Overall, these results confirm that Ca 2+ Chelation with the sequence shown in SEQ ID NO:1 was successful.

[0093] (2) UV, fluorescence and XRD analysis

[0094] Prepare an aqueous solution of the peptide-calcium chelate (0.5 mg / mL) and the sequence shown in SEQ ID NO:1 (0.5 mg / mL). Use a spectrophotometer to scan the two samples from 190 to 800 nm. Scan the solution of the peptide-calcium chelate (0.25 mg / mL) and the sequence shown in SEQ ID NO:1 (0.25 mg / mL) with a fluorescence spectrophotometer. The emission wavelength range is 200-500 nm, and the excitation wavelength is 295 nm. Analyze the secondary structure of the peptide-calcium chelate (0.1 mg / mL) and the polypeptide shown in SEQID NO:1 (0.1 mg / mL) using a circular dichroism spectrometer in the ultraviolet region of 190-260 nm. Analyze the crystal structure of the peptide-calcium chelate using an X-ray diffractometer at a speed of 2° / min.

[0095] WYEEGTDKA exhibits a UV absorption peak at 270 nm (e.g. Figure 7 After chelation, the absorption peak shifted to 264 nm and the intensity weakened.

[0096] Peptides containing aromatic amino acids will produce intrinsic fluorescence under 295nm excitation light. WYEEGTDKA shows a strong fluorescence absorption peak at 304nm, which is mainly caused by the Y residue (such as Figure 8 After chelation, the intensity of the peak decreased significantly, which means that the chromophore was involved in the binding with Ca 2+ of chelation, and the conformation of the peptide changed after chelation.

[0097] The sequence shown in SEQ ID NO:1 and the crystal structure of the peptide-calcium chelate were analyzed using XRD. 2 As an ionic compound, it shows significant and sharp diffraction peaks (such as Fig. 9 The sequence shown in SEQ ID NO:1 exhibits a broad and flat diffraction peak, indicating its amorphous structure. After chelation, the sharp diffraction peak disappears, indicating that CaCl 2 The crystal structure of Ca2+ was changed and calcium existed in an amorphous form. These results further confirmed that Ca 2+ Successful chelation with WYEEGTDKA.

[0098] (3) Infrared spectroscopy analysis

[0099] WYEEGTDKA-Ca or WYEEGTDKA (5 mg) was mixed with 100 mg KBr. The samples were measured using a Fourier transform infrared spectrometer (FT-IR) to obtain the wavelengths of 400-4000 cm -1 FT-IR spectrum in the range.

[0100] The solution of WYEEGTDKA-Ca or WYEEGTDKA was transferred to an NMR tube and the NMR spectrometer was used to obtain 1 H NMR spectroscopy.

[0101] like Fig.10 As shown in Figure 2, FT-IR analysis revealed the functional groups involved in chelation in WYEEGTDKA. After chelation, the absorption peak due to NH in WYEEGTDKA increased from 3291 cm -1 Move to 3294cm -1 , which indicates that Ca 2+ The amide I band of WYEEGTDKA (C=O, 1634 cm -1 ) and band II (NH, 1518cm -1 ) shifted to 1651 cm after chelation. -1 and 1560cm -1 , indicating that Ca 2+Chemical interaction with C=O. After chelation, the 1439 cm -1 The peak shifted to 1410 cm -1 , indicating the formation of -COO-Ca. After chelation, the stretching vibration of CN in WYEEGTDKA shifted from 1140 cm -1 Move to 1106cm -1 , indicating that CN participated in the reaction. After chelation, 1202 cm -1 The CO stretching vibration peak shifted to 1244 cm -1 , and the intensity is weakened, which indicates the interaction between CO and Ca. WYEEGTDKA is at 500-1000cm -1 The peak of -COO- is attributed to NH vibration. After chelation, the wave number and intensity of the peak decrease, indicating that this group participates in the reaction. Overall, these results indicate that -COO-, NH, CN, CO, and C=O participate in the reaction with Ca 2+ Chelation.

[0102] (4) In vitro digestion stability

[0103] The WYEEGTDKA-Ca solution (1 mg / mL) was incubated at 37°C, and pepsin (enzyme / LEVEIHA-Ca=1:50, w / w) was added to the solution. After adjusting the pH to 2, in vitro gastric digestion (GD) of WYEEGTDKA-Ca was performed for 2 h under stirring conditions. The pH was then adjusted to 7, trypsin was added to the solution, and intestinal digestion (ID) lasted for 2 h. During the digestion process, 2 mL samples were taken every 0.5 h, and the calcium retention rate (CRR) was measured according to formula (b).

[0104] CRR (%) = (1- (amount of calcium released) / (total amount of calcium in the sample)) × 100 (b)

[0105] The simulated digestion stability of WYEEGTDKA-Ca was analyzed (eg Fig.11 After digestion, most of the calcium (80.67 ± 1.78%) remained in the WYEEGTDKA-Ca chelate, while calcium was mainly released during GD.

[0106] Example 5

[0107] Example 5 evaluated the calcium absorption capacity of WYEEGTDKA-Ca.

[0108] Caco-2 cells were used to verify whether WYEEGTDKA-Ca has a promoting effect on calcium absorption. To determine the optimal concentration of WYEEGTDKA-Ca used in the experiment, the effects of different concentrations of WYEEGTDKA-Ca on cell viability were evaluated. Caco-2 cells were cultured in DMEM medium containing 15% FBS, 1% NEAA, and 1% penicillin-streptomycin. The cells were seeded into 96-well plates and incubated at 37°C, 5% CO 2 The cells were cultured overnight under the same conditions. Then, different concentrations (0-35 mg / mL) of WYEEGTDKA-Ca were added to the cells and treated for 24 hours. The cell viability was determined using the CCK8 kit.

[0109] The calcium absorption was then studied. Caco-2 cells were treated with WYEEGTDKA-Ca (5-15 mg / mL) for 2 h. Unabsorbed WYEEGTDKA-Ca was washed away with HBSS, and then the cells were treated with Fluo-3AM (100 μL, 5 μM) for 1 h at 37 °C. The absorbed calcium specifically binds to the fluorescent probe Fluo-3AM. The fluorescence intensity of each group was measured using a flow cytometer. CaCl 2 and CPP-Ca (calcium content equivalent to WYEEGTDKA-Ca) as blank and positive controls. The percentage of fluorescence intensity of the treated group relative to the blank group represents the amount of calcium absorption.

[0110] like Fig.12 As shown in the results, 2.5-20 mg / mL of WYEEGTDKA-Ca had no effect on cell growth (p<0.05), so a medium concentration of WYEEGTDKA-Ca (10 mg / mL) was selected in subsequent studies. Fig.13 As shown in Figure 2, the fluorescence intensity of the peptide-Ca chelate groups (WYEEGTDKA-Ca and CPP-Ca) was significantly higher than that of the control group (CaCl 2 )(p<0.05), which indicates that these two groups have a promoting effect on calcium absorption. In addition, the promoting effect of peptide-calcium chelate on calcium absorption is stronger than that of CPP-Ca (p<0.05).

[0111] The present invention identifies and screens Hericium erinaceus polypeptides SEQ ID NO:1 to SEQ ID NO:8 through traditional separation methods and molecular docking, which are promising CCP and CaSR activators. Taking SEQ ID NO:1 as an example, the WYGTDKA-Ca conjugate has two microstructures: a compact plate-like structure and a regular spherical structure. The secondary structure of the polypeptide changes after chelation. The W, E, T, D, K and A residues are involved in the binding with Ca 2+ Chelation of -COO-, NH and C=O groups in peptides with Ca2+ An ionic bond or a coordination bond is formed. After simulated digestion, most of the calcium is retained in the peptide-calcium chelate. The peptide-calcium chelate effectively promotes calcium absorption in Caco-2 cells. In short, the peptide-calcium chelate provided by the present invention is a novel peptide-Ca chelate from Hericium erinaceus and is a promising calcium supplement.

[0112] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific implementation", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0113] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An isolated calcium ion binding peptide, characterized in that It has at least one of the sequences shown in SEQ ID NO:1 to SEQ ID NO:

8.

2. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the isolated calcium ion binding peptide of claim 1.

3. A construct, characterized in that The construct comprises the isolated nucleic acid of claim 2.

4. A host cell, characterized in that The host cell comprises the construct of claim 3.

5. A peptide-calcium chelate, characterized in that By at least one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 8 and Ca 2+ Chelated.

6. The method for preparing the peptide-calcium chelate according to claim 5, characterized in that: include: providing a calcium ion-binding peptide solution, wherein the calcium ion-binding peptide solution is obtained by dissolving the isolated calcium ion-binding peptide according to claim 1 in water; The calcium ion binding peptide solution and Ca 2+ reacting at a predetermined temperature, and after the reaction is completed, precipitating with anhydrous ethanol and centrifuging to obtain a peptide-calcium chelate; Optionally, the mass concentration of the calcium ion binding peptide solution is 1 to 5 wt %; Optionally, the Ca 2+ is calcium chloride, and the mass ratio of the calcium ion binding peptide to the calcium chloride is (2-3):1; Optionally, the predetermined temperature is 45 to 60 degrees Celsius.

7. A method for promoting calcium absorption by a sample, characterized in that: include: incubating the sample with the isolated calcium ion binding peptide of claim 1 or with the peptide-calcium chelate of claim 5; Optionally, the sample is an ex vivo biological sample.

8. A calcium absorbent, characterized in that: The invention comprises the isolated calcium ion binding peptide according to claim 1 or the peptide-calcium chelate according to claim 5, and an acceptable excipient.

9. Use of the isolated calcium ion binding peptide of claim 1 or the peptide-calcium chelate of claim 5 in the preparation of a medicament or an agent for enhancing calcium absorption in a subject.

10. Use of the isolated calcium ion binding peptide according to claim 1 or the peptide-calcium chelate according to claim 5 in the preparation of a medicament for treating diseases or complications caused by calcium deficiency; Optionally, the disease or complication caused by calcium deficiency is selected from at least one of osteoporosis, rickets, and muscle spasm.

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