Egg-derived calcium absorption promoting polypeptide as well as preparation method and application thereof

Abstract

The invention discloses an egg source calcium absorption promoting polypeptide as well as a preparation method and application thereof, and belongs to the technical field of polypeptides. The egg source calcium absorption promoting polypeptide comprises phosvitin source polypeptide or shell membrane source polypeptide; the amino acid sequence of the phosvitin source polypeptide is LSKIWGR, and the amino acid sequence of the shell membrane source polypeptide is GPAGFPGAP. Two egg source polypeptides LSKIWGR and GPAGFPGAP are screened and identified for the first time, a physicochemical activity binding cell model is adopted for evaluating the calcium absorption promoting function of the polypeptides, and it is found that the two polypeptides not only can be efficiently bound with calcium ions, but also have certain capacity of improving the transcellular membrane transport activity of calcium elements, and can promote absorption of the calcium ions. The two egg source polypeptides provided by the invention have the potential of being developed and prepared into related products for supplementing calcium and / or promoting calcium absorption.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, in particular to an egg-derived polypeptide promoting calcium absorption and a preparation method and application thereof. Background Art

[0002] Calcium is an essential mineral for the human body and plays an important role in bone development, blood coagulation, nerve signaling and enzyme activation. Adequate calcium intake is essential for maintaining calcium balance and bone density in the body, especially for developing adolescents, postmenopausal women and the elderly. However, insufficient dietary calcium intake is common due to eating habits or low calcium content in food. Severe calcium deficiency can lead to rickets, osteoporosis and even bone loss, greatly damaging the health of the body. Therefore, calcium supplements are often used to prevent or treat such diseases. At present, the mainstream calcium supplements are inorganic calcium and organic calcium, among which inorganic calcium is mainly represented by calcium carbonate, and organic calcium mainly refers to calcium gluconate and calcium citrate. After ingesting inorganic calcium or organic calcium, calcium ions are released in the low pH gastric environment, and then the calcium ions are transported to the intestinal environment and absorbed to achieve the purpose of calcium supplementation. However, studies have shown that these two calcium supplements are not effective in supplementing calcium. This may be because the content of soluble calcium in the intestine is affected by other substances in the intestine, such as phytic acid and oxalic acid. These substances can react with soluble calcium to form insoluble calcium, thereby inhibiting calcium absorption and weakening the calcium supplementation effect of inorganic or organic calcium. In addition, studies have found that long-term intake of inorganic calcium puts a burden on the gastrointestinal tract of the body and is very likely to cause constipation, affecting the health of the body.

[0003] In order to optimize the above problems, a new generation of peptide-calcium calcium supplements has gradually gained favor in the market. Peptides prepare peptide-calcium chelates by exerting their chelating effect, thereby increasing the soluble calcium content in the intestine, promoting calcium absorption and improving calcium bioavailability. In addition, food-derived peptides have the advantages of rich sources, high biocompatibility and safety, and have broad application prospects in the development and preparation of peptide-calcium supplements. At present, researchers have identified and prepared peptides with calcium absorption activity from raw materials such as milk, soybeans and fish products, which greatly enriched the application value of peptides and promoted the development of the calcium supplement product market.

[0004] Poultry eggs are rich in high-quality protein and are one of the sources of active peptides. Poultry egg-derived peptides have functional activities such as antibacterial, antioxidant, mineral chelation, and promoting its absorption. In addition, the shell membrane is a component of the eggshell of poultry eggs, which is mainly a protein network structure composed of keratin and collagen. In the actual production of the poultry egg industry, the shell membrane is usually not utilized and is directly discarded, which is not only a serious waste of resources, but also pollutes the environment. Therefore, in order to promote the development of the poultry egg industry, improve the high-value utilization of poultry egg protein, and meet the market demand for new and efficient calcium supplements, it is necessary to develop egg-derived peptides with calcium chelation activity and calcium absorption promotion effects. Summary of the invention

[0005] The purpose of the present invention is to provide an egg-derived polypeptide promoting calcium absorption and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention screens and identifies two egg-derived polypeptides, LSKIWGR and GPAGFPGAP, for the first time. Both polypeptides have the potential to be developed into related products for calcium supplementation and / or promoting calcium absorption.

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

[0007] The present invention provides an egg-derived polypeptide for promoting calcium absorption, wherein the egg-derived polypeptide for promoting calcium absorption comprises a polypeptide derived from phosvitin or a polypeptide derived from shell membrane;

[0008] The amino acid sequence of the phosvitin-derived polypeptide is LSKIWGR, and the amino acid sequence of the shell membrane-derived polypeptide is GPAGFPGAP.

[0009] The present invention also provides a method for preparing the egg-derived calcium absorption promoting polypeptide, comprising the following steps:

[0010] Extract and prepare phosvitin phosphopeptide and shell membrane polypeptide;

[0011] The phosvitin phosphopeptide and shell membrane polypeptide were docked with the key calcium absorption receptors by molecular simulation, and the phosvitin-derived polypeptide LSKIWGR and the shell membrane-derived polypeptide GPAGFPGAP were screened and identified.

[0012] Furthermore, the key receptor for calcium absorption includes TRPV6.

[0013] Furthermore, the phosvitin phosphopeptide is extracted and prepared by the following method:

[0014] The egg yolk is diluted with water, and the egg yolk particles are obtained by centrifugation, and the egg yolk particles are subjected to salting out to obtain phosvitin;

[0015] The phosvitin solution was prepared, pretreated at 121°C for 30 min, and then trypsin was added for enzymatic hydrolysis to obtain the phosvitin phosphopeptide.

[0016] Furthermore, the volume ratio of the egg yolk to the water is 1:(2-4); and the conditions for the enzymatic hydrolysis are: enzymatic hydrolysis at 37° C. for 6-8 hours.

[0017] Furthermore, the shell membrane polypeptide is extracted and prepared by the following method:

[0018] The eggshells are crushed to obtain eggshell powder, and the shell membrane is obtained by a moisture method;

[0019] The shell membrane is added into water, and alkaline protease is added for enzymatic hydrolysis to obtain the shell membrane polypeptide.

[0020] Furthermore, the moisture method comprises: placing eggshell powder in water at a mass ratio of 9%-12%, stirring at 1500-3000r / min to separate the shell membrane and the outer shell crystals to obtain the shell membrane; the material-liquid ratio of the shell membrane to the water is 1g: (15-20)mL; the enzymatic hydrolysis conditions are: enzymatic hydrolysis at 55°C for 4-9h.

[0021] The present invention also provides the use of the egg-derived calcium absorption promoting polypeptide in the preparation of products for calcium supplementation and / or promoting calcium absorption.

[0022] The present invention also provides a product for supplementing calcium and / or promoting calcium absorption, comprising the egg-derived polypeptide for promoting calcium absorption.

[0023] The present invention discloses the following technical effects:

[0024] The present invention screened and identified two egg-derived polypeptides, LSKIWGR and GPAGFPGAP, for the first time, and evaluated their calcium absorption promoting functions by combining physical and chemical activities with cell models, and found that the two egg-derived polypeptides can not only efficiently bind to calcium ions, but also have a certain ability to improve the transcellular membrane transport activity of calcium elements, and can promote the absorption of calcium ions. Both egg-derived polypeptides provided by the present invention have the potential to be developed into products related to calcium supplementation and / or calcium absorption promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 The interaction forces between peptides LSKIWGR (A) and GPAGFPGAP (B) and receptor proteins;

[0027] Figure 2 The in vitro calcium chelating activity of the peptides LSKIWGR and GPAGFPGAP;

[0028] Figure 3 The effects of peptides LSKIWGR and GPAGFPGAP on calcium transport in Caco-2 cells. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Preparation and screening of polypeptides

[0035] The present invention adopts the idea of ​​proteolysis identification to screen the target polypeptide.

[0036] 1. Extraction and preparation of phosvitin phosphopeptide

[0037] Fresh egg yolk was used as the raw material. The fresh egg yolk was first diluted with water (the volume ratio of egg yolk to pure water = 1:3), and then centrifuged at 4500r / min for 30min to obtain egg yolk particles. Subsequently, the egg yolk particles were treated with 2mol / L NaCl solution, and the pH of the solution was adjusted to 4.5, and yolk phosphoprotein was obtained by salting out.

[0038] A 2 mg / mL phosvitin solution was accurately prepared and then pretreated at 121°C for 30 min. After the pretreatment, trypsin was added and enzymatically digested at 37°C for 6 h to obtain phosvitin phosphopeptides.

[0039] 2. Extraction and preparation of shell membrane polypeptides

[0040] Collect fresh eggshells and rinse the egg white residue with clean water. Crush the clean eggshells to obtain eggshell powder. Use the water method to obtain the shell membrane: 12% (m / m) eggshell powder is placed in water, stirred at 2500r / min to separate the shell membrane and the outer shell crystals, and obtain the shell membrane.

[0041] 5 g of shell membrane was weighed and added into pure water at a solid-liquid ratio of 1 g:20 mL, and then alkaline protease was added to perform enzymolysis at 55° C. for 9 h to obtain shell membrane polypeptide.

[0042] 3. Screening and identification of target peptides

[0043] The phosvitin phosphopeptide and shell membrane polypeptide obtained above were screened and identified.

[0044] Phosvitin phosphopeptide and shell membrane polypeptide were prepared into polypeptide solutions, identified by HPLC-MS / MS and analyzed by bioinformatics. The peptide property calculator online tool was used to predict the function of the polypeptide, and the Autodock1.5.7 software was used to perform molecular simulation docking with the key calcium absorption receptor (TRPV6). The phosvitin-derived highly active peptide LSKIWGR (Leu-Ser-Lys-Ile-Trp-Gly-Arg) and the shell membrane polypeptide-derived polypeptide GPAGFPGAP (Gly-Pro-Ala-Gly-Phe-Pro-Gly-Ala-Pro) were screened and identified. The molecular docking results of the polypeptides are shown in Table 1, and the interactions between the polypeptides and protein receptors are shown in Table 1. Figure 1 shown.

[0045] Table 1 Molecular docking results

[0046]

[0047] Example 2 Determination of in vitro activity of active polypeptides

[0048] LSKIWGR and GPAGFPGAP were prepared by solid phase chemical synthesis, and the in vitro calcium chelating activity of the peptides was evaluated.

[0049] 10 mL of 2 mg / mL polypeptide solution was prepared, 1 mL of 0.1 mol / L CaCl2 solution was added, and the pH was adjusted to 7.0. Chelation was carried out for 1 h at room temperature. After the chelation reaction, the polypeptide was precipitated with anhydrous ethanol and the supernatant was obtained by centrifugation at 1000 r / min. The unchelated calcium content was determined by flame atomic absorption. At the same time, the phosphoprotein phosphopeptide group was set as control group 1 and the shell membrane polypeptide group was set as control group 2. The results are shown in Figure 2. Figure 2As shown, the calcium chelation rates of the two polypeptides were finally determined to be 91.42% (LSKIWGR) and 98.11% (GPAGFPGAP), which were significantly higher than those of the two control groups (the calcium chelation rate of the phosvitin phosphopeptide group was 63.21%, and the calcium chelation rate of the shell membrane polypeptide group was 64.35%).

[0050] Example 3: Determination of calcium transport activity of active polypeptides

[0051] A Caco-2 monolayer cell model was constructed and the calcium absorption promoting activity of LSKIWGR and GPAGFPGAP was evaluated based on this cell model.

[0052] Caco-2 cells seeded into TransWell TW The cell resistance meter was used to measure the transmembrane resistance of the cells. 2 This indicated that the Caco-2 cell monolayer model had been constructed and could be used for calcium transport experiments.

[0053] After Caco-2 cells were treated with medium containing 100 μg / mL LSKIWGR and GPAGFPGAP for 3 h, the medium on the permeation side was taken and the calcium content in the medium was determined by atomic absorption spectroscopy. At the same time, the phosvitin phosphopeptide group was set as control group 1 and the shell membrane polypeptide group was set as control group 2. Figure 3 As shown, the calcium transport amounts in the LSKIWGR and GPAGFPGAP groups were 10.55 μg / well and 10.58 μg / well, respectively, which were significantly higher than those in the two control groups (9.19 μg / well and 9.51 μg / well).

[0054] Based on the above results, LSKIWGR and GPAGFPGAP both have the activity of promoting calcium absorption and have the potential to be used in the development and preparation of calcium supplement related products.

[0055] 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-derived polypeptide promoting calcium absorption, characterized in that: The egg-derived polypeptide for promoting calcium absorption includes a phosvitin-derived polypeptide or a shell membrane-derived polypeptide; The amino acid sequence of the phosvitin-derived polypeptide is LSKIWGR, and the amino acid sequence of the shell membrane-derived polypeptide is GPAGFPGAP.

2. The method for preparing the egg-derived calcium absorption promoting polypeptide according to claim 1, characterized in that: The following steps are involved: Extract and prepare phosvitin phosphopeptide and shell membrane polypeptide; The phosvitin phosphopeptide and shell membrane polypeptide were docked with the key calcium absorption receptors by molecular simulation, and the phosvitin-derived polypeptide LSKIWGR and the shell membrane-derived polypeptide GPAGFPGAP were screened and identified.

3. The preparation method according to claim 2, characterized in that: The key receptors for calcium absorption include TRPV6.

4. The preparation method according to claim 2, characterized in that: The phosvitin phosphopeptide is extracted and prepared by the following method: The egg yolk is diluted with water, and the egg yolk particles are obtained by centrifugation, and the egg yolk particles are subjected to salting out to obtain phosvitin; The phosvitin solution was prepared, pretreated at 121°C for 30 min, and then trypsin was added for enzymatic hydrolysis to obtain the phosvitin phosphopeptide.

5. The preparation method according to claim 4, characterized in that: The volume ratio of the egg yolk to the water is 1:(2-4); The enzymatic hydrolysis conditions are: 37° C. for 6-8 hours.

6. The preparation method according to claim 2, characterized in that: The shell membrane polypeptide is extracted and prepared by the following method: The eggshells are crushed to obtain eggshell powder, and the shell membrane is obtained by a moisture method; The shell membrane is added into water, and alkaline protease is added for enzymatic hydrolysis to obtain the shell membrane polypeptide.

7. The preparation method according to claim 6, characterized in that: The water method comprises: placing eggshell powder in water at a mass ratio of 9% to 12%, stirring at 1500 to 3000 r / min to separate the shell membrane and the outer crystals of the shell, and obtaining the shell membrane; The material-liquid ratio of the shell membrane to the water is 1 g: (15-20) mL; The enzymatic hydrolysis conditions are: 55° C. for 4-9 hours.

8. Use of the egg-derived calcium absorption promoting polypeptide according to claim 1 in the preparation of products for calcium supplementation and / or promoting calcium absorption.

9. A product for supplementing calcium and / or promoting calcium absorption, characterized in that: The invention comprises the egg-derived calcium absorption promoting polypeptide according to claim 1.

Images