Egg yolk-derived polypeptide capable of promoting calcium and magnesium absorption and its application

Abstract

The present invention discloses an egg yolk-derived polypeptide having the effect of promoting calcium and magnesium absorption and its application, and belongs to the field of biotechnology. The amino acid sequence of the egg yolk-derived polypeptide of the present invention is shown in SEQ ID NO.1, specifically Arg-Ser-Ala-His-Arg-Gln-Glu-Phe-Pro-Lys. The present invention screens out an egg yolk-derived polypeptide from egg yolk hydrolysate, and the egg yolk-derived polypeptide can promote cell 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. It can also chelate calcium and magnesium ions, providing a new material resource for the research and development of calcium and magnesium chelators.

Description

Technical Field

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

[0002] Calcium and magnesium are essential minerals for the human body. Calcium's primary function in the body is to support bone and tooth health and participate in physiological processes such as blood coagulation, muscle contraction, and nerve conduction. Magnesium catalyzes over 300 enzyme reactions, playing a particularly important role in energy metabolism, protein synthesis, nerve signaling, and muscle relaxation. A balanced balance of calcium and magnesium is crucial for maintaining bone health and normal neuromuscular function. The two not only work together during bone mineralization but also interact to regulate muscle contraction and relaxation during neuromuscular activity.

[0003] Inadequate calcium and magnesium intake is a common problem among modern people, and this phenomenon is particularly prominent among special populations such as the elderly, pregnant women, and nursing mothers. Studies have shown that the bioavailability of calcium and magnesium is low, and both intake and absorption rates are affected by a variety of factors, including the presence of anti-nutrients in food, intestinal health, and vitamin D levels. Chronic calcium deficiency can lead to low blood calcium levels and increased parathyroid hormone secretion, which in turn causes bones to release calcium into the bloodstream, leading to gradual bone brittleness. Magnesium deficiency can exacerbate osteoporosis and affect heart function, potentially causing arrhythmias, muscle spasms, and increased nerve excitability. Maintaining a healthy balance between calcium and magnesium is also crucial. Magnesium deficiency disrupts calcium metabolism, increases neuromuscular excitability, and can lead to neurological abnormalities. A variety of calcium and magnesium supplements are currently available on the market, including calcium preparations such as calcium carbonate, calcium lactate, and calcium citrate, and magnesium preparations such as magnesium sulfate, magnesium chloride, and magnesium citrate. These supplements can alleviate calcium and magnesium deficiencies to a certain extent. However, these conventional supplements not only suffer from low absorption efficiency and gastrointestinal discomfort, but also struggle to ensure balanced absorption of calcium and magnesium due to their single-ingredient nature. This can easily lead to health problems caused by an imbalanced calcium and magnesium intake, resulting in reduced bioavailability of the individual elements in the human body. Therefore, developing a novel bioactive substance that ensures a synergistic effect of calcium and magnesium is an important direction in current nutritional research.

[0004] In recent years, bioactive peptides have garnered widespread attention as a novel form of mineral supplementation. Bioactive peptides are small peptide fragments produced by enzymatic hydrolysis of proteins and exhibit a variety of biological functions, including enhanced mineral absorption, antioxidant activity, and immunomodulatory properties. Studies have found that bioactive peptides derived from natural foods such as eggs, milk, and soybeans can bind to minerals to form stable chelates, enhancing their absorption in the intestine. These peptides not only effectively prevent free minerals from forming insoluble precipitates in the intestine but also serve as bridges between minerals and ion channels, promoting mineral absorption. Poultry eggs, as a renewable biological resource, provide an economical and sustainable raw material for the production of bioactive peptides. Egg yolks contain a variety of high-quality proteins and are an important source of high-quality bioactive peptides. Therefore, developing highly active, side-effect-free, and low-cost egg yolk-derived peptides that can synergize calcium and magnesium absorption is of great significance. Summary of the Invention

[0005] The present invention aims to provide an egg yolk-derived polypeptide that promotes calcium and magnesium absorption, and its use, to address the problems of the prior art. The present invention screens an egg yolk-derived polypeptide from egg yolk hydrolysate. This egg yolk-derived polypeptide can promote cellular absorption and transport of calcium and magnesium, providing a new auxiliary means for promoting calcium and magnesium absorption in the human body. It can be used to develop new calcium and magnesium supplements and can also chelate calcium and magnesium ions, providing a new material resource for the 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-derived polypeptide in the preparation of a calcium and magnesium supplement.

[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-magnesium supplement has the effect of synergistically promoting the absorption and transportation of calcium and magnesium elements by the human body.

[0012] Furthermore, the synergistic promotion of the absorption and transport of calcium and magnesium by the human body refers to increasing the absorption and transport amount of calcium under the action of magnesium and maintaining the absorption and transport 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-derived polypeptide in the preparation of a calcium-magnesium chelating agent.

[0015] The present invention also provides a calcium-magnesium chelating agent, which contains the 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 simultaneously promotes the absorption and transport of calcium and magnesium from egg yolks. The present invention adopted physical and chemical activity and cell models to evaluate its calcium and magnesium absorption-promoting function. The results showed that the egg yolk-derived polypeptide has an excellent chelating effect on calcium and magnesium ions, and also has the effect of promoting cellular absorption and transport of calcium and magnesium elements. It shows 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 to develop 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0020] Figure 1 This is the MS / MS identification image of the egg yolk-derived polypeptide RSAHRQEFPK;

[0021] Figure 2 Interaction diagram of the yolk-derived peptide RSAHRQEFPK with 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-derived polypeptide RSAHRQEFPK;

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

[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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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 solutions of the present invention are as follows:

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

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

[0033] 1. Take the yolk of a fresh egg, collect the yolk liquid, dilute the 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 yolk particles.

[0034] 2. Prepare the egg yolk particles obtained in step 1 into a 1-5 mg / mL solution, then perform ultrasonic homogenization (power: 400-600 W; time: 10-15 min). After homogenization, add trypsin for enzymatic hydrolysis (enzymatic hydrolysis temperature: 30-40°C; enzymatic hydrolysis time: 3-8 h) to obtain egg yolk enzymatic peptide fragments.

[0035] Example 1 Preparation of Egg Yolk-derived Polypeptide

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

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

[0038] 2. Mix the egg yolk particles obtained in step 1 with water to prepare a 4 mg / mL solution, 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 peptide fragments.

[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 peptide were predicted using the online prediction tools Peptide Property Calculator and ToxinPred, and the function of the peptide was predicted using the bioinformatics tool Discovery Studio software, and molecular docking was performed with the small intestinal epithelial cell calcium transport channel protein TRPV6 and magnesium transport channel proteins TRPM6 and TRPM7, respectively. The polypeptide 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 The interaction between the yolk-derived polypeptide and TRPV6, TRPM6 and TRPM7 proteins is shown in 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] 2 mg of egg yolk-derived polypeptide was dissolved in 10 mL of water, and 2.0 mL of CaCl2 (5 mmol / L) was added. The mixture was chelated at pH 7.5 and 70°C for 2 h. After the chelation reaction, 40 mL of anhydrous ethanol was added to precipitate the polypeptide. The supernatant was obtained by centrifugation at 8000 r / min for 15 min. 1 mL of the supernatant was dried to remove the ethanol and the volume was adjusted to 200 mL. The unchelated calcium content was determined by flame atomic absorption spectroscopy, and the calcium chelation rate of the egg yolk-derived polypeptide was finally determined to be 89.92% (as shown in Figure 2). Figure 3 ).

[0045] 2 mg of egg yolk polypeptide was dissolved in 10 mL of water, 3.3 mL of MgCl2 (5 mmol / L) was added, and the mixture was chelated at pH 7.5 and 70°C for 2 h. After the chelation reaction, 40 mL of anhydrous ethanol was added to precipitate the polypeptide, and the supernatant was obtained by centrifugation at 8000 r / min for 15 min. 1 mL of the supernatant was dried to remove the ethanol and the volume was adjusted to 200 mL. The unchelated magnesium content was determined by flame atomic absorption spectroscopy, and the magnesium chelation rate of the egg yolk polypeptide was finally determined to be 71.44% (as shown in Figure 2). Figure 3 ).

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

[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 phase and treated with 100 μL of egg yolk-derived peptide solution at different concentrations (0-800 μg / mL). The cell viability after 24 hours of peptide solution treatment was measured using the CCK8 kit (purchased from Biyuntian Biotechnology Co., Ltd.). The results are as follows: Figure 4 As shown, the cell activity of the polypeptide can be maintained above 90% within the concentration range of 0-800 μg / mL, indicating that the egg yolk-derived polypeptide has no toxic effect on cells and is safe and reliable.

[0049] 2. Calcium and magnesium absorption activity test

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

[0051] After treating Caco-2 cells with different cell culture media for 3 hours, the culture medium on the permeable side was taken and the calcium and magnesium contents in the culture medium were measured. Figure 5 As shown, the calcium transport capacity (peptide calcium group) and magnesium transport capacity (peptide magnesium group) of the egg yolk-derived polypeptide were significantly higher than those of the control group (control group CaCl2, control group MgCl2); in the peptide mixed group, the calcium transport capacity of the egg yolk-derived polypeptide was significantly higher than that of the peptide calcium group, and the magnesium transport capacity was not significantly different from that of the peptide magnesium group. This shows that the egg yolk-derived polypeptide has the effect of promoting the absorption and transport of calcium and magnesium by cells; in the presence of calcium and magnesium at the same time, the egg yolk-derived polypeptide can further improve the absorption and transport of calcium by cells, showing an excellent 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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 preparation for promoting calcium and magnesium absorption.

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

4. The preparation for promoting calcium and magnesium absorption according to claim 3, characterized in that The preparation for promoting calcium and magnesium absorption also includes pharmaceutically acceptable carriers or excipients.

5. The preparation for promoting calcium and magnesium absorption according to claim 3, characterized in that The dosage forms of the preparation for promoting calcium and magnesium absorption include tablets, granules, mixtures and capsules.

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

Images