Yolk source polypeptide with effect of promoting calcium and magnesium absorption and application thereof

Abstract

The invention discloses a yolk source polypeptide with a calcium and magnesium absorption promoting effect and application thereof, and belongs to the technical field of biology. The amino acid sequence of the yolk source polypeptide disclosed by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence is specifically as follows: Arg-Ser-Ala-His-Arg-Gln-Glu-Phe-Pro-Lys. According to the invention, a yolk-source polypeptide is screened from egg yolk zymolyte, can promote cell absorption and transport of calcium and magnesium elements, provides a new auxiliary means for promoting absorption of calcium and magnesium elements by human bodies, can be used for research and development of novel calcium and magnesium supplements, can also chelate calcium and magnesium ions, and can be used for preparing calcium and magnesium supplements. And a new material resource is provided for research and development of the calcium-magnesium chelating agent.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an egg yolk-derived polypeptide capable of promoting calcium and magnesium absorption and application thereof. Background Art

[0002] Calcium and magnesium are essential minerals for the human body. The main function of calcium in the body is to support the health of bones and teeth, and participate in physiological processes such as blood coagulation, muscle contraction, and nerve conduction. Magnesium plays a catalytic role in more than 300 enzyme reactions, especially in energy metabolism, protein synthesis, nerve signal transmission, and muscle relaxation. The balance between calcium and magnesium is essential for maintaining bone health and normal neuromuscular function. The two not only work together in the process of bone mineralization, but also regulate muscle contraction and relaxation through interaction in neuromuscular activities.

[0003] Modern people generally have insufficient calcium and magnesium intake, which is particularly prominent in special groups such as the elderly, pregnant women and nursing mothers. Studies have shown that the bioavailability of calcium and magnesium is low, and the intake and absorption rate are affected by many factors, including the interference of anti-nutritional factors in food, intestinal health, vitamin D levels, etc. Long-term calcium deficiency will lead to a decrease in blood calcium levels and increased secretion of parathyroid hormone, which will prompt the bones to release calcium into the blood, causing the bones to gradually become brittle. Magnesium deficiency will aggravate osteoporosis and affect heart function, and may cause arrhythmia, muscle spasms and increased nerve excitability. The balance between calcium and magnesium is also very important. When magnesium is lacking, calcium metabolism in the body will be disturbed, increasing the excitability of nerves and muscles and leading to abnormalities in the nervous system. There are currently a variety of calcium and magnesium supplements on the market, such as calcium carbonate, calcium lactate, calcium citrate and other calcium preparations, as well as magnesium sulfate, magnesium chloride, magnesium citrate and other magnesium preparations. These supplements can alleviate calcium and magnesium deficiency to a certain extent. However, these conventional supplements not only have disadvantages such as low absorption efficiency and gastrointestinal discomfort, but also due to their single nature, it is difficult to ensure the balanced absorption of calcium and magnesium, which can easily cause health problems caused by unbalanced calcium and magnesium intake, resulting in reduced bioavailability of single elements in the human body. Therefore, the development of a new type of bioactive substance that ensures the synergistic effect of calcium and magnesium is an important direction of current nutrition research.

[0004] In recent years, bioactive peptides have received widespread attention as a new form of mineral supplementation. Bioactive peptides are small molecule peptides produced by protein enzymatic hydrolysis, which have multiple biological functions such as enhancing mineral absorption, anti-oxidation, and immunomodulation. Studies have found that bioactive peptides derived from natural foods such as eggs, milk, and soybeans can combine with minerals to form stable chelates, enhancing their absorption in the intestine. These peptides can not only effectively prevent free minerals from forming insoluble precipitation in the intestine, but also serve as a bridge between minerals and ion channels to promote mineral absorption. As a renewable biological resource, poultry eggs provide an economical and sustainable raw material for the production of bioactive peptides. Egg yolks contain a variety of high-quality proteins and are one of the important sources of high-quality bioactive peptides. Therefore, it is of great significance to develop a yolk-derived polypeptide that is highly active, has no side effects, and is low-cost for synergistic calcium and magnesium absorption. Summary of the invention

[0005] The purpose of the present invention is to provide an egg yolk-derived polypeptide having the function of promoting calcium and magnesium absorption and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention screens out an egg yolk-derived polypeptide from egg yolk hydrolysate, which can promote cell absorption and transport of calcium and magnesium elements, provides a new auxiliary means for promoting human body absorption of calcium and magnesium elements, can be used for the research and development of new calcium and magnesium supplements, and can also chelate calcium and magnesium ions, providing a new material resource for the research and development of calcium and magnesium chelators.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an egg yolk-derived polypeptide having the function of promoting calcium and magnesium absorption. The amino acid sequence of the egg yolk-derived polypeptide is shown in SEQ ID NO.1, specifically Arg-Ser-Ala-His-Arg-Gln-Glu-Phe-Pro-Lys (RSAHRQEFPK).

[0008] The present invention also provides the use of the egg yolk source polypeptide in the preparation of calcium and magnesium supplements.

[0009] The present invention also provides a calcium-magnesium supplement, which contains the egg yolk-derived polypeptide.

[0010] Furthermore, the calcium and magnesium supplement also includes a pharmaceutically acceptable carrier or excipient.

[0011] Furthermore, the calcium and magnesium supplement has the function of synergistically promoting the absorption and transportation of calcium and magnesium by the human body.

[0012] Furthermore, the synergistic promotion of the absorption and transportation of calcium and magnesium in the human body refers to increasing the absorption and transportation amount of calcium under the action of magnesium and maintaining the absorption and transportation amount of magnesium.

[0013] Optionally, the dosage forms of the calcium and magnesium supplement include tablets, granules, mixtures and capsules.

[0014] The present invention also provides the use of the egg yolk source polypeptide in the preparation of a calcium-magnesium chelating agent.

[0015] The present invention also provides a calcium-magnesium chelating agent, which contains the above-mentioned egg yolk-derived polypeptide.

[0016] Furthermore, the calcium-magnesium chelating agent has the function of chelating calcium ions and magnesium ions in the environment.

[0017] The present invention discloses the following technical effects:

[0018] The present invention screened and identified for the first time an egg yolk-derived polypeptide RSAHRQEFPK that has the function of promoting calcium and magnesium absorption and transport from egg yolk. The present invention adopted physical and chemical activities and cell models to evaluate its calcium and magnesium absorption-promoting function. The results showed that the egg yolk-derived polypeptide has excellent chelating effects on calcium and magnesium ions, and also has the function of promoting cellular absorption and transport of calcium and magnesium elements, and exhibits obvious synergistic effects in the process of promoting absorption and transport. On the one hand, the egg yolk-derived polypeptide of the present invention can promote cellular absorption and transport of calcium and magnesium elements, providing a new auxiliary means for promoting the absorption of calcium and magnesium elements by the human body, and can be used for the research and development of new calcium and magnesium supplements. On the other hand, the egg yolk-derived polypeptide of the present invention can chelate calcium and magnesium ions, providing a new material resource for the research and development of calcium and magnesium chelators. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the secondary mass spectrometry identification diagram of the egg yolk-derived polypeptide RSAHRQEFPK;

[0021] Figure 2 The diagrams are the interaction diagrams between the yolk-derived polypeptide RSAHRQEFPK and TRPV6 (A), TRPM7 (B), and TRPM6 (C) proteins;

[0022] Figure 3 This is the test result of the chelation rate of calcium and magnesium by the egg yolk peptide RSAHRQEFPK;

[0023] Figure 4 The results of the cytotoxicity experiment of the egg yolk-derived peptide RSAHRQEFPK;

[0024] Figure 5 These are the test results of the effect of egg yolk-derived polypeptide RSAHRQEFPK on the calcium and magnesium transport in Caco-2 cells. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solution of the present invention is as follows:

[0031] The egg yolk is composed of pulp and particles. The yolk phospholipids in the egg yolk particles are the most phosphorylated proteins in nature, and have the effect of capturing metal ions and promoting mineralization. The present invention uses egg yolk as raw material, adopts enzymatic purification technology to prepare egg yolk enzymatic peptides, and identifies a yolk source polypeptide Arg-Ser-Ala-His-Arg-Gln-Glu-Phe-Pro-Lys (RSAHRQEFPK) through HPLC-MS / MS technology, online prediction tools Peptide Property Calculator, ToxinPred, bioinformatics tools Discovery Studio software and molecular docking technology. Further through calcium and magnesium chelation experiments and cell experiments, it is verified that the yolk source polypeptide has calcium and enzyme chelation, as well as the effect of promoting cell absorption and transportation of calcium and magnesium elements, and the yolk source polypeptide has no toxic effect on cells, safe and reliable.

[0032] The preparation process of egg yolk enzymatic peptide fragments is as follows:

[0033] 1. Take the yolk of fresh eggs, collect the egg yolk liquid, dilute the egg yolk liquid with 1-4 times the volume of 4℃ distilled water, and centrifuge at 3500-6000r / min at 4℃ for 20-40min to separate the egg yolk particles.

[0034] 2. The egg yolk particles obtained in step 1 are prepared into a 1-5 mg / mL solution, and then subjected to ultrasonic homogenization (power: 400-600 W; time: 10-15 min). After homogenization, trypsin is added for enzymatic hydrolysis (enzymatic hydrolysis temperature: 30-40° C.; enzymatic hydrolysis time: 3-8 h) to obtain egg yolk enzymatic peptides.

[0035] Example 1 Preparation of egg yolk-derived polypeptide

[0036] This example uses the idea of ​​enzymatic purification of egg yolk to screen egg yolk-derived polypeptides. The specific process is as follows:

[0037] 1. Take the yolk of fresh eggs, collect the egg yolk liquid, dilute the egg yolk liquid with 2 times the volume of 4℃ distilled water, and centrifuge at 5000r / min at 4℃ for 30min to separate the egg yolk particles.

[0038] 2. Mix the egg yolk particles obtained in step 1 with water to prepare a 4 mg / mL solution, and then perform ultrasonic homogenization (power: 500 W; time: 15 min). After homogenization, add trypsin for enzymatic hydrolysis (enzymatic hydrolysis temperature: 37° C.; enzymatic hydrolysis time: 8 h) to obtain egg yolk enzymatic peptides.

[0039] 3. The egg yolk enzymatic peptide obtained in step 2 was identified by HPLC-MS / MS. Subsequently, the water solubility and toxicity of the peptides were predicted using the online prediction tools Peptide Property Calculator and ToxinPred, and the function of the peptides was predicted using the bioinformatics tool Discovery Studio software, which was then molecularly docked with the intestinal epithelial cell calcium transport channel protein TRPV6 and the magnesium transport channel proteins TRPM6 and TRPM7, respectively. The peptide RSAHRQEFPK with a molecular weight of 1522.53 Da that promotes calcium and magnesium absorption was identified. The water solubility, toxicity and molecular docking results of the egg yolk-derived polypeptide are shown in Table 1; the secondary mass spectrum of the egg yolk-derived polypeptide is shown in Table 1. Figure 1 As shown; the interaction between the yolk-derived polypeptide and TRPV6, TRPM6 and TRPM7 proteins is shown Figure 2 shown.

[0040] Table 1 Water solubility, toxicity and molecular docking results of egg yolk peptides

[0041]

[0042] Example 2 Determination of in vitro activity of egg yolk derived polypeptides

[0043] The egg yolk-derived peptide RSAHRQEFPK was prepared by solid phase synthesis and its calcium and magnesium chelating activities were evaluated. The specific process is as follows:

[0044] Take 2 mg of egg yolk polypeptide and dissolve it in 10 mL of water, add 2.0 mL of CaCl2 (5 mmol / L), and chelate at pH 7.5 and 70°C for 2 hours. After the chelation reaction is completed, add 40 mL of anhydrous ethanol to precipitate the polypeptide, centrifuge at 8000 r / min for 15 minutes to obtain the supernatant, take 1 mL of the supernatant, dry it to remove the ethanol, and then adjust the volume to 200 mL. The unchelated calcium content was determined by flame atomic absorption, and the calcium chelation rate of the egg yolk polypeptide was finally determined to be 89.92% (such as Figure 3 ).

[0045] Take 2mg of egg yolk polypeptide and dissolve it in 10mL of water, add 3.3mL of MgCl2 (5mmol / L), and chelate at pH 7.5 and 70℃ for 2h. After the chelation reaction, add 40mL of anhydrous ethanol to precipitate the polypeptide, centrifuge at 8000r / min for 15min to obtain the supernatant, take 1mL of the supernatant, dry it to remove the ethanol, and then make the volume to 200mL. The unchelated magnesium content was determined by flame atomic absorption, and the magnesium chelation rate of the egg yolk polypeptide was finally determined to be 71.44% (such as Figure 3 ).

[0046] Example 3 Determination of cytotoxicity and calcium and magnesium absorption promoting activity of egg yolk derived polypeptide

[0047] 1. Cytotoxicity assay

[0048] The passaged Caco-2 cells (human cloned colon adenocarcinoma cells, similar in structure and function to differentiated intestinal epithelial cells) were cultured to the logarithmic growth phase, and 100 μL of egg yolk-derived polypeptide solution (0-800 μg / mL) was added to treat the cells. The CCK8 kit (purchased from Biyuntian Biotechnology Co., Ltd.) was used to measure the cell activity after 24 hours of polypeptide solution treatment. The results are as follows: Figure 4 As shown, the cell activity of the polypeptide can be maintained at more than 90% within the concentration range of 0-800 μg / mL, indicating that the egg yolk polypeptide has no toxic effect on cells and is safe and reliable.

[0049] 2. Experiment on promoting calcium and magnesium absorption activity

[0050] Caco-2 cells were inoculated onto TransWellTW plates to construct Caco-2 cell monolayer models. The Caco-2 cell monolayer models were divided into control group CaCl2, control group MgCl2, peptide calcium group, peptide magnesium group, and peptide mixed group. The basal cell culture medium formula was DMEM containing 20% ​​fetal bovine serum (FBS) and 1% double antibody. The control group CaCl2 and the control group MgCl2 added 270μg / mL CaCl2 and 200μg / mL MgCl2 to the basal cell culture medium, respectively; the peptide calcium group added 100μg / mL egg yolk polypeptide and 270μg / mL CaCl2 to the basal cell culture medium; the peptide magnesium group added 100μg / mL egg yolk polypeptide and 200μg / mL MgCl2 to the basal cell culture medium; the peptide mixed group added 100μg / mL egg yolk polypeptide, 270μg / mL CaCl2, and 200μg / mL MgCl2 to the basal cell culture medium.

[0051] After treating Caco-2 cells with different cell culture media for 3 hours, the culture media on the permeable side were taken to measure the calcium and magnesium contents in the culture media. Figure 5 As shown, the calcium transport amount (peptide calcium group) and magnesium transport amount (peptide magnesium group) of the egg yolk polypeptide were significantly higher than those of the control group (control group CaCl2, control group MgCl2); in the peptide mixed group, the calcium transport amount of the egg yolk polypeptide was significantly higher than that of the peptide calcium group, and the magnesium transport amount was not significantly different from that of the peptide magnesium group. This shows that the egg yolk polypeptide has the effect of promoting the cell absorption and transport of calcium and magnesium; in the presence of calcium and magnesium at the same time, the egg yolk polypeptide can further improve the cell absorption and transport of calcium, showing a superior synergistic absorption-promoting effect.

[0052] In summary, RSAHRQEFPK can synergistically promote the transport of calcium and magnesium into the small intestinal epithelial cells, has good calcium and magnesium absorption and transport activity, and can be used to promote the absorption of calcium and magnesium in the human body.

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An egg yolk-derived polypeptide having the effect of promoting calcium and magnesium absorption, characterized in that: The amino acid sequence of the egg yolk-derived polypeptide is shown in SEQ ID NO.

1.

2. Use of the egg yolk-derived polypeptide according to claim 1 in the preparation of a calcium and magnesium supplement.

3. A calcium and magnesium supplement, characterized in that: The calcium and magnesium supplement comprises the egg yolk-derived polypeptide according to claim 1.

4. The calcium and magnesium supplement according to claim 3, characterized in that The calcium and magnesium supplement also includes a pharmaceutically acceptable carrier or auxiliary material.

5. The calcium and magnesium supplement according to claim 3, characterized in that The calcium-magnesium supplement has the function of synergistically promoting the absorption and transportation of calcium and magnesium elements by the human body.

6. The calcium and magnesium supplement according to claim 5, characterized in that The synergistic promotion of the absorption and transportation of calcium and magnesium by the human body refers to increasing the absorption and transportation amount of calcium under the action of magnesium and maintaining the absorption and transportation amount of magnesium.

7. The calcium and magnesium supplement according to claim 3, characterized in that The dosage forms of the calcium and magnesium supplement include tablets, granules, mixtures and capsules.

8. Use of the egg yolk-derived polypeptide according to claim 1 in the preparation of a calcium-magnesium chelating agent.

9. A calcium-magnesium chelating agent, characterized in that: The calcium-magnesium chelator comprises the egg yolk-derived polypeptide according to claim 1.

10. The calcium-magnesium chelating agent according to claim 9, characterized in that The calcium-magnesium chelating agent has the function of chelating calcium ions and magnesium ions in the environment.

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