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

Abstract

This invention discloses egg-derived calcium absorption-promoting peptides, their preparation methods, and applications, belonging to the field of peptide technology. The egg-derived calcium absorption-promoting peptides include egg yolk high-phosphorus protein-derived peptides or shell membrane-derived peptides; the amino acid sequence of the egg yolk high-phosphorus protein-derived peptide is LSKIWGR, and the amino acid sequence of the shell membrane-derived peptide is GPAGFPGAP. This invention is the first to screen and identify two egg-derived peptides, LSKIWGR and GPAGFPGAP, and evaluates their calcium absorption-promoting function using physicochemical activity combined with cell models. It was found that these two peptides not only bind efficiently to calcium ions but also have a certain ability to enhance the transmembrane transport activity of calcium, thus promoting calcium ion absorption. Both egg-derived peptides provided by this invention have the potential to be developed into calcium supplements and / or calcium absorption-promoting products.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to egg-derived calcium absorption-promoting polypeptides, their preparation methods, and applications. Background Technology

[0002] Calcium is an essential mineral for the human body, playing a vital role in bone development, blood clotting, nerve signal transduction, and enzyme activation. Adequate calcium intake is crucial for maintaining calcium balance and bone density, especially for developing adolescents, postmenopausal women, and the elderly. However, insufficient dietary calcium intake is common due to dietary habits or low calcium content in food. Severe calcium deficiency can lead to rickets, osteoporosis, and even osteoporosis, significantly harming health. Therefore, calcium supplements are often used to prevent or treat these diseases. Currently, the mainstream calcium supplements are inorganic and organic calcium. Inorganic calcium is mainly represented by calcium carbonate, while organic calcium mainly refers to calcium gluconate and calcium citrate. After ingestion, inorganic or organic calcium releases calcium ions in the low pH environment of the stomach. These ions are then transported to the intestinal environment and absorbed, achieving the purpose of calcium supplementation. However, studies have shown that these two calcium supplements are not very effective. This may be because the content of soluble calcium in the intestines is affected by other substances such as phytic acid and oxalic acid. These substances can combine with soluble calcium to form insoluble calcium, thereby inhibiting calcium absorption and weakening the calcium supplementation effect of inorganic or organic calcium. Furthermore, studies have found that long-term intake of inorganic calcium can burden the gastrointestinal tract and easily cause constipation, affecting overall health.

[0003] To address these issues, next-generation peptide-calcium supplements are gaining market favor. Peptides utilize their chelating effect to prepare peptide-calcium chelates, thereby increasing the content of soluble calcium in the intestine, promoting calcium absorption, and improving calcium bioavailability. Furthermore, food-derived peptides offer advantages such as abundant sources, high biocompatibility, and safety, making them promising candidates for the development and preparation of peptide-calcium supplements. Currently, researchers have identified and prepared peptides with calcium absorption-enhancing activity from raw materials such as milk, soybeans, and fish products, greatly enriching the application value of peptides and promoting the development of the calcium supplement market.

[0004] Poultry eggs are rich in high-quality protein and are a source of bioactive peptides. Egg-derived peptides possess functional activities such as antibacterial, antioxidant, mineral chelation, and absorption-promoting effects. Furthermore, the shell membrane, a component of the eggshell, is a protein network structure mainly composed of keratin and collagen. In actual poultry egg production, shell membranes are often discarded without being utilized, which is not only a serious waste of resources but also pollutes the environment. Therefore, to promote the development of the poultry egg industry, improve the high-value utilization of egg protein, and meet the market demand for novel and efficient calcium supplements, it is necessary to develop egg-derived peptides with calcium chelation activity and calcium absorption-promoting effects. Summary of the Invention

[0005] The purpose of this invention is to provide egg-derived calcium absorption-promoting peptides, their preparation methods, and applications, in order to solve the problems existing in the prior art. This invention has for the first time screened and identified two egg-derived peptides, LSKIWGR and GPAGFPGAP, both of which have the potential to be developed into calcium supplements and / or calcium absorption-promoting products.

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

[0007] This invention provides egg-derived calcium absorption-promoting peptides, which include egg yolk high-phosphorus protein-derived peptides or shell membrane-derived peptides;

[0008] The amino acid sequence of the egg yolk high-phosphorus protein-derived polypeptide is LSKIWGR, and the amino acid sequence of the shell membrane-derived polypeptide is GPAGFPGAP.

[0009] This invention also provides a method for preparing the aforementioned egg-derived calcium absorption-promoting peptide, comprising the following steps:

[0010] Extraction and preparation of egg yolk high-phosphoprotein phosphopeptides and shell membrane polypeptides;

[0011] The egg yolk high-phosphoprotein phosphopeptide and shell membrane polypeptide were respectively subjected to molecular simulation docking with key calcium absorption receptors, and the egg yolk high-phosphoprotein-derived polypeptide LSKIWGR and shell membrane-derived polypeptide GPAGFPGAP were screened and identified.

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

[0013] Furthermore, the egg yolk high-phosphoprotein phosphopeptide is prepared by the following method:

[0014] Egg yolks are diluted with water and centrifuged to obtain egg yolk particles. The egg yolk particles are then salted out to obtain egg yolk high-phosphorus protein.

[0015] Egg yolk high-phosphoprotein solution was prepared, pretreated at 121℃ for 30 min, and then trypsin was added for enzymatic hydrolysis to obtain egg yolk high-phosphoprotein phosphopeptides.

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

[0017] Furthermore, the shell membrane polypeptide is prepared by extraction using the following method:

[0018] Eggshells are crushed to obtain eggshell powder, and shell membranes are obtained using a moisture method.

[0019] Take the shell membrane and add it to water, then add alkaline protease to hydrolyze it to obtain shell membrane polypeptides.

[0020] Further, the water method includes: placing eggshell powder in water at a mass ratio of 9%-12%, stirring at 1500-3000 r / min to separate the shell membrane from the outer crystals, and obtaining 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℃ for 4-9h.

[0021] The present invention also provides the application of the egg-derived calcium absorption-promoting peptide in the preparation of calcium supplements and / or products that promote calcium absorption.

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

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

[0024] This invention identifies two egg-derived peptides, LSKIWGR and GPAGFPGAP, for the first time. Their calcium absorption-promoting function was evaluated using a combination of physicochemical activity and cell models. The results showed that these two peptides not only bind efficiently to calcium ions but also enhance the transmembrane transport of calcium, thus promoting calcium absorption. Both egg-derived peptides provided by this invention have the potential to be developed into calcium supplements and / or products that promote calcium absorption. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[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 peptides LSKIWGR and GPAGFPGAP was evaluated.

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

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

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0034] Example 1: Preparation and Screening of Polypeptides

[0035] This invention employs a protease digestion identification approach for screening target peptides.

[0036] 1. Extraction and preparation of egg yolk high-phosphoprotein phosphopeptides

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

[0038] A 2 mg / mL solution of egg yolk high-phosphoprotein was accurately prepared, followed by pretreatment at 121 °C for 30 min. After pretreatment, trypsin was added, and the solution was enzymatically hydrolyzed at 37 °C for 6 h to obtain egg yolk high-phosphoprotein phosphopeptides.

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

[0040] Collect fresh eggshells and rinse them with clean water to remove any egg white residue. Crush the clean eggshells to obtain eggshell powder. Obtain the shell membrane using a water method: place 12% (m / m) eggshell powder in water and stir at 2500 r / min to separate the shell membrane from the outer crystals, thus obtaining the shell membrane.

[0041] Weigh 5g of shell membrane and add it to pure water at a material-to-liquid ratio of 1g:20mL. Then add alkaline protease and enzymatically hydrolyze at 55℃ for 9h to obtain shell membrane polypeptide.

[0042] 3. Screening and identification of target peptides

[0043] The egg yolk high-phosphoprotein phosphopeptides and shell membrane polypeptides obtained above were screened and identified.

[0044] Egg yolk high-phosphoprotein phosphopeptide and shell membrane polypeptide were prepared into polypeptide solutions, identified by HPLC-MS / MS, and analyzed by bioinformatics. The peptides were functionally predicted using the online peptide property calculator, and molecular simulation docking with the key calcium absorption receptor (TRPV6) was performed using Autodock 1.5.7 software. The highly active peptide LSKIWGR (Leu-Ser-Lys-Ile-Trp-Gly-Arg) from egg yolk high-phosphoprotein and the shell membrane polypeptide GPAGFPGAP (Gly-Pro-Ala-Gly-Phe-Pro-Gly-Ala-Pro) from shell membrane polypeptide were screened and identified. The molecular docking results of the peptides are shown in Table 1, and the interactions between the peptides and protein receptors are as follows: Figure 1 As shown.

[0045] Table 1 Molecular docking results

[0046]

[0047] Example 2: Determination of the in vitro activity of active peptides

[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 a 2 mg / mL peptide solution was prepared, and 1 mL of 0.1 mol / L CaCl2 solution was added. The pH was adjusted to 7.0, and chelation was carried out at room temperature for 1 h. After the chelation reaction, the peptide was precipitated with anhydrous ethanol, and the supernatant was obtained by centrifugation at 1000 r / min. The content of unchelated calcium was determined by flame atomic absorption spectrometry. The egg yolk high-phosphoprotein phosphopeptide group served as control group 1, and the shell membrane peptide group served as control group 2. The results are as follows: Figure 2As shown, the final calcium chelation rates of the two peptides were 91.42% (LSKIWGR) and 98.11% (GPAGFPGAP), which were significantly higher than those of the two control groups (the calcium chelation rate of the egg yolk high phosphoprotein phosphopeptide group was 63.21%, and the calcium chelation rate of the shell membrane peptide group was 64.35%).

[0050] Example 3: Determination of the calcium transport-promoting activity of active peptides

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

[0052] Caco-2 cells were seeded into TransWell cells TW The cells were placed on a plate, and their transmembrane resistance was measured using a cell osmometer. When the resistance was greater than 300 Ω × cm⁻¹... 2 This indicates that the Caco-2 cell monolayer membrane model has been established and can be used for calcium transport experiments.

[0053] After treating Caco-2 cells with a medium containing 100 μg / mL LSKIWGR and GPAGFPGAP for 3 h, the permeate surface of the medium was collected, and the calcium content in the medium was determined by atomic absorption spectrometry. Simultaneously, a yolk high-phosphoprotein phosphopeptide group was set up as control group 1, and a shell membrane polypeptide group was set up as control group 2. Results are as follows: Figure 3 As shown, the calcium transport capacity of the LSKIWGR and GPAGFPGAP groups was 10.55 μg / well and 10.58 μg / well, respectively, which was significantly higher than that of the two control groups (9.19 μg / well and 9.51 μg / well).

[0054] Based on the above results, both LSKIWGR and GPAGFPGAP possess the activity of promoting calcium absorption and have the potential to be applied to the development and preparation of calcium supplements.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An egg-derived calcium absorption-enhancing polypeptide, characterized in that, The egg-derived calcium absorption-promoting polypeptide is a high-phosphorus protein-derived polypeptide from egg yolk or a shell membrane-derived polypeptide. The amino acid sequence of the egg yolk high-phosphorus protein-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-enhancing polypeptide as described in claim 1, characterized in that, Includes the following steps: Extraction and preparation of egg yolk high-phosphoprotein phosphopeptides and shell membrane polypeptides; Functional predictions were performed on yolk high-phosphoprotein phosphopeptide and shell membrane polypeptide, respectively. Molecular simulation docking was performed between yolk high-phosphoprotein phosphopeptide and shell membrane polypeptide and key calcium absorption receptor, respectively, and yolk high-phosphoprotein-derived polypeptide LSKIWGR and shell membrane-derived polypeptide GPAGFPGAP were screened and identified. The egg yolk high-phosphorus protein phosphopeptide was extracted and prepared by the following method: egg yolk was diluted with water and centrifuged to obtain egg yolk particles, and the egg yolk particles were salted out to obtain egg yolk high-phosphorus protein; an egg yolk high-phosphorus protein solution was prepared, pretreated at 121℃ for 30 min, and then trypsin was added for enzymatic hydrolysis to obtain egg yolk high-phosphorus protein phosphopeptide. The shell membrane polypeptide was extracted and prepared by the following method: eggshells were crushed to obtain eggshell powder, and shell membranes were obtained by water method; the shell membranes were added to water, and alkaline protease was added for enzymatic hydrolysis to obtain shell membrane polypeptides. The key receptor for calcium absorption is TRPV6.

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

4. The preparation method according to claim 2, characterized in that, The water method includes: placing eggshell powder in water at a mass ratio of 9%-12%, stirring at 1500-3000 r / min to separate the shell membrane from the outer crystals, and obtaining the shell membrane; The ratio of the membrane to the water is 1g:(15-20)mL; The enzymatic hydrolysis conditions are: 55℃ for 4-9 hours.

5. The use of the egg-derived calcium absorption-promoting polypeptide as described in claim 1 in the preparation of calcium supplements and / or products that promote calcium absorption.

6. A product for supplementing calcium and / or promoting calcium absorption, characterized in that, It contains the egg-derived calcium absorption-promoting polypeptide as described in claim 1.

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