Calcium ion chelating peptide, preparation method and application thereof, peptide calcium chelate and composition

Abstract

The invention relates to the technical field of bioactive peptides, in particular to a calcium ion chelating peptide, a preparation method and application thereof, a peptide calcium chelate and a composition. The oligopeptides GVDDQPLFH and PQFEE are identified by taking vital gluten as a raw material through enzymolysis, purification and separation, and both the oligopeptides GVDDQPLFH and PQFEE have high calcium ion chelating activity which is higher than 75%, have excellent calcium carrying capacity and have the potential of developing a novel biological calcium supplement. Experiments verify that the peptide-calcium chelate prepared from the oligopeptide GVDDQPLFH separated and identified by the invention has an excellent calcium supplement effect and higher bioavailability compared with a traditional calcium supplement reagent, and can be used for preparing calcium supplement medicines or health foods and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to calcium ion chelating peptides, preparation methods and applications thereof, peptide calcium chelates and compositions. Background Art

[0002] Calcium is an essential nutrient for the human body and plays an important role in various physiological and metabolic activities, such as muscle contraction, blood coagulation, nerve transmission and bone development. Insufficient calcium intake often leads to metabolic bone diseases, such as rickets, osteomalacia, osteoarthritis and osteoporosis. Calcium exists in the form of ions and is easy to form precipitation during gastrointestinal digestion. There are obvious defects in calcium absorption and bioavailability. Long-term use may cause side effects and increase the burden on the human body. Therefore, calcium supplements with better absorption and high bioavailability are needed. Peptide calcium chelates, as a new type of calcium supplement, have independent chelation and transportation systems, which can prevent phytate from forming precipitation in the intestine and promote its absorption by the human body, thus becoming a good carrier for calcium absorption and transportation. Peptide calcium chelates have dual nutritional functions and can be used as calcium supplements and can also supplement the body with bioactive peptides. Casein phosphopeptide is the earliest calcium ion chelating peptide reported and studied. Later, active peptides with calcium ion chelating ability were isolated from proteins from various sources such as grains, animal bones, and seafood.

[0003] In recent years, the research on calcium ion chelating peptides from plant sources, especially agricultural by-products, has become increasingly popular. Wheat oligopeptides have multiple physiological functions, with the characteristics of low cost, good water solubility, easy absorption, strong biological activity, and good processability. Wheat oligopeptides are usually composed of 2-6 amino acid molecules, with a molecular weight of less than 1000Da and a balanced amino acid composition. Reports on wheat oligopeptides have shown that they have the effects of improving immunity, anti-oxidation, and regulating the immune system. Natural oligopeptides prepared from wheat are a good source for preparing chelated calcium. There are also studies on the separation of peptides with calcium chelating activity from wheat protein. However, they generally involve directly chelating the peptide mixture obtained by enzymatic hydrolysis with calcium ions, and the chelation rate of wheat peptide chelates is low. Therefore, it is necessary to screen and prepare oligopeptides with high calcium chelating activity. Summary of the invention

[0004] In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a calcium ion chelating peptide with high calcium chelating activity.

[0005] The second object of the present invention is to provide a method for preparing a calcium ion chelating peptide, which is simple to operate and suitable for industrial application.

[0006] The third object of the present invention is to provide a peptide calcium chelate, which has excellent calcium supplementation effect and high bioavailability and can be used as a calcium supplement.

[0007] A fourth object of the present invention is to provide a calcium supplement composition, comprising the peptide calcium chelate provided by the present invention, which has improved body absorption and bioavailability performance.

[0008] A fifth object of the present invention is to provide an application of the calcium ion chelating peptide provided by the present invention in the preparation of a calcium supplement.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The calcium ion chelating peptide comprises oligopeptide 1 and / or oligopeptide 2; wherein the amino acid sequence of oligopeptide 1 is GVDDQPLFH, as shown in the sequence table SEQ ID NO.1; and the amino acid sequence of oligopeptide 2 is PQFEE, as shown in the sequence table SEQ ID NO.2.

[0011] The preparation method of calcium ion chelating peptide comprises the following steps:

[0012] 1) using gluten powder as a raw material, and sequentially performing enzymatic hydrolysis treatment with neutral protease and alkaline protease to prepare an enzymatic hydrolyzate;

[0013] 2) purifying the enzymatic hydrolysate to obtain an enzymatic hydrolysate with a molecular weight of ≤1 kDa;

[0014] 3) Separating and identifying oligopeptides with high calcium chelating activity from the enzymatic hydrolysate prepared in step 2).

[0015] Optionally, the specific method of step 1) enzymatic hydrolysis is: first prepare a gluten suspension, heat it to 45-50°C, adjust the pH to 7.0, add neutral protease, and after enzymatic hydrolysis, heat it to 55-65°C, adjust the pH to 8.5, add alkaline protease, and after enzymatic hydrolysis, inactivate the enzyme, centrifuge and take the supernatant to obtain an enzymatic solution.

[0016] In a specific embodiment of the present invention, the mass volume concentration of the gluten suspension is 5-10%; the specific preparation method of the gluten suspension is: adding gluten to deionized water to dissolve and stir to mix evenly, ultrasonically applying a power of 400W for 20min to fully disperse, and magnetically stirring for 1h to form a uniformly dispersed suspension.

[0017] In a specific embodiment of the present invention, the dosage of neutral protease is 3000-4000 U / g; the dosage of alkaline protease is 6000-8000 U / g; the enzymolysis time of neutral protease is 2-3 hours; and the enzymolysis time of alkaline protease is 3-4 hours.

[0018] Optionally, the specific method for purifying the enzymatic hydrolysate in step 2) is: purification using a filter membrane with a molecular weight cutoff of 1 kDa, a processing pressure of 1.0-2.0 MPa, a processing volume of 2-5 L / h, and spray drying to obtain an enzymatic hydrolysate.

[0019] Optionally, the specific method of step 3) is to separate and identify the oligopeptides with high calcium chelating activity: separation and identification by LC-MS / MS technology, and the liquid chromatography conditions are: buffer: liquid A is 0.1% formic acid aqueous solution, liquid B is a mixed solution of 0.1% formic acid, 80% acetonitrile and water; the chromatographic column is balanced with 100% liquid A; after the sample is injected, it is gradient separated through the chromatographic analysis column at a flow rate of 300nl / min; the liquid separation gradient is as follows: 0-2min, liquid B linear gradient from 2% to 5%; 2-44min, liquid B linear gradient from 5% to 28%; 44-51min, liquid B linear gradient from 28% to 40%; 51-53min, liquid B linear gradient from 40% to 100%; 53-60min, liquid B is maintained at 100%;

[0020] The calcium chelating activities of the isolated peptides were compared to screen out the oligopeptides with high calcium chelating activity.

[0021] Before the liquid chromatography injection separation, the enzymatic hydrolysate to be separated is also subjected to sample preparation, including desalting using a C18 Stage Tip, vacuum drying, re-dissolving with 0.1% (v / v) formic acid, and filtering through a 0.45 μm membrane.

[0022] The calcium ion chelating peptide provided by the present invention can be used to prepare calcium supplement drugs or health foods, including first chelating the calcium ion chelating peptide with calcium ions to prepare peptide calcium chelate, and then using it as a core active ingredient to prepare a calcium supplement composition.

[0023] Beneficial effects of the present invention:

[0024] 1) The present invention uses gluten powder as a raw material, separates and identifies oligopeptides with calcium ion chelating activity, which are GVDDQPLFH (oligopeptide 1) and PQFEE (oligopeptide 2), and the calcium ion chelating rates are 77.81% and 75.05%, respectively. Compared with oligopeptide 2, oligopeptide 1 has a better calcium carrying capacity;

[0025] 2) In the specific embodiment of the present invention, the peptide calcium chelate prepared by the oligopeptide with calcium ion chelating activity isolated and identified by the present invention is verified through in vitro simulation test, which has excellent calcium absorption effect and higher bioavailability, and can be used as a raw material source for the preparation of a new calcium supplement;

[0026] 3) The preparation method of the present invention adopts two enzyme combination enzymolysis, combined with molecular weight screening separation, and liquid chromatography-mass spectrometry technology to separate and identify oligopeptides with high calcium ion chelating activity, which is simple to operate and easy to promote and apply in industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the comparison results of the calcium chelation rates of chelated calcium 1, chelated calcium 2, and chelated calcium 3 in Example 1;

[0028] Figure 2 Schematic diagram of the molecular docking simulation results of GVDDQPLFH and calcium;

[0029] Figure 3 Schematic diagram of the molecular docking simulation results of PQFEE and calcium;

[0030] Figure 4 is the liquid chromatogram of solid phase synthesized GVDDQPLFH;

[0031] Figure 5 is the liquid chromatogram of solid phase synthesized PQFEE;

[0032] Figure 6 is the mass spectrum of solid phase synthesized GVDDQPLFH;

[0033] Figure 7 is the mass spectrum of solid phase synthesized PQFEE;

[0034] Figure 8 Schematic diagram of the comparison of bone volume fraction (BV / TV) of the tibia of mice in different experimental groups;

[0035] Fig. 9 This is a schematic diagram of the comparison of trabecular bone thickness (Tb.Th) of the tibia of mice in different experimental groups;

[0036] Fig.10 Micro-CT images of the tibiae of mice in different experimental groups. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained from commercial sources.

[0038] The evaluation method of calcium ion chelating activity in the following examples is:

[0039] Weigh the oligopeptide to be tested and anhydrous calcium chloride according to the mass ratio of 2:1, add 10 times the mass of water after mixing, mix and dissolve thoroughly, adjust pH 8.5, place in a 65℃ constant temperature water bath and stir continuously, and perform chelation reaction for 3 hours. After cooling to room temperature, centrifuge at 4000r / min for 10 minutes to remove the precipitate, and obtain the supernatant as peptide chelated calcium solution. Put the reaction solution into a 200Da dialysis bag and dialyze for 24 hours to remove free calcium ions in the product. Replace pure water 3 times in the middle, and the dialyzate is concentrated under reduced pressure, spray dried to obtain wheat oligopeptide chelated calcium, and the chelation rate is measured.

[0040] The peptide chelated calcium was prepared into a 0.2 mg / mL aqueous solution, and the calcium content was determined by flame atomic absorption spectrometry according to GB 5009.92-2016, and the chelation rate was calculated using the following formula:

[0041]

[0042] Where:

[0043] W-chelation rate, in %;

[0044] m 1 -Calcium content in peptide calcium chelate, in mg;

[0045] m 2 -The amount of calcium added during the reaction, in mg.

[0046] Example 1

[0047] This example provides a method for preparing a calcium ion chelating peptide, and the specific steps are as follows:

[0048] 1) Enzymatic hydrolysis:

[0049] Take gluten powder and add deionized water to dissolve and stir to prepare an 8% (w / v) mixed solution, ultrasonically disperse the wheat protein in the water at a power of 400W for 20 minutes, and magnetically stir for 1 hour to form a uniform suspension;

[0050] The first stage: heat the water bath to 45°C and adjust the pH to 7.0. Add 4000U / g of neutral protease according to the substrate mass and perform enzymolysis for 2.5h.

[0051] The second stage: heating to 60°C and adjusting the pH to 8.5, adding 8000U / g alkaline protease according to the mass of the substrate, enzymolysis for 3h, inactivating the enzyme in a boiling water bath for 10min, centrifuging at 4000r / min for 10min after cooling, taking the supernatant to obtain an enzymolysis solution, and conducting a preliminary chelation reaction between the enzymolysis solution and anhydrous calcium chloride according to the above-mentioned evaluation method for calcium ion chelation activity to obtain chelated calcium 1;

[0052] 2) Purification treatment:

[0053] Purification was performed using a filter membrane with a molecular weight cutoff of 1 kDa, a processing pressure of 1.5 MPa, and a processing volume of 3 L / h. The retentate was collected and spray-dried to obtain an enzymatic hydrolyzate with a molecular weight of ≤1 kDa; and the enzymatic hydrolyzate with a molecular weight of ≥1 kDa was collected;

[0054] According to the above-mentioned evaluation method of calcium ion chelating activity, the enzymatic hydrolysates with a molecular weight of ≤1 kDa and a molecular weight of ≥1 kDa were subjected to chelation reaction with anhydrous calcium chloride to obtain chelated calcium 2 and chelated calcium 3;

[0055] The comparative analysis results of the chelation rates of chelated calcium 1, chelated calcium 2, and chelated calcium 3 are as follows: Figure 1 As shown, it is shown that the enzymatic hydrolysates with molecular weight ≤1 kDa have higher calcium chelating activity;

[0056] 3) Identification and screening of amino acid sequences with high calcium chelating activity:

[0057] 2) The purified and separated hydrolysate with a molecular weight of ≤1 kDa was desalted using a C18 StageTip, vacuum dried, and the peptides were re-dissolved with 0.1% (v / v) formic acid and filtered through a 0.45 μm membrane for detection;

[0058] LC-MS / MS detection liquid chromatography conditions: Use nanoliter flow rate Easy nLC 1200 chromatography system (ThermoScientific) for chromatographic separation. Buffer: Liquid A is 0.1% formic acid aqueous solution, and liquid B is a mixed solution of 0.1% formic acid, acetonitrile and water (including 80% acetonitrile). The chromatographic column is balanced with 100% liquid A. The sample is injected into the Trap Column (100μm*20mm, 5μm, C18, Dr.Maisch GmbH) and then passed through the chromatographic analysis column (75μm*150mm, 3μm, C18, Dr.MaischGmbH) for gradient separation at a flow rate of 300nl / min;

[0059] The liquid phase separation gradient is as follows: 0-2min, liquid B linear gradient from 2% to 5%; 2-44min, liquid B linear gradient from 5% to 28%; 44-51min, liquid B linear gradient from 28% to 40%; 51-53min, liquid B linear gradient from 40% to 100%; 53-60min, liquid B is maintained at 100%.

[0060] After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). The analysis time was 60 min, and the detection mode was positive ion. The parent ion scan range was 350-1800 m / z, the primary mass spectrometry resolution was 120,000@m / z 200, the secondary mass spectrometry resolution was 15,000@m / z 200, and the collision energy was CE28 eV.

[0061] Two peptides with high calcium chelating activity and high biosafety were screened out, PQFEE (oligopeptide 2) and GVDDQPLFH (oligopeptide 1). The physicochemical properties of the screened peptides are shown in Table 1:

[0062] Table 1 Sequence information and physicochemical properties of peptides screened out with potential high calcium chelating activity

[0063] Peptide sequence Hydrophilicity toxicity Charge pI Molecular weight 1 PQFEE 0.74 Non-toxic -2.00 3.80 648.28 2 GDQ -0.01 Non-toxic -1.50 4.20 1026.48

[0064] It should be noted that, in the preparation and screening process of the oligopeptide with calcium ion chelating activity of the present invention, the parameters in the enzymatic hydrolysis and purification steps can be adjusted within a certain range, and the oligopeptide of the present invention can also be separated and identified. The specific mass concentration of the gluten powder solution is 5%-10%; the parameters such as temperature, enzyme dosage and enzymatic hydrolysis time are as follows: after heating to 45-50°C in the first stage, neutral protease is added at a dosage of 3000-4000U / g, and enzymatic hydrolysis is performed for 2-3h; in the second stage, it is heated to 55-65°C, alkaline protease is added at a dosage of 6000-8000U / g, and enzymatic hydrolysis is performed for 3-4h; membrane purification, the processing pressure is 1.0-2.0MPa, and the processing volume is 2-5L / h.

[0065] Example 2 Performance Evaluation

[0066] 1. Evaluation of the stability of calcium ion chelating molecular structure:

[0067] The peptide structure was optimized using Chem 3D software. After minimizing the energy of the peptide structure using the MM2 algorithm, the batch molecular docking simulation of GVDDQPLFH (oligopeptide 1) and PQFEE (oligopeptide 2) with calcium was performed using Autodock software. The peptides with low energy were screened according to the docking energy. The results were processed using PyMol software. The molecular docking results are shown in Figure 2. Figure 2 (GVDDQPLFH) and Figure 3 (PQFEE) shown.

[0068] The chelation calcium mode was predicted by molecular docking. The results showed that PQFEE-Ca chelates through the carboxyl oxygen atom of Gln2, which belongs to the monodentate mode of the calcium chelation model. GVDDQPLFH-Ca is connected to the carboxyl oxygen atom of Val2 through the amino nitrogen atom of Asp4, which belongs to the α mode in the chelation mode. The α mode of GVDDQPLFH and calcium chelates with the amino acid residues of the peptide through two chelate bonds, with a shorter bond length and stronger bond energy, and is more firmly bound than the monodentate mode.

[0069] 2. Calcium ion chelating activity:

[0070] Solid phase synthesis of active sequences and activity verification

[0071] According to the screening results in Example 1, Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the polypeptides GVDDQPLFH and PQFEE using a solid phase synthesis method. The liquid phase characterization results are as follows Figure 4 (GVDDQPLFH), Figure 5 (PQFEE) as shown; mass spectrometry characterization results are shown Figure 6 (GVDDQPLFH), Figure 7(PQFEE) shown.

[0072] The calcium chelation rate of the synthetic peptides was detected according to the detection method provided in the specific embodiment of the present invention. The results are shown in Table 2. Compared with the chelated calcium 1 prepared from the preliminary enzymatic hydrolysate, the calcium chelation rates of the peptides after purification and screening were greatly improved, among which the peptide GVDDQPLFH had the highest calcium chelation activity.

[0073] Table 2 Calcium chelation rate of synthetic peptide sequences

[0074] Serial number Peptide sequence Chelation rate (%) 1 GDQ 77.81±0.21 2 PQFEE 75.05±0.34 3 Chelated calcium 1 35.72±0.18

[0075] 3. Evaluation of the application value of calcium supplement preparation:

[0076] (1) Preparation of peptide chelated calcium aqueous solution:

[0077] Weigh the oligopeptide 1GVDDQPLFH and anhydrous calcium chloride in a mass ratio of 2:1, add 10 times the mass of water after mixing, mix and dissolve thoroughly, adjust the pH to 8.5, place in a 65℃ constant temperature water bath and stir continuously to carry out chelation reaction for 3h. Take out and cool to room temperature, centrifuge at 4000r / min for 10min, remove the precipitate, and obtain the supernatant as the peptide chelated calcium aqueous solution;

[0078] (2) Experimental process:

[0079] The experimental animals KM male mice were 4 weeks old and purchased from Henan Sikebes Biotechnology Co., Ltd. After one week of adaptive feeding, 6 groups were set up with 10 mice in each group. After 4 weeks of feed feeding, gavage treatment was started. The experimental groupings are shown in Table 3.

[0080] Table 3 Animal experiment groups

[0081]

[0082]

[0083] In order to explore the improvement effect of peptide-calcium chelate on osteoporosis, the tibia was observed by staining 3D images of micro-CT to determine the microstructure of trabeculae. The distal femur was scanned using a Micro-CT system under specific conditions of 70 kV voltage and 114 μA current. The scan covered 3 mm of the distal femur with a resolution of 10 μm. According to the Micro-CT images (such as Fig.10 Calculate morphometric parameters, including trabecular volume percentage (BV / TV) (as shown in Figure 8 As shown), trabecular thickness (Tb.Th) (as shown Fig. 9 shown).

[0084] The results showed that the trabeculae of the osteoporosis model group fed with a low-calcium diet showed sparse interconnectivity. After the intervention of peptide-calcium chelate, the symptoms of bone loss in mice were alleviated. At the same time, the bone volume fraction and trabecular thickness of the peptide calcium chelate treatment group increased. In summary, there was a more obvious calcium supplementation effect than the calcium chloride drug control group, indicating that the oligopeptide with high calcium ion chelating activity separated and identified by the present invention can be used as a key material to prepare peptide calcium chelate for the preparation of calcium supplement drugs or health foods.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A calcium chelating peptide, characterized in that: It comprises oligopeptide 1 and / or oligopeptide 2; wherein the amino acid sequence of oligopeptide 1 is GVDDQPLFH, as shown in the sequence table SEQ ID NO.1; and the amino acid sequence of oligopeptide 2 is PQFEE, as shown in the sequence table SEQ ID NO.

2.

2. A method for preparing a calcium ion chelating peptide, characterized in that: The steps include: 1) using gluten powder as a raw material, and sequentially performing enzymatic hydrolysis treatment with neutral protease and alkaline protease to prepare an enzymatic hydrolyzate; 2) purifying the enzymatic hydrolysate to obtain an enzymatic hydrolysate with a molecular weight of ≤1 kDa; 3) Separating and identifying oligopeptides with high calcium chelating activity from the enzymatic hydrolysate prepared in step 2).

3. The method for preparing the calcium chelating peptide according to claim 2, characterized in that: The specific method of step 1) enzymatic hydrolysis is as follows: firstly, a gluten suspension is prepared, heated to 45-50°C, pH is adjusted to 7.0, neutral protease is added, and after enzymatic hydrolysis, the suspension is heated to 55-65°C, pH is adjusted to 8.5, alkaline protease is added, and after enzymatic hydrolysis, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain an enzymatic hydrolyzate.

4. The method for preparing the calcium chelating peptide according to claim 3, characterized in that: The mass volume concentration of the gluten suspension is 5-10%. The specific preparation method of the gluten suspension is: adding gluten to deionized water to dissolve and stir to mix evenly, ultrasonically treating at a power of 400W for 20 minutes to fully disperse, and magnetically stirring for 1 hour to form a uniformly dispersed suspension.

5. The method for preparing the calcium chelating peptide according to any one of claims 2 to 4, characterized in that: The dosage of neutral protease is 3000-4000U / g; the dosage of alkaline protease is 6000-8000U / g; the enzymolysis time of neutral protease is 2-3h; the enzymolysis time of alkaline protease is 3-4h.

6. The method for preparing the calcium chelating peptide according to any one of claims 2 to 4, characterized in that: Step 2) The specific method for purifying the enzymatic hydrolysate is: purifying with a filter membrane with a molecular weight cutoff of 1 kDa, a processing pressure of 1.0-2.0 MPa, a processing volume of 2-5 L / h, and spray drying to obtain an enzymatic hydrolysate.

7. The method for preparing the calcium chelating peptide according to any one of claims 2 to 4, characterized in that: Step 3) The specific method for separating and identifying the oligopeptides with high calcium chelating activity is as follows: separation and identification by LC-MS / MS technology, and the liquid chromatography conditions are as follows: buffer: liquid A is a 0.1% formic acid aqueous solution, and liquid B is a mixed solution of 0.1% formic acid, 80% acetonitrile and water; the chromatographic column is balanced with 100% liquid A; after the sample is injected, it is subjected to gradient separation through the chromatographic analysis column at a flow rate of 300nl / min; the liquid phase separation gradient is as follows: 0-2min, liquid B linear gradient from 2% to 5%; 2-44min, liquid B linear gradient from 5% to 28%; 44-51min, liquid B linear gradient from 28% to 40%; 51-53min, liquid B linear gradient from 40% to 100%; 53-60min, fluid B is maintained at 100%; The calcium chelating activities of the isolated peptides were compared to screen out oligopeptides with high calcium chelating activity; The amino acid sequence of oligopeptide 1 is GVDDQPLFH, as shown in SEQ ID NO.1 in the sequence list; the amino acid sequence of oligopeptide 2 is PQFEE, as shown in SEQ ID NO.2 in the sequence list.

8. Use of the calcium ion chelating peptide according to claim 1 in the preparation of calcium supplement drugs or health foods.

9. A peptide calcium chelate, characterized in that The invention comprises the calcium ion chelating peptide as claimed in claim 1 and calcium ion chelating.

10. A calcium supplement composition, characterized in that The method comprises the peptide calcium chelate as claimed in claim 9.

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