A polypeptide derived from shiitake mushrooms that has calcium-binding capacity and promotes calcium absorption.

Abstract

This invention proposes a polypeptide derived from shiitake mushrooms that possesses calcium ion binding capacity and promotes calcium absorption, relating to the field of biotechnology. Specifically, this invention proposes an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, and / or 4. This polypeptide not only binds efficiently to calcium ions but also exhibits activity that promotes calcium ion absorption. This allows for further development into functional foods such as polypeptide-calcium supplements to meet the demand for novel supplements.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a polypeptide derived from shiitake mushrooms that has calcium ion binding capacity and promotes calcium absorption. Background Technology

[0002] Calcium is the most abundant inorganic element in the human body, forming bones, teeth, and other strong tissues. It also plays a crucial role in regulating hormone secretion, blood pressure, and nerve conduction, making it an indispensable element. However, calcium deficiency is widespread, especially among children during growth and development, pregnant women, the elderly, and postmenopausal women. Severe calcium deficiency can lead to rickets, osteoporosis, and other diseases, significantly harming health and reducing quality of life. Currently, the mainstream calcium supplements on the market are primarily first- and second-generation calcium supplements based on inorganic calcium (calcium carbonate) and organic calcium (calcium gluconate, calcium citrate, etc.). However, these two types of calcium supplements suffer from poor absorption, significant gastrointestinal irritation, and the tendency to form calcium soaps in the small intestine, leading to calcium loss and constipation.

[0003] To avoid the aforementioned problems, a new generation of peptide-calcium supplements is gaining popularity. These supplements utilize the chelation effect of peptides with calcium ions to prepare peptide-calcium conjugates. Because calcium ions are bound to peptides, they are less likely to be bound by substances such as phytic acid in the intestines to form insoluble substances. Simultaneously, the calcium bound to the peptides can directly enter the body through endocytosis by the small intestinal epithelial cells, effectively improving calcium absorption. From a safety perspective, food-derived peptides have the most promising application prospects in the preparation of peptide-calcium supplements and in promoting calcium absorption. Currently, researchers have used raw materials such as milk, eggs, fish, and soybeans to enzymatically prepare food-derived peptides that can bind to calcium ions, developing corresponding peptide-calcium conjugates. They have also found that these food-derived peptides can effectively promote calcium absorption. These peptides have broad market prospects and application value in binding calcium ions, promoting calcium absorption, and developing novel calcium supplements.

[0004] Shiitake mushrooms are a nutritious fungus used in both medicine and food, with a wide cultivation area and large annual yield. Currently, they are mostly sold as dried shiitake mushrooms. Shiitake mushrooms are rich in protein, and the protein in shiitake mushrooms is high in amino acids such as glutamic acid and aspartic acid, which can bind to metal ions. Therefore, they have nutritional advantages for developing calcium-binding peptides and polypeptide-calcium supplements. However, to date, there have been no relevant reports in either the research or market fields.

[0005] Therefore, in order to promote the diversified and functional development of the shiitake mushroom industry and meet people's demand for new calcium supplements, it is necessary to develop shiitake mushroom-derived peptides with calcium ion binding capacity and calcium absorption-promoting effects. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0007] Shiitake mushrooms are a protein-rich plant, and their protein contains high levels of acidic amino acids such as glutamic acid and aspartic acid. These high levels of acidic amino acids endow shiitake peptides with the ability to bind to metal ions. Based on this characteristic, the inventors isolated a peptide from shiitake mushrooms that can efficiently bind to calcium ions through extensive experiments and developed a method for the efficient separation of this peptide. Furthermore, the inventors chelated this peptide with calcium ions to develop a novel calcium supplement—a peptide-calcium supplement. In vitro experiments have shown that, compared to ionic calcium, this calcium supplement promotes the efficient absorption of calcium ions by cells. Therefore, this calcium supplement has broad market prospects and can be widely used for calcium supplementation in the body.

[0008] Based on this, in a first aspect, the present invention provides an isolated polypeptide. According to embodiments of the present invention, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, 2, 3 and / or 4. The polypeptide of the present invention not only binds efficiently to calcium ions but also possesses activity that promotes calcium ion absorption. This allows the polypeptide to be further developed into functional foods such as polypeptide-calcium supplements to meet the demand for novel supplements.

[0009] In a second aspect, the present invention provides a polypeptide-calcium conjugate. According to embodiments of the invention, the polypeptide-calcium conjugate comprises the polypeptide described in the first aspect, and elemental calcium. By binding the polypeptide to calcium ions, the bioavailability of calcium can be improved, and the body's ability to absorb calcium can be enhanced. Therefore, this polypeptide-calcium supplement can serve as an innovative product to meet people's needs for efficient calcium absorption, providing an innovative solution for efficient calcium absorption.

[0010] In a third aspect, the present invention provides the use of the polypeptide described in the first aspect in a preparation kit for chelating calcium ions. The polypeptide, as a chelating agent in the kit, possesses the characteristic of highly efficient binding to calcium ions. This high efficiency ensures the kit's high efficiency and selectivity in the chelation reaction, effectively chelating the target calcium ions and binding them to form a polypeptide-calcium supplement. Therefore, the kit of the present invention has advantages such as ease of use, high efficiency, and wide applicability. This kit-form polypeptide chelating agent provides users with a convenient, reliable, and efficient method for preparing polypeptide-calcium supplements.

[0011] In a fourth aspect, the present invention provides a method for preparing the polypeptide described in the first aspect. According to an embodiment of the present invention, the method includes: extracting protein from shiitake mushrooms to obtain shiitake protein; enzymatically hydrolyzing the shiitake protein to obtain shiitake protein hydrolysate; and ultrafiltration the shiitake protein hydrolysate to obtain the polypeptide. The method described in this invention can separate the polypeptide from other macromolecules while maintaining its activity. This method has undergone detailed step design and optimization, enabling controllable separation and extraction of the polypeptide. This method provides an effective and feasible strategy for extracting and purifying active polypeptides from shiitake mushrooms.

[0012] In a fifth aspect, the present invention provides a polypeptide. According to embodiments of the present invention, the polypeptide is prepared using the method described in the third aspect. The polypeptide prepared by the above method has excellent calcium ion binding capacity and can promote the absorption of calcium ions by cells in vivo and in vitro, providing a good strategy for developing novel polypeptide-calcium supplements.

[0013] In a sixth aspect, the present invention provides a method for preparing the polypeptide-calcium conjugate described in the second aspect. According to an embodiment of the present invention, the method includes: chelating the polypeptide described in the first aspect with a calcium salt to obtain the polypeptide-calcium conjugate. As mentioned above, the polypeptide can bind efficiently to calcium ions. By employing this method, the binding of the polypeptide to calcium ions in the calcium salt can be effectively promoted, forming a stable polypeptide-calcium conjugate. Furthermore, this method has the advantages of being simple to operate, controllable, and highly adjustable.

[0014] In a seventh aspect, the present invention provides a polypeptide-calcium conjugate. According to embodiments of the present invention, the polypeptide-calcium conjugate is prepared using the method described in the fourth aspect. The polypeptide-calcium conjugate prepared by the above method has broad application potential in the food and pharmaceutical fields. As a component of functional foods, the polypeptide-calcium conjugate can provide calcium supplementation and meet people's calcium needs. In the pharmaceutical field, the polypeptide-calcium conjugate can also be used as a calcium supplement, bone density modulator, and other drugs for the treatment of osteoporosis and related diseases.

[0015] In an eighth aspect, the present invention provides a method for promoting the absorption of calcium ions by cells. According to embodiments of the present invention, the method includes: co-incubating cells with a polypeptide described in the first or fourth aspect, or a polypeptide-calcium conjugate described in the second or sixth aspect. As previously mentioned, the polypeptide has a strong calcium ion binding capacity and calcium absorption-promoting activity; therefore, the method described in this invention can promote the absorption of calcium ions by cells.

[0016] In a ninth aspect of the invention, the invention provides for the use of the polypeptide described in the first or fourth aspect, or the polypeptide-calcium conjugate described in the second or sixth aspect, in the preparation of a product having at least one of the following uses: as a calcium supplement; or to promote the absorption of calcium ions in vivo or in vitro cells.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 This is the elution curve of lentinanone chromatographic separation of peptides in Example 1 of the present invention;

[0020] Figure 2 This is a graph showing the calcium ion binding rate of each component after ultrafiltration of shiitake polypeptide in Example 1 of the present invention.

[0021] Figure 3 This is a graph showing the calcium ion binding rate of each component after anion chromatography separation of lentinan polypeptide in Example 1 of the present invention.

[0022] Figure 4 This is the secondary mass spectrum of the lentinan polypeptide Leu-Glu-Val-Glu-IlE-His-Ala (LEVEIHA) obtained in Example 1 of the present invention;

[0023] Figure 5 This is the secondary mass spectrum of the lentinan polypeptide Ala-Tyr-Trp-Glu-Val-Glu-Leu-Glu-Lys (AYWEVELEK) obtained in Example 1 of this invention;

[0024] Figure 6 This is the secondary mass spectrum of the lentinan polypeptide Leu-Thr-IlE-Glu-Glu-Gly-IlE-Phe-Glu (LTIEEGIFE) obtained in Example 1 of the present invention;

[0025] Figure 7 This is the secondary mass spectrum of the lentinan polypeptide Leu-Leu-Arg-IlE-Glu-Glu-Glu-Thr((LLRIEEET)) obtained in Example 1 of the present invention;

[0026] Figure 8 This is a graph showing the binding rates of different polypeptides to calcium ions in Example 2 of the present invention;

[0027] Figure 9The graph shows the calcium ion binding rate of lentinan polypeptides obtained by enzymatic hydrolysis with different proteases in Example 3 of the present invention.

[0028] Figure 10 This is a scanning electron microscope image of the lentinan polypeptide-calcium (LEVEIHA-Ca) prepared in Example 4 of the present invention;

[0029] Figure 11 The elemental energy spectrum of lentinan polypeptide-calcium (LEVEIHA-Ca) prepared in Example 4 of this invention;

[0030] Figure 12 The fluorescence spectrum of lentinan polypeptide-calcium (LEVEIHA-Ca) prepared in Example 4 of this invention;

[0031] Figure 13 This is a diagram showing how shiitake mushroom polypeptide promotes calcium absorption in Caco2 cells in Example 4 of the present invention. Detailed Implementation

[0032] The present invention will be further described below through specific embodiments. It should be noted that the embodiments described below are only for explaining the present invention and are not intended to limit the present invention.

[0033] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] In this application, unless otherwise stated, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this application but do not exclude other contents.

[0035] In this application, unless otherwise stated, the terms “optionally,” “optionally,” or “optionally” generally mean that the event or condition described below may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0036] In this application, unless otherwise stated, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0037] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0038] This invention proposes an isolated polypeptide, its preparation method and uses, a polypeptide-calcium conjugate, its preparation method and uses, and a method for promoting the absorption of calcium ions by cells, which will be described in detail below.

[0039] Polypeptides, their preparation methods and uses

[0040] This invention proposes an isolated polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, and / or 4. The polypeptide of this invention not only binds efficiently to calcium ions but also exhibits activity that promotes calcium ion absorption. This allows the polypeptide to be further developed into functional foods such as polypeptide-calcium supplements to meet the demand for novel supplements.

[0041] LEVEIHA (SEQ ID NO:1)

[0042] AYWEVELEK (SEQ ID NO:2)

[0043] LTIEEGIFE (SEQ ID NO:3)

[0044] LLRIEEET(SEQ ID NO:4)

[0045] This invention proposes the use of the aforementioned peptide in a kit for chelating calcium ions. The peptide, acting as a chelating agent in the kit, exhibits highly efficient binding to calcium ions. This high efficiency ensures the kit's high efficiency and selectivity in the chelation reaction, effectively chelating the target calcium ions and forming a peptide-calcium supplement. Therefore, the kit of this invention offers advantages such as ease of use, high efficiency, and wide applicability. This kit-based peptide chelating agent provides users with a convenient, reliable, and efficient method for preparing peptide-calcium supplements.

[0046] This invention proposes a method for preparing the aforementioned polypeptide. According to an embodiment of the invention, the method includes: extracting protein from shiitake mushrooms to obtain shiitake protein; enzymatically hydrolyzing the shiitake protein to obtain shiitake protein hydrolysate; and ultrafiltration the shiitake protein hydrolysate to obtain the polypeptide. The method described in this invention can separate polypeptides from other macromolecules while maintaining their activity. This method has undergone detailed step design and optimization, enabling controllable separation and extraction of polypeptides. This method provides an effective and feasible strategy for extracting and purifying active polypeptides from shiitake mushrooms.

[0047] According to some embodiments of the present invention, the method may further include at least one of the following additional technical features:

[0048] According to some embodiments of the present invention, the enzymatic hydrolysis is carried out in the presence of a protease.

[0049] According to some embodiments of the present invention, the protease is selected from at least one of neutral protease, alkaline protease, trypsin, pepsin, complex protease, and papain. The inventors have found that adding different proteases for hydrolysis can affect the properties of the final polypeptide, thereby affecting its calcium ion binding efficiency. Specifically, hydrolysis with neutral proteases results in polypeptides with higher calcium ion binding efficiency, followed by trypsin.

[0050] According to some embodiments of the present invention, the protease is selected from neutral proteases. By using neutral proteases for hydrolysis, this method can achieve protein degradation while ensuring the high activity of the polypeptide. Neutral proteases hydrolyze proteins under neutral pH conditions, avoiding potential loss of enzyme activity or destruction of polypeptide structure during hydrolysis. Therefore, the obtained polypeptides retain high activity and biological function.

[0051] According to some embodiments of the present invention, the ultrafiltration treatment includes a first ultrafiltration treatment and a second ultrafiltration treatment. After two ultrafiltration treatments, the obtained peptide can have a high calcium-binding capacity.

[0052] According to some embodiments of the present invention, the first ultrafiltration treatment is carried out in an ultrafiltration membrane of 45 to 55 kDa.

[0053] According to some embodiments of the present invention, the second ultrafiltration treatment is carried out in an ultrafiltration membrane with a density of 1 to 10 kDa.

[0054] According to some embodiments of the present invention, the pressures of the first ultrafiltration treatment and the second ultrafiltration treatment are selected from 0.1 to 0.2 MPa, respectively.

[0055] According to some embodiments of the present invention, the ultrafiltration process further includes purifying the ultrafiltration product to obtain the polypeptide. Purification further enhances the calcium ion binding capacity of the polypeptide.

[0056] According to some embodiments of the present invention, the purification process is carried out by anion exchange chromatography.

[0057] According to some embodiments of the present invention, the packing material of the anion exchange chromatography column is selected from Diamond Q.

[0058] According to some embodiments of the present invention, the eluent used in the anion exchange chromatography is selected from a Tris-HCl solution containing NaCl. After elution with the salt solution, the calcium ion binding rate of lentinan polypeptides is significantly improved, and the calcium ion binding rate of the eluted polypeptide gradually increases with the increase of the ionic strength of the eluent solution.

[0059] According to some embodiments of the present invention, the concentration of NaCl in the Tris-HCl solution is 0 to 1.5 mol / L.

[0060] It should be noted that the "anion exchange chromatography" described in this invention involves sequentially eluting with a Tris-HCl solution containing 0 mol / L NaCl, a Tris-HCl solution containing 0.25 mol / L NaCl, a Tris-HCl solution containing 0.5 mol / L NaCl, and a Tris-HCl solution containing 1 mol / L NaCl, for elution times of 26 min, 49 min, 17 min, and 38 min, respectively. The peptides with the amino acid sequences shown in SEQ ID NO: 1, 2, 3, and 4 are all present in the fraction eluted with the Tris-HCl solution containing 1 mol / L NaCl.

[0061] According to some embodiments of the present invention, the anion exchange chromatography method employs isocratic elution.

[0062] This invention proposes a polypeptide. According to embodiments of the invention, the polypeptide is prepared using the method described above. The polypeptide prepared by the above method exhibits excellent calcium ion binding capacity and can promote the absorption of calcium ions by cells in vivo and in vitro, providing a good strategy for developing novel polypeptide-calcium supplements.

[0063] This invention proposes the use of the aforementioned polypeptide in the preparation of products for promoting the absorption of calcium ions in cells, either in vivo or in vitro.

[0064] According to some embodiments of the present invention, the product is selected from pharmaceuticals, food, or health food.

[0065] According to some embodiments of the present invention, the product further includes pharmaceutically acceptable excipients or carriers, or food or health food acceptable excipients or carriers.

[0066] In this article, "acceptable in food" refers to substances or compositions that are edible for human consumption, which may be adjusted according to the food requirements of different countries.

[0067] In this article, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0068] In this document, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0069] In this document, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof known to those skilled in the art. It covers the use of any conventional carrier in therapeutic or pharmaceutical compositions, except in cases where any conventional carrier is incompatible with the active ingredient.

[0070] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for a particular target dosage form. The use of any conventional excipients is also within the scope of consideration for this disclosure, except for any range of incompatibilities with the polypeptides of this disclosure, such as any adverse biological effects produced or harmful interactions with any other component of the pharmaceutically acceptable composition.

[0071] Polypeptide-calcium conjugates, their preparation methods and uses

[0072] This invention proposes a polypeptide-calcium conjugate, comprising the aforementioned polypeptide and elemental calcium. By binding the polypeptide to calcium ions, the bioavailability of calcium can be improved, and the body's ability to absorb calcium can be enhanced. Therefore, this polypeptide-calcium supplement can serve as an innovative product to meet people's needs for efficient calcium absorption, providing an innovative solution for efficient calcium absorption.

[0073] According to some embodiments of the present invention, the polypeptide-calcium conjugate may further include at least one of the following technical features:

[0074] According to some embodiments of the present invention, the calcium element is provided by a calcium salt.

[0075] According to some embodiments of the present invention, the calcium salt is selected from at least one of calcium gluconate, calcium hydrogen phosphate, calcium lactate, and calcium chloride.

[0076] This invention proposes a method for preparing the aforementioned polypeptide-calcium conjugate. According to an embodiment of the invention, the method includes: chelating the aforementioned polypeptide with a calcium salt to obtain the polypeptide-calcium conjugate. As mentioned earlier, the polypeptide can bind efficiently to calcium ions. By employing this method, the binding of the polypeptide to calcium ions in the calcium salt can be effectively promoted, forming a stable polypeptide-calcium conjugate. Furthermore, this method has the advantages of being simple to operate, controllable, and highly adjustable.

[0077] According to some embodiments of the present invention, the method may further include at least one of the following additional technical features:

[0078] According to some embodiments of the present invention, the polypeptide is provided in solution form.

[0079] According to some embodiments of the present invention, the concentration of the polypeptide in the solution is 0.5 to 1.5 mg / mL, for example 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, and 1.5 mg / mL.

[0080] According to some embodiments of the present invention, the solution is selected from deionized water.

[0081] According to some embodiments of the present invention, the calcium salt is selected from at least one of calcium chloride, calcium gluconate, calcium hydrogen phosphate, and calcium lactate.

[0082] According to some embodiments of the present invention, the mass ratio of the polypeptide to the calcium salt is (1-3):1.

[0083] According to some embodiments of the present invention, the chelation treatment is carried out at 50-60°C for 45-55 minutes.

[0084] According to some embodiments of the present invention, the chelation treatment further includes: evaporating the chelation product; precipitating the evaporation product with alcohol; and centrifuging the alcohol-precipitated product to obtain the polypeptide-calcium conjugate.

[0085] According to some embodiments of the present invention, the evaporation process is carried out using a rotary evaporator.

[0086] According to some embodiments of the present invention, the temperature of the rotary evaporator is 60–70°C.

[0087] According to some embodiments of the present invention, the alcohol precipitation treatment is carried out in the presence of ethanol.

[0088] According to some embodiments of the present invention, the centrifugation process is carried out at a speed of 3500-4500 r / min for a time of 5-15 min.

[0089] This invention proposes a polypeptide-calcium conjugate. According to embodiments of the invention, the polypeptide-calcium conjugate is prepared using the method described above. The polypeptide-calcium conjugate prepared by the above method has broad application potential in the food and pharmaceutical fields. As a component of functional foods, the polypeptide-calcium conjugate can provide calcium supplementation and meet people's calcium needs. In the pharmaceutical field, the polypeptide-calcium conjugate can also be used as a calcium supplement, bone density modulator, and other drugs for the treatment of osteoporosis and related diseases.

[0090] This invention proposes the use of the aforementioned polypeptide-calcium conjugate in the preparation of products having at least one of the following uses: as a calcium supplement; or to promote the absorption of calcium ions in vivo or in vitro cells.

[0091] According to some embodiments of the present invention, the product is selected from pharmaceuticals, food, or health food.

[0092] According to some embodiments of the present invention, the product further includes pharmaceutically acceptable excipients or carriers, or food or health food acceptable excipients or carriers.

[0093] Methods to promote cellular calcium ion absorption

[0094] This invention proposes a method for promoting calcium ion absorption by cells. According to an embodiment of the invention, the method includes co-incubating cells with the aforementioned polypeptide or the aforementioned polypeptide-calcium conjugate. As mentioned above, the polypeptide has a strong calcium ion binding capacity and calcium absorption-promoting activity; therefore, the method described in this invention can promote calcium ion absorption by cells.

[0095] It should be noted that the aforementioned "method for promoting cellular calcium ion absorption" can be applied both in vivo and in vitro. When applied in vivo, it can promote the absorption of calcium ions by cells, thereby increasing the calcium content in the body. This provides an effective strategy for preventing or treating calcium deficiency diseases (such as rickets, osteoporosis, etc.), promoting bone health, increasing bone density, and reducing fracture risk, and has potential clinical application value. When applied in vitro, it can be used for experimental research, such as studying changes in intracellular calcium ion concentration to assess the enhancing effect of peptides or peptide-calcium binders on cellular calcium ion absorption, and studying the activity of specific signaling pathways and changes in the expression of related genes or proteins in cells treated with peptides or peptide-calcium binders.

[0096] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0097] Example 1: Extraction of Lentinan polypeptides

[0098] 1. Shiitake mushroom protein extraction

[0099] Shiitake mushrooms were dried at 50℃ and then pulverized, passing through a 40-mesh sieve to obtain shiitake mushroom powder. The powder was added to deionized water at a liquid-to-solid ratio of 4 mL:1 g, and sonicated at 700 W for 10 min. The pH of the solution was adjusted to 9.5 with 4 mol / L NaOH, and the mixture was extracted by stirring in a water bath at 60℃ for 120 min. Then, it was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The pH of the supernatant was adjusted to 3.5 with 1 mol / L HCl. After standing for 4 h, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was collected and freeze-dried under vacuum for 24 h to obtain shiitake mushroom protein.

[0100] 2. Proteolytic enzyme digestion

[0101] The freeze-dried shiitake mushroom protein was prepared into a 5% (w / w) shiitake mushroom protein solution using deionized water and ultrasonically dispersed at 500 W for 8 min. The pH was adjusted to 7, and neutral protease was added at a ratio of 3% of the shiitake mushroom protein mass. Enzymatic hydrolysis was carried out at 65℃ for 3 h. The enzyme was inactivated by boiling water bath for 10 min, cooled, and centrifuged at 8000 r / min for 10 min. The supernatant was collected and freeze-dried to obtain the shiitake mushroom protein hydrolysate.

[0102] 3. Ultrafiltration separation of shiitake mushroom peptides

[0103] Take the shiitake mushroom protein hydrolysate obtained above, add deionized water to prepare a 2%-8% solution, disperse by ultrasonication, and then ultrafilter with a 50KD ultrafiltration membrane at an ultrafiltration pressure of 0.15Mpa. Collect the filtrate with less than 50KD, and then ultrafilter with a 5KD ultrafiltration membrane to collect the ultrafiltration fraction with less than 5KD. Freeze-dry to obtain shiitake mushroom polypeptide with less than 5KD.

[0104] 4. Separation of lentinan polypeptides by anion exchange chromatography

[0105] Anion exchange chromatography was used to separate and purify the lentinan polypeptide samples smaller than 5KD in step 3. The specific steps are as follows: the chromatography column size is Φ24×200mm, the anion exchange chromatography packing material is Diamond Q strong anion exchange medium, the equilibration buffer is 0.02mol / L Tris-HCl buffer at pH 9.0, the eluent is 0.02mol / L Tris-HCl solution at pH 9.0 containing 0, 0.25, 0.5 and 1mol / L NaCl, respectively, the elution method is isocratic elution (elution times for each eluent are 26 min, 49 min, 17 min and 38 min, respectively), the eluent flow rate is 1-6 mL / min, the sample loading volume is 200-600 mg, the sample solution needs to be filtered through a 0.45 μm filter membrane, the concentration is 1.2-4.0 mg / mL, and the detection wavelength is 220 nm. The fraction collected for 92-130 min (i.e., the fraction eluted with 1 mol / L NaCl eluent) was dialyzed to remove salt and then freeze-dried to obtain lentinan polypeptides with calcium ion binding capacity and calcium absorption promotion effect. The separation and elution curves obtained are shown in the figure. Figure 1 ,Depend on Figure 1 It can be seen that it is divided into F 31 F 32 F 33 and F 34 Four components.

[0106] An evaluation study was conducted on the calcium ion binding rates of each component separated by ultrafiltration and anion exchange chromatography of lentinan polypeptides.

[0107] (1) Evaluation and analysis of calcium ion binding rate of each component separated by ultrafiltration.

[0108] The experimental results are shown in Figure 2 (Where F0 represents peptides that have not been separated by ultrafiltration, F1 represents components larger than 50 kDa, F2 represents components between 5 and 50 kDa, and F3 represents components smaller than 5 kDa), from Figure 2 It was found that molecular weight was significantly related to the calcium ion binding rate of lentinan polypeptides. Among them, the calcium ion binding rate of F3 component with a molecular weight of less than 5 kDa was 90.02%, which was significantly higher than that of other groups (p<0.05).

[0109] (2) Evaluation and analysis of calcium ion binding rate of each component separated by anion exchange chromatography

[0110] The experimental results are shown in Figure 3 .Depend on Figure 3 It can be seen that after elution with salt solution, the calcium ion binding rate of lentinan peptides is significantly increased, and the calcium ion binding rate of the eluted peptides gradually increases with the increase of the ionic strength of the elution solution. Among them, the lentinan peptide F eluted with 1 mol / L NaCl eluent is the highest. 34 The calcium ion concentration was highest in the group and significantly higher than in the other groups (p<0.05).

[0111] (3) For F 34 Components were analyzed and identified by Nano-LC-ESI-MS / MS.

[0112] F obtained by anion exchange chromatography 34 The components were analyzed and identified by Nano-LC-ESI-MS / MS. The amino acid sequences of the lentinan polypeptides obtained were: Leu-Glu-Val-Glu-IlE-His-Ala (LEVEIHA), Ala-Tyr-Trp-Glu-Val-Glu-Leu-Glu-Lys (AYWEVELEK), Leu-Thr-IlE-Glu-Glu-Gly-IlE-Phe-Glu (LTIEEGIFE), and Leu-Leu-Arg-IlE-Glu-Glu-Glu-Thr (LLRIEEET). The secondary mass spectrometry of the LEVIEHA polypeptide and its position in the parent protein are shown below. Figure 4 The secondary mass spectrometry of the AYWEVELEK peptide and its location within its parent protein are shown in [reference needed]. Figure 5 The secondary mass spectrometry of the LTIEEGIFE peptide and its location within its parent protein are shown in [reference needed]. Figure 6 The secondary mass spectra of the LLRIEEET peptide and its location within its parent protein are shown in [reference needed]. Figure 7 .

[0113] Example 2: Binding of different shiitake mushroom polypeptides to calcium ions

[0114] The isolated and identified lentinan polypeptides LEVIEHA, AYWEVELEK, LTIEEGIFE, and LTIEEGIFE were each prepared into 1% solutions using deionized water. Calcium chloride was then added to each polypeptide solution for chelation. The basic conditions for the chelation reaction were: a polypeptide to calcium salt mass ratio of 2:1 (polypeptide:calcium salt), an initial solution pH of 8.0, a chelation time of 50 min, and a temperature of 55℃. After the reaction, half the volume of water was removed using a rotary evaporator (temperature 65℃, rotation speed). Ten times the volume of ethanol was added for precipitation for 2 h. The precipitate was then centrifuged at 4000 r / min for 10 min. The lower precipitate was freeze-dried to obtain lentinan polypeptide chelated calcium. Its mass was weighed, and the calcium content was determined using atomic absorption spectroscopy. The calcium ion binding rate was calculated using the following formula:

[0115] Calcium ion binding rate (%) = 100 × M1 × C / M2

[0116] Where M1 is the mass (g) of lentinan polypeptide chelated calcium; C is the calcium content (mg / g) of the detected lentinan polypeptide chelated calcium; and M2 is the amount of calcium added in the reaction.

[0117] The binding rates of different peptides to calcium are shown in the figure. Figure 8 .Depend on Figure 8 It can be seen that the four polypeptides all have a calcium ion binding capacity of over 85%, with the polypeptide LEVEIHA having the highest calcium ion binding rate.

[0118] Example 3: Experiment to study the effect of different proteases on the calcium ion binding rate of lentinan polypeptides

[0119] The freeze-dried shiitake mushroom protein from Example 1 was used to prepare a 4% (w / w) shiitake mushroom protein solution with deionized water and ultrasonically dispersed at 700 W for 10 min. The pH was adjusted to 7, and different proteases listed in Table 1 were added at a ratio of 3% of the shiitake mushroom protein mass. Enzymatic hydrolysis was carried out for 3 h under optimal conditions (see Table 1). The enzymes were inactivated by boiling in a water bath for 10 min, cooled, and centrifuged at 8000 r / min for 10 min. The supernatant was collected and freeze-dried to obtain the shiitake mushroom protein hydrolysate. The shiitake mushroom protein hydrolysates obtained from the hydrolysis of each protease were prepared into a 2% solution, and calcium chloride was added to chelate the peptides. The basic conditions for the chelation reaction were: a peptide to calcium salt mass ratio of 2:1 (peptide: calcium salt), an initial solution pH of 8.0, a chelation time of 50 min, and a temperature of 55 °C. After the reaction was complete, half the volume of water was removed using a rotary evaporator (temperature 65℃, rotation speed). Ten times the volume of ethanol was added to precipitate the precipitate for 2 hours. The precipitate was then centrifuged at 4000 r / min for 10 minutes. The lower precipitate was freeze-dried to obtain lentinan polypeptide chelated calcium. Its mass was weighed, and the calcium content was determined using atomic absorption spectroscopy. The calcium ion binding rate was calculated using the following formula:

[0120] Calcium ion binding rate (%) = 100 × M1 × C / M2

[0121] Where M1 is the mass (g) of lentinan polypeptide chelated calcium; C is the calcium content (mg / g) of the detected lentinan polypeptide chelated calcium; and M2 is the amount of calcium added in the reaction.

[0122] The effects of different proteases on the calcium binding rate of shiitake peptides are shown in the figure. Figure 9 .Depend on Figure 9 It can be seen that the peptides obtained by hydrolyzing shiitake mushroom protein with different proteases have significant differences in calcium ion binding capacity. Among them, the shiitake mushroom peptides obtained by hydrolysis with neutral protease have the highest calcium binding rate, which is significantly higher than that of the other proteases (p<0.05).

[0123] Table 1. Optimal temperature and pH for each protease

[0124]

[0125]

[0126] Example 4: Preparation of lentinan polypeptide-calcium conjugate (LEVEIHA-Ca)

[0127] The isolated and identified lentinan polypeptide LEVIEHA was prepared into a 1% solution using deionized water. Calcium chloride was added to chelate the polypeptide. The basic conditions for the binding reaction were: a polypeptide to calcium salt mass ratio of 2:1 (polypeptide:calcium salt), an initial solution pH of 8.0, a chelation time of 50 min, and a temperature of 55℃. After the reaction, half the volume of water was removed using a rotary evaporator (temperature 65℃, rotation speed). Ten times the volume of ethanol was added to precipitate the product for 2 h. The product was then centrifuged at 4000 r / min for 10 min, and the lower precipitate was freeze-dried to obtain the lentinan polypeptide-calcium conjugate (LEVEIHA-Ca).

[0128] Characterization of lentinan polypeptide-calcium conjugate (LEVEIHA-Ca)

[0129] (1) Scanning electron microscopy analysis of lentinan polypeptide-calcium conjugate (LEVEIHA-Ca)

[0130] Experimental results are as follows Figure 10 As shown, the lentinan polypeptide LEVIEHA exhibits a relatively loose and porous sheet-like structure (left image). Once it binds with calcium ions, the structure becomes a more compact bird's nest-like structure (right image), indicating that after the intervention of calcium ions, it can interact with the lentinan polypeptide groups to generate a new lentinan polypeptide-calcium conjugate.

[0131] (2) Elemental energy dispersive spectroscopy analysis of lentinan polypeptide-calcium conjugate (LEVEIHA-Ca)

[0132] Experimental results are as follows Figure 11 As shown, the lentinan polypeptide LEVIEHA is mainly composed of C, O and N elements. After reacting with calcium ions, a significant calcium signal appeared in the product, with a calcium content of 3.3%. This result further confirms that the isolated and identified lentinan polypeptide LEVIEHA has a strong calcium ion binding capacity and is a potential polypeptide that can be used to prepare lentinan polypeptide-calcium conjugates.

[0133] (3) Fluorescence spectroscopy analysis of lentinan polypeptide-calcium conjugate (LEVEIHA-Ca)

[0134] The lentinan polypeptide LEVEIHA and lentinan polypeptide-calcium conjugate (LEVEIHA-Ca) were prepared into a solution with a concentration of 0.025 mg / mL using deionized water, and fluorescence spectroscopy was performed under the conditions of excitation wavelength of 210 nm and emission wavelength of 200-500 nm.

[0135] Experimental results are as follows Figure 12 As shown, the lentinan peptide LEVIEHA exhibits a maximum absorption peak at 250 nm, but the absorption peak intensity significantly decreases after interaction with calcium ions. This result indicates that the introduction of calcium ions can induce conformational folding in the lentinan peptide, resulting in a more compact peptide structure and inducing fluorescence quenching in LEVIEHA. This result further demonstrates that the isolated and identified lentinan peptide LEVIEHA can effectively bind to calcium ions, making it an excellent food-derived calcium-binding peptide.

[0136] (4) Analysis of the cellular absorption effect of lentinan polypeptide-Ca (LEVEIHA-Ca)

[0137] a. Caco2 cell culture: Caco2 cells were cultured in DMEM medium supplemented with 15% fetal bovine serum, 1% non-essential amino acids and 1% penicillin and antibiotics at a temperature of 37°C and a CO2 concentration of 5%.

[0138] b. The specific steps for studying the absorption of calcium from lentinan polypeptide-Ca (LEVEIHA-Ca) by cells are as follows: CaCO2 cells in the logarithmic growth phase are seeded into 6-well plates at a concentration of 2 × 10⁻⁶. 5Cells were cultured at a density of 10 cells / mL. After confluence, lentinan-derived peptide-Ca (LEVEIHA-Ca) solution was added (final calcium concentration in 6-well plates was 0.2-0.6 mg / mL). After incubation at 37°C for 2 hours, 3 mL of HBSS buffer was added to wash away any unabsorbed lentinan-derived peptide-Ca (LEVEIHA-Ca). Then, 100 μL of 5 μmol / L calcium-binding fluorescent dye Fluo-3AM was added, and the cells were incubated at 37°C for 1 hour. Another 100 μL of HBSS buffer was added, and the cells were incubated at 37°C for 0.5 hours. The fluorescence intensity of the cells was then detected using flow cytometry. Cells without lentinan-derived peptide-Ca (LEVEIHA-Ca) served as a blank control, cells with casein phosphopeptide-calcium (CPP-Ca) added served as a positive control, and ionic calcium (CaCl2) served as a negative control. The percentage of fluorescence intensity in each group relative to the blank control represents the calcium absorption effect.

[0139] Experimental results are as follows Figure 13 The results show that, compared with ionic calcium CaCl2, cells exhibit higher calcium absorption efficiency from both peptide-calcium conjugates (p<0.05). Specifically, at a concentration of 0.4 mg / mL, the cellular absorption rate of lentinan peptide-Ca (LEVEIHA-Ca) was significantly higher than that of commercially available CPP-Ca (p<0.05). At concentrations of 0.6 and 0.2 mg / mL, the cellular absorption rates of lentinan peptide-Ca (LEVEIHA-Ca) were comparable to those of CPP-Ca. These results confirm that the lentinan peptide-calcium (LEVEIHA-Ca) prepared in this study has superior cellular absorption performance and can be used for calcium supplementation, demonstrating broad market potential.

[0140] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0141] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A polypeptide-calcium conjugate, characterized in that, It comprises: the polypeptide of claim 1 and calcium.

3. The polypeptide-calcium conjugate according to claim 2, characterized in that, The calcium element is provided by calcium salts; Optionally, the calcium salt is selected from at least one of calcium gluconate, calcium hydrogen phosphate, calcium lactate, and calcium chloride.

4. A method for preparing the polypeptide of claim 1, characterized in that, The method includes: The shiitake mushrooms are processed to extract protein, resulting in shiitake mushroom protein; The shiitake mushroom protein was enzymatically hydrolyzed in the presence of neutral protease to obtain shiitake mushroom protease hydrolysate. The shiitake mushroom protein hydrolysate was subjected to ultrafiltration. The ultrafiltration product was purified, and the fraction collected after 92-130 min was obtained to yield the polypeptide. The amount of neutral protease added is 3% of the protein content of the shiitake mushroom; The enzymatic hydrolysis treatment was carried out at 65°C for 3 hours. The ultrafiltration process includes a first ultrafiltration process and a second ultrafiltration process; The first ultrafiltration treatment is carried out on an ultrafiltration membrane of 45~55KD; The second ultrafiltration process is carried out using an ultrafiltration membrane with a density of 1~10 kDa; The pressures for the first and second ultrafiltration processes are selected from 0.1 to 0.2 MPa, respectively. The purification process was performed using anion exchange chromatography. The packing material for the anion exchange chromatography column was selected from Diamond Q; The eluent used in the anion exchange chromatography was a 0.02 mol / L Tris-HCl solution with pH 9.0 containing 0, 0.25, 0.5, and 1 mol / L NaCl, respectively, and the elution times were 26 min, 49 min, 17 min, and 38 min, respectively. The anion exchange chromatography method used isocratic elution.

5. Use of the polypeptide of claim 1 in a kit for preparing a calcium ion chelate.

6. A method for preparing the polypeptide-calcium conjugate according to claim 2 or 3, characterized in that, include: The polypeptide described in claim 1 is chelated with a calcium salt to obtain the polypeptide-calcium conjugate.

7. The method according to claim 6, characterized in that, The polypeptide is provided in solution form; Optionally, the concentration of the polypeptide in the solution is 0.5~1.5 mg / mL; Optionally, the solution is selected from deionized water; Optionally, the calcium salt is selected from at least one of calcium chloride, calcium gluconate, calcium hydrogen phosphate, and calcium lactate; Optionally, the mass ratio of the polypeptide to the calcium salt is (1~3):1; Optionally, the chelation treatment is carried out at 50-60°C for 45-55 minutes.

8. A polypeptide-calcium conjugate, characterized in that, It is prepared by the method described in any one of claims 6 to 7.

9. Use of the polypeptide-calcium conjugate according to any one of claims 2-3 and 8 in the preparation of a product, said product being a calcium supplement.

10. The use according to claim 9, characterized in that, The products are selected from pharmaceuticals and food.

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