Ion-modified cubilose peptide compound for improving bone mineral density and promoting growth and development as well as preparation method and application of ion-modified cubilose peptide compound

Abstract

The invention relates to the technical field of food and bioactive peptide preparation, in particular to an ion-modified cubilose peptide compound for improving bone mineral density and promoting growth and development as well as a preparation method and application of the ion-modified cubilose peptide compound. The cubilose peptide compound comprises cubilose peptide modified by metal ions, and the metal ion loading capacity of the cubilose peptide compound is 15-25%. According to the ion-modified cubilose peptide compound disclosed by the invention, through metal ion modification, the binding capacity of cubilose peptide to metal ions is improved, so that the ion-modified cubilose peptide compound has antioxidant activity and biological activity for promoting osteoblast differentiation, and the nutritional function characteristics of a product are improved. Aiming at the particularity of the cubilose peptide structure, the metal ion modification method provided by the invention can improve the modification stability and batch consistency, improve the metal ion loading amount, and reduce the influence on the cubilose peptide nutritional ingredients. The preparation method is simple and convenient to operate and suitable for large-scale production and application.

Description

Ion-modified bird's nest peptide complex for improving bone density and promoting growth and development, its preparation method and application Technical Field

[0001] This application relates to the field of food and bioactive peptide preparation technology, and in particular to an ion-modified bird's nest peptide complex that enhances bone density and promotes growth and development, as well as its preparation method and application. Background Technology

[0002] Bird's nest, a traditional tonic, is rich in nutrients such as protein and amino acids. Bird's nest peptides, obtained through enzymatic hydrolysis, have the characteristics of small molecular weight and good solubility, making them easier for the body to absorb and utilize, and have attracted attention in the functional food field in recent years. In the research field of food-derived peptides, modifying peptides by introducing metal ions is a technical means to enhance their functional activity. Metal ions can complex with carboxyl and amino groups in the peptide chain, thereby improving their biological activity and physicochemical properties. However, due to the unique source and structural composition of bird's nest peptides, their amino acid composition, spatial conformation, and hydrophilic-hydrophobic ratio all affect their binding with metal ions, leading to problems such as low efficiency, insufficient complex stability, and easy precipitation in food systems, thus affecting product quality and application effects.

[0003] Therefore, it is necessary to develop a metal ion modification technology suitable for bird's nest peptide systems to obtain bird's nest peptide complexes with better stability and higher metal ion loading. Summary of the Invention

[0004] This application provides an ion-modified bird's nest peptide complex, its preparation method, and its application, addressing the problems of low binding efficiency of metal ions to bird's nest peptides, insufficient complex stability, and easy precipitation in food systems in existing technologies. It combines antioxidant capacity with bioactivity that promotes osteoblast differentiation, enhancing the nutritional functionality of bird's nest peptide products, promoting user growth and development, and increasing bone density to strengthen and toughen bones. The preparation method provided in this application effectively increases the metal ion loading of bird's nest peptides through metal ion modification, maintaining the stability of the original nutritional components while improving the metal ion modification effect and batch consistency, making it suitable for large-scale production applications.

[0005] In a first aspect, this application proposes an ion-modified bird's nest peptide complex that enhances bone density and promotes growth and development. According to embodiments of this application, the ion-modified bird's nest peptide complex comprises bird's nest peptides modified with metal ions; wherein the metal ion loading of the ion-modified bird's nest peptide complex is 15%–25%; and the metal ion is calcium ion. According to embodiments of this application, the ion-modified bird's nest peptide complex, after modification, is loaded with a high content of metal ions, thereby increasing its nutritional content. Simultaneously, combining the advantages of both metal elements and bird's nest peptides, the complex can be endowed with antioxidant capacity and bioactivity that promotes osteoblast differentiation, meeting the current needs of multifunctional foods and biomaterials. Furthermore, in the structure of the ion-modified bird's nest peptide complex, the metal is fully coordinated, not easily dissociated or interfered with, and possesses strong stability. This ion-modified bird's nest peptide complex exhibits in vitro stability. The chelation structure reduces the dissociation rate of metal ions, preventing the loss of metal ions due to oxidation and precipitation during food processing (such as high-temperature sterilization and pH fluctuations) or formulation storage. At the same time, it protects the glycosyl and sialic acid groups of the bird's nest peptide from degradation, maintaining batch-to-batch consistency of product activity.

[0006] According to embodiments of this application, the bird's nest peptide comprises polypeptides with a molecular weight between 500 Da and 3000 Da. Bird's nest polypeptides meeting these molecular weight requirements are beneficial for improving the binding efficiency of bird's nest peptides with metal ions and for improving the solubility and application stability of the complex.

[0007] In a second aspect of this application, a method for preparing an ion-modified bird's nest peptide complex is provided. According to an embodiment of this application, the method includes: pretreating bird's nest to obtain bird's nest peptides; modifying the bird's nest peptides with a metal salt to obtain a metal ion-modified bird's nest peptide complex; wherein the modification treatment includes pulsed electric field treatment; and the metal salt is calcium chloride. According to the preparation method of the bird's nest peptide complex according to the embodiments of this application, the inventors further discovered through research that introducing pulsed electric field-assisted treatment during the chelation process of bird's nest peptides and metal ions achieves multiple technical advantages through the non-thermal effect and directional regulation of the electric field: On the one hand, the pulsed electric field can relax the molecular structure of bird's nest peptides, fully exposing the chelating active sites such as amino and carboxyl groups that are occupied and blocked by sugars and sialic acid. At the same time, the electric field force accelerates the directional migration of metal ions to the active sites, improves the ion collision efficiency, and thus improves the chelation rate and chelation ratio of bird's nest peptides and metal ions; on the other hand, this treatment can promote orderly intermolecular interactions without significantly increasing the temperature, avoiding peptide chain degradation and loss of sugars and sialic acid caused by high temperature, effectively protecting the components with important nutritional functions in the bird's nest peptide complex, and improving the consistency and stability of the chelated product. Furthermore, the pulsed electric field can reduce the bitterness and fishy taste caused by the exposure of hydrophobic amino acids by inducing the hydrophobic groups of bird's nest peptide molecules to fold inward and the hydrophilic groups to arrange outward. At the same time, it can inhibit the off-flavors caused by the random polymerization of peptides and regulate the uniformity of product molecular weight distribution to improve taste. It can also enhance the hydrogen and ionic cross-linking between the active sites of bird's nest peptides and metal ions, sugars, and sialic acid to form a dense and stable molecular network structure, improve the stability of chelates, and induce the exposure of antioxidant active sites to form a natural antioxidant barrier, reducing the rate of oxidative degradation during storage. Ultimately, it can achieve a synergistic improvement in chelation efficiency, product flavor and taste, and storage stability.

[0008] According to embodiments of this application, the preparation method of the above-mentioned ion-modified bird's nest peptide complex may further include the following additional technical features: According to embodiments of this application, the pretreatment includes stewing, enzymatic hydrolysis, and optional ultrafiltration. The bird's nest peptide obtained after the above pretreatment can be effectively used for the preparation of metal ion-modified complexes.

[0009] According to embodiments of this application, the enzymatic hydrolysis process employs a complex enzyme, which includes trypsin and flavor protease. This complex enzyme helps hydrolyze bird's nest protein into smaller polypeptides, improving the efficiency of subsequent metal ion modification, and the addition of flavor protease helps optimize the molecular weight distribution of the product.

[0010] According to embodiments of this application, the ratio of the trypsin to the flavor protease is (1~5):1. Proteases meeting this ratio can efficiently prepare bird's nest peptides with a uniform molecular weight distribution, which is beneficial to the metal ion modification efficiency and stability of the bird's nest peptides.

[0011] According to embodiments of this application, the amount of the compound enzyme added is 4000 U / g to 8000 U / g. Protease added at the above-mentioned amount can efficiently prepare bird's nest peptides, ensuring the enzymatic hydrolysis reaction proceeds fully.

[0012] According to the embodiments of this application, the pH value of the enzymatic hydrolysis treatment is 7.0~8.0. Meeting the above pH environment is beneficial to maintaining enzyme activity and ensuring the smooth progress of the enzymatic hydrolysis reaction, thereby improving the enzymatic hydrolysis efficiency.

[0013] According to the embodiments of this application, the temperature of the enzymatic hydrolysis treatment is 50 ℃~55 ℃. Meeting the above treatment temperature is beneficial to maintaining enzyme activity and ensuring that the enzymatic hydrolysis reaction proceeds smoothly, thereby improving the enzymatic hydrolysis efficiency.

[0014] According to the embodiments of this application, the enzymatic hydrolysis time is 4 h to 6 h, thereby fully hydrolyzing the bird's nest to obtain bird's nest peptides, thus improving the yield of bird's nest peptides, while avoiding the loss of nutrients due to excessive enzymatic hydrolysis.

[0015] According to embodiments of this application, the molar ratio of the bird's nest peptide to the metal ion of the metal salt is (1~5):1. This facilitates obtaining an ion-modified bird's nest peptide complex with a high loading rate and reduces the residue of unbound metal ions.

[0016] According to an embodiment of this application, before the modification treatment, the pH value of the mixture of bird's nest peptide and metal salt is 6.5~8.5. Therefore, a modification environment satisfying the above pH value ensures that the key coordinating groups on the peptide chain are in a deprotonated charged state, providing electron pairs for coordination with metal ions, thus providing reaction conditions for subsequent modification treatment. This facilitates the efficient loading of metal ions onto the bird's nest peptide, while avoiding the precipitation of hydroxides due to a high pH, ​​or the competition for binding sites between hydrogen ions and metal ions due to a low pH.

[0017] According to embodiments of this application, the electric field strength of the pulsed electric field treatment is 5 kV / cm to 30 kV / cm. Pulsed electric field treatment meeting these strengths can more effectively open peptide aggregates, expose internal coordination sites, and promote the chelation modification efficiency of metal ions and bird's nest peptides. Simultaneously, it avoids reduced modification efficiency due to insufficient electric field strength, or peptide denaturation or aggregation due to excessively high electric field strength.

[0018] According to embodiments of this application, the pulse frequency of the pulsed electric field treatment is 50 Hz to 200 Hz. Pulsed electric field treatment meeting this frequency can more effectively open peptide aggregates, expose internal coordination sites, and promote the chelation modification efficiency of metal ions and bird's nest peptides. Simultaneously, it avoids the discontinuous auxiliary effect caused by lower pulse frequencies, or the thermal denaturation of peptide chains or metal ions caused by the significant thermal effect of higher pulse frequencies.

[0019] According to embodiments of this application, the pulse width of the pulsed electric field treatment is 2 μs to 10 μs. Electric field treatment with a pulse width meeting these requirements can improve the chelation modification efficiency of metal ions and bird's nest peptides. Simultaneously, it avoids situations where a short pulse width is insufficient to induce conformational changes for metal ion modification, or where a long pulse width causes localized overheating, altering the reaction pathway.

[0020] According to embodiments of this application, the pulsed electric field treatment time is 4 s to 10 s. Electric field treatment meeting this time can promote the chelation modification efficiency of metal ions and bird's nest peptides. This avoids situations where a shorter treatment time leads to poor treatment results, or a longer treatment time leads to product degradation or adverse side reactions.

[0021] According to an embodiment of this application, the preparation method of the bird's nest peptide complex further includes: purifying the treatment solution obtained from the modification treatment to obtain the metal ion modified bird's nest peptide complex; the purification treatment includes ultrafiltration treatment and drying treatment.

[0022] In a third aspect of this application, this application proposes a food or biological material comprising: the ion-modified bird's nest peptide complex described in the first aspect embodiment or the ion-modified bird's nest peptide complex prepared by the method described in the second aspect embodiment.

[0023] In a fourth aspect of this application, the application of the ion-modified bird's nest peptide complex described in the first aspect embodiment or the ion-modified bird's nest peptide complex prepared by the method described in the second aspect embodiment in the preparation of food or biomaterials that promote bone health is proposed.

[0024] Beneficial Effects: The ion-modified bird's nest peptide complex preparation method of this application can effectively prepare metal ion-modified bird's nest peptide complexes. The pulsed electric field treatment method utilizes the directional control of the electric field to influence the secondary and tertiary structures of bird's nest peptides, reducing the occupancy and shielding effects of sugars and sialic acid, fully exposing the metal ion binding sites, promoting efficient coordination between metal ions and the carboxyl and amino groups of bird's nest peptides, increasing calcium loading, protecting the inherent nutritional components of bird's nest peptides, and endowing the complex with multiple functions such as antioxidant properties, thus solving the problems of low modification efficiency and poor product stability. The metal ion-modified bird's nest peptide complex can overcome the functional limitations of traditional bird's nest peptides and metal fortifiers, expanding its application to food, pharmaceuticals, skincare products, and biomaterials. Attached Figure Description

[0025] 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: Figure 1 is a schematic diagram of the preparation method of the ion-modified bird's nest peptide complex; Figure 2 is the ultraviolet spectrum of bird's nest peptide and bird's nest peptide-calcium complex; Figure 3 is the fluorescence spectrum of bird's nest peptide and bird's nest peptide-calcium complex; Figure 4 is the infrared spectrum of bird's nest peptide and bird's nest peptide-calcium complex; Figure 5 is the Zeta potential of bird's nest peptide and bird's nest peptide-calcium complex; Figure 6 is a scanning electron microscope image of bird's nest peptide and bird's nest peptide-calcium complex; Figure 7 is an energy dispersive spectroscopy (EDS) analysis diagram of bird's nest peptide and bird's nest peptide-calcium complex. Figure 8. Effect of different concentrations of bird's nest peptide-calcium complex on the proliferation rate of MC3T3-E1 cells; Figure 9. Effect of different concentrations of bird's nest peptide-calcium complex on ALP activity in MC3T3-E1 cells; Figure 10. Effect of different concentrations of bird's nest peptide-calcium complex on mineralization of MC3T3-E1 cells; Figure 11. Quantitative analysis of the binding of alizarin red dye to MC3T3-E1 cells; Figure 12. Effect of bird's nest peptide-calcium complex on gene expression in MC3T3-E1 cells; Figure 13. Stability of bird's nest peptide-calcium complex in simulated gastric and intestinal fluids at different times. Detailed Implementation

[0026] The embodiments of this application are described in detail below, with examples of the embodiments illustrated in the accompanying drawings. Those skilled in the art will understand that the embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0027] It should be noted that the terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in sequences other than those illustrated or described herein.

[0028] First, let me explain the technical terms used in this application: In the embodiments of this application, "bird's nest peptide" refers to a mixture of small molecule active peptides obtained by hydrolyzing bird's nest with protease. It combines the nutritional characteristics of bird's nest with the absorption advantages of small molecule peptides and is an important product form in the field of deep processing of bird's nest.

[0029] In this embodiment of the application, "metal ion modification" refers to the technical means of introducing metal ions into the structure of the target substance bird's nest peptide through chemical coordination or covalent bonding to modify its physicochemical properties or functional activities.

[0030] In this embodiment of the application, "metal ion loading" refers to the quantity of metal ions actually bound or carried by the target carrier bird's nest peptide through chemical coordination or covalent binding. It is used to quantify the efficiency of the metal ion modification process and is directly related to the functional performance of the carrier.

[0031] In this embodiment of the application, "pulse electric field" is abbreviated as PEF. It is a physical technology that applies a short-duration, high-intensity pulsed electric field to a target system through a specific device. It uses the electric field to act on charged particles in the system and releases energy in the form of pulses at the nanosecond to millisecond level, thereby achieving the regulation of the structure, function or state of matter.

[0032] In the embodiments of this application, "enzymatic hydrolysis" refers to the biochemical process of specifically breaking down macromolecules into small molecular fragments under relatively mild conditions by utilizing the catalytic activity of enzymes.

[0033] In this embodiment of the application, "ultrafiltration" refers to a pressure-driven separation technology based on porous membranes, which achieves the separation of small molecules from large molecules or colloidal particles in a mixture under relatively mild pressure through the pore size sieving effect of the membrane.

[0034] In this embodiment of the application, "freeze-drying" refers to a physical dehydration technique that freezes water-containing materials into solid ice under low temperature and vacuum conditions, then sublimates the ice directly into water vapor and removes it, ultimately obtaining a dried product.

[0035] In this embodiment of the application, "spray drying" refers to a continuous drying technology that disperses liquid or slurry materials into tiny droplets through an atomizer, which then come into direct contact with high-temperature air in a hot airflow, causing the moisture in the droplets to evaporate instantly and quickly transform into powdery or granular products.

[0036] In the embodiments of this application, "antioxidant capacity" refers to the ability of a substance to remove or inhibit oxidative reactive substances such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body or system, or to block the oxidative damage reactions they cause.

[0037] In the embodiments of this application, "promoting osteogenic activity" refers to the ability of a material or drug to promote bone tissue formation, repair bone defects, or enhance bone density by regulating the proliferation and differentiation of osteogenic-related cells or modulating osteogenic signaling pathways.

[0038] In the embodiments of this application, "chelation modification" refers to the technical means of using multiple coordinating atoms in a chelating agent molecule to form a stable cyclic chelate with a metal ion through coordination bonds, thereby regulating the existence form, activity or function of the metal ion, or endowing the target substance with new properties.

[0039] In the embodiments of this application, "enzyme-catalyzed reaction rate" refers to an efficiency indicator for measuring the conversion of enzyme substrate into product. It is usually defined as the amount of substrate consumed or product generated per unit time, reflecting the catalytic activity of enzyme under specific conditions.

[0040] In this embodiment of the application, "deprotonation" refers to the loss of one or more protons (H atoms) by a molecule, atom, or ion. + ) chemical processes.

[0041] In the embodiments of this application, "thermal denaturation of metal ions" refers to the process by which metal ions, under conditions of increased temperature, interact with specific groups of biological macromolecules, thereby exacerbating or regulating the destruction of the spatial conformation and loss of function of biological macromolecules.

[0042] In this embodiment of the application, "Zeta potential analysis" refers to a characterization technique that quantifies the surface charge state of particles by measuring the electrokinetic potential (i.e., Zeta potential) on the surface of colloidal particles or dispersion systems, thereby assessing system stability, predicting aggregation risk, or resolving interfacial interactions.

[0043] In the field of food-derived peptide research, metal ion modification technology is widely used to improve the physicochemical properties and application performance of peptides. However, research on metal ion modification of peptides derived from bird's nest is still relatively limited, especially in the preparation process that balances modification efficiency and system stability, where further optimization is needed. How to achieve effective metal ion modification while maintaining the unique structural characteristics of bird's nest peptides has become an urgent technical problem to be solved.

[0044] The inventors, while researching metal ion modification of bird's nest peptides, discovered that traditional metal ion modification methods, when applied to bird's nest peptide systems, may suffer from unstable modification efficiency, easy precipitation of the composite system, and insufficient product stability. Further analysis revealed that bird's nest peptides differ from conventional food-derived peptides, exhibiting a high degree of glycosylation and sialylation modification in their molecular structure, forming peptide-glycosylation-sialic acid complex structural units. This structural feature may affect the contact and binding behavior between metal ions and coordinating groups on the peptide backbone, thereby influencing the composite efficiency and product stability.

[0045] Furthermore, conventional simple mixing methods are insufficient to achieve high binding efficiency in bird's nest peptide systems, while high-temperature or organic solvent-assisted modification methods may adversely affect the original structure of bird's nest peptides, leading to decreased system stability. Therefore, it is necessary to provide a metal ion modification method suitable for bird's nest peptide systems that can improve the metal ion loading level while maintaining the structural stability and processing adaptability of the composite system.

[0046] Based on this, in a first aspect, this application provides an ion-modified bird's nest peptide complex. The bird's nest peptide complex comprises bird's nest peptides modified with metal ions, wherein the metal ion loading is 15%–25%, and the metal ion is a calcium ion.

[0047] In the embodiments of this application, when the metal ion loading is controlled within the range of 15% to 25%, a relatively stable binding state is formed between the metal ions and the bird's nest peptides in the complex system. This helps to increase the binding ratio of metal ions and reduce the precipitation of free metal ions. Furthermore, the bird's nest peptide complex with this loading exhibits good stability in terms of molecular structure and particle size distribution, which is beneficial for improving the stability of the product during processing and storage. Simultaneously, within this loading range, the complex system demonstrates batch-to-batch consistency and good gastrointestinal digestive tolerance, maintaining a high metal ion binding rate in a simulated gastrointestinal digestive environment, making it suitable for application in food and related product systems.

[0048] In the embodiments of this application, the bird's nest peptide includes polypeptides with a molecular weight of 500 Da to 3000 Da.

[0049] It should be noted that peptides with the above-mentioned molecular weight can promote the efficiency of chelation modification between bird's nest peptides and metal ions, as well as the degree of nutrient absorption and utilization of bird's nest peptide complexes by the human body. Avoid peptides with a molecular weight greater than 3000 Da, as excessively long peptide chains can obscure active sites and increase absorption difficulty; or peptides with a molecular weight less than 500 Da, as excessively short peptide chains can significantly reduce activity.

[0050] Based on this, in a second aspect, this application provides a method for preparing an ion-modified bird's nest peptide complex. According to an embodiment of this application, referring to Figure 1, the preparation method includes the following steps: S100, pretreating bird's nest to obtain bird's nest peptides.

[0051] S200 involves modifying bird's nest peptides with metal salts to obtain a metal ion-modified bird's nest peptide complex.

[0052] It should be noted that the modification treatment includes pulsed electric field treatment, and the metal salt is calcium chloride.

[0053] According to the preparation method of the ion-modified bird's nest peptide complex according to the embodiments of this application, the inventors have discovered through research that the pulsed electric field-assisted modification treatment can significantly expand the molecular structure of bird's nest peptides, allowing amino acid sites that are occupied or blocked by sugars and sialic acid to be fully exposed, thereby improving the ion modification efficiency of bird's nest peptides. At the same time, combined with the directional regulation of the electric field, the interaction between molecules is promoted without significantly increasing the temperature, protecting the peptides, sugars and sialic acid with important nutritional functions in the bird's nest peptide complex, and improving the consistency and stability of the ion modification of bird's nest peptides.

[0054] According to embodiments of this application, the pretreatment includes stewing, enzymatic hydrolysis, and optional ultrafiltration. The bird's nest peptides obtained after the above pretreatment can be effectively used in the preparation of metal ion-modified complexes.

[0055] According to embodiments of this application, the enzymatic hydrolysis process employs a complex enzyme, which includes trypsin and flavor protease. The complex enzyme system facilitates the hydrolysis of bird's nest protein into smaller polypeptides, improving the efficiency of subsequent metal ion modification. The addition of flavor protease helps optimize the molecular weight distribution of the product.

[0056] According to the embodiments of this application, the ratio of trypsin to flavor protease in the complex enzyme is (1~5):1. Proteases meeting the above ratio can efficiently prepare bird's nest peptides. Within the above ratio range, it helps to obtain bird's nest peptides with a more uniform molecular weight distribution, thereby facilitating subsequent modification processing.

[0057] According to the embodiments of this application, the amount of compound enzyme added is 4000 U / g to 8000 U / g. Protease added at the above-mentioned amount can efficiently prepare bird's nest peptides, and within the above-mentioned range, the enzymatic hydrolysis reaction can be ensured to proceed fully.

[0058] According to the embodiments of this application, the pH value of the enzymatic hydrolysis treatment is 7.0~8.0, which is beneficial to maintain enzyme activity and ensure the smooth progress of the enzymatic hydrolysis reaction, thereby improving the enzymatic hydrolysis efficiency.

[0059] According to the embodiments of this application, the temperature of the enzymatic hydrolysis treatment is 50 ℃~55 ℃, which is beneficial to maintain enzyme activity and ensure that the enzymatic hydrolysis reaction proceeds smoothly, thereby improving the enzymatic hydrolysis efficiency.

[0060] According to the embodiments of this application, the enzymatic hydrolysis time is 4 h to 6 h, so that the bird's nest is fully hydrolyzed to obtain bird's nest peptides, thereby increasing the yield of bird's nest peptides and avoiding the loss of nutrients due to excessive enzymatic hydrolysis.

[0061] According to embodiments of this application, the molar ratio of bird's nest peptide to metal ions of the metal salt is (1~5):1. This facilitates obtaining ion-modified bird's nest peptide complexes with higher loading rates and reduces the residue of unbound metal ions.

[0062] According to the embodiments of this application, before the modification treatment, the pH value of the mixture of bird's nest peptide and metal salt is 6.5~8.5, which provides a reaction strip for the subsequent modification treatment and is conducive to efficient loading onto bird's nest peptide.

[0063] According to embodiments of this application, the electric field strength of the pulsed electric field treatment is 5 kV / cm to 30 kV / cm, which helps to improve the binding efficiency of metal ions and bird's nest peptides, while avoiding the adverse effects of too low or too high electric field strength.

[0064] According to embodiments of this application, the pulse frequency of the pulsed electric field treatment is 50 Hz to 200 Hz, which helps to improve the modification efficiency while avoiding the adverse effects caused by parameters that are too low or too high.

[0065] According to embodiments of this application, the pulse width of the pulsed electric field treatment is 2 μs to 10 μs. Electric field treatment within this pulse width range can improve the chelation modification efficiency of metal ions and bird's nest peptides, thus facilitating the binding of metal ions.

[0066] According to embodiments of this application, the processing time for pulsed electric field treatment is 4 s to 10 s. Applying treatment within this range can obtain a relatively uniformly distributed composite system, which helps to improve the modification efficiency while avoiding structural instability that may result from over-treatment.

[0067] According to an embodiment of this application, the preparation method of the ion-modified bird's nest peptide complex further includes: purifying the treatment solution obtained from the modification treatment to obtain the metal ion-modified bird's nest peptide complex; wherein, the purification treatment includes ultrafiltration treatment and drying treatment.

[0068] In summary, considering the high glycosylation and sialylation modification characteristics of bird's nest peptide molecules, the ion-modified bird's nest peptide complex provided in this application can form a relatively stable binding state within a certain metal ion loading range, which helps to increase the binding ratio of metal ions and improve the stability of the composite system in the gastrointestinal digestive environment. Furthermore, the preparation method of the metal ion-modified bird's nest peptide complex provided in this application effectively improves the stability and repeatability of the modification process through optimized control of process parameters, thereby obtaining composite products with good batch consistency. Compared with traditional simple mixing methods, this method is beneficial for improving the binding efficiency of metal ions and completing the modification process under milder conditions, thus reducing potential adverse effects during processing. In addition, the preparation method has a relatively simple process flow and controllable operating conditions, showing potential for industrial application.

[0069] Based on this, in a third aspect, this application proposes a food or biological material, including the ion-modified bird's nest peptide complex of the first aspect embodiment or the ion-modified bird's nest peptide complex prepared by the method of the second aspect embodiment.

[0070] Based on this, in a fourth aspect, this application proposes the application of the ion-modified bird's nest peptide complex of the first aspect embodiment or the ion-modified bird's nest peptide complex prepared by the method of the second aspect embodiment in the preparation of food or biomaterials that promote bone health.

[0071] The technical solutions in the embodiments of this application will be described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0072] Example 1 In this example, the bird's nest peptide complex was prepared according to the following steps: 1. Preparation of bird's nest peptides Weigh a certain amount of dried bird's nest, add purified water at a material-to-liquid ratio of 1:30, stew at 100 ℃ for 60 min, add a complex enzyme of trypsin and flavor protease at a rate of 6000 U / g (trypsin:flavor protease ratio of 3:1), enzymatically hydrolyze at pH 7.5 and temperature 55 ℃ for 5 h, then inactivate at 100 ℃ for 10 min, cool, centrifuge at 12000 rpm for 10 min, collect the supernatant, ultrafilter at 3000 Da, and freeze dry to obtain bird's nest peptides.

[0073] 2. Pulsed electric field (PEF) modification treatment: Using a 3:1 molar ratio of bird's nest peptide to calcium and calcium chloride as the calcium source, calcium chloride was added to a 5% (w / v) bird's nest peptide solution and mixed thoroughly. After adjusting the pH to 7.5, the mixture was pumped into a high-voltage pulsed electric field treatment chamber. The electric field parameters were: electric field strength 20 kV / cm, pulse width 6 μs, pulse frequency 100 Hz, and electric field treatment time 7s. Multiple rounds of high-voltage pulsed electric field treatment were used, with each round including the electric field treatment time and the interval time, for a total of 60 rounds of electric field treatment for 20 min. After completion, the material was collected, purified by 10000 Da ultrafiltration, and finally freeze-dried to obtain the ion-modified bird's nest peptide-calcium complex.

[0074] Examples 2 and 3 were prepared according to the method of Example 1, except that the electric field strength was replaced with 5 kV / cm and 30 kV / cm, respectively.

[0075] Examples 4 and 5 prepared bird's nest peptide complexes according to the method of Example 1, except that the pulse widths were replaced with 2 μs and 10 μs, respectively.

[0076] Examples 6 and 7 prepared bird's nest peptide complexes according to the method of Example 1, except that the pulse frequency was replaced with 50 Hz and 200 Hz respectively.

[0077] Examples 8 and 9 prepared bird's nest peptide complexes according to the method of Example 1, except that the electric field treatment time was replaced with 4s and 10s, respectively.

[0078] Examples 10-11 prepared bird's nest peptide complexes according to the method of Example 1, except that the molar ratio of bird's nest peptides to calcium was replaced with 5:1 and 1:1, respectively.

[0079] Comparative Examples 1-3 prepared bird's nest peptide complexes according to the method of Example 1, except that calcium chloride was replaced with calcium lactate, calcium gluconate, and calcium carbonate, respectively.

[0080] Comparative Example 4 prepared bird's nest peptide complex according to the method of Example 1, except that the electric field strength was replaced with 1 kV / cm.

[0081] Comparative Example 5 prepared the bird's nest peptide complex according to the method of Example 1, except that the pulse width was replaced with 1 μs.

[0082] Comparative Example 6 prepared bird's nest peptide complex according to the method of Example 1, except that the pulse frequency was replaced with 40 Hz.

[0083] Comparative Example 7 prepared bird's nest peptide complex according to the method of Example 1, except that the electric field treatment time was replaced with 3 s.

[0084] Comparative Example 8 prepared a bird's nest peptide complex according to the method of Example 1, except that it was modified by heating in an 80 ℃ water bath.

[0085] Comparative Example 9 prepared a bird's nest peptide complex according to the method of Example 1, except that it was not modified by a pulsed electric field.

[0086] The variable parameters and their specific settings in Examples 2-11 and Comparative Examples 1-9 compared to Example 1 are shown in Table 1: Table 1

[0087] Test Example 1: Calcium loading was determined according to the second method of inductively coupled plasma optical emission spectrometry (ICP-OES) in GB 500268.2-2016 "National Food Safety Standard - Determination of Multiple Elements in Food". The calcium loading in the products prepared in Examples 1-11 and Comparative Examples 1-9 was detected. The results are shown in Table 2 (in Table 2, # indicates a highly statistically significant difference compared to Example 1, showing a highly significant decrease; ns indicates no statistically significant difference compared to Example 1).

[0088] Table 2

[0089] Conclusion: The products prepared in the embodiments of this application have good calcium loading. Without the use of calcium chloride (Comparative Examples 1-3), without pulsed electric field treatment (Comparative Examples 8-9), and when high-voltage pulsed electric field treatment is used, any one of the electric field strength, pulse width, pulse frequency, and treatment time is not within the parameter range proposed in this application (Comparative Examples 1-7). The calcium loading of the products prepared by this application is significantly lower than that of the products prepared by the preparation method of this application, which proves the effectiveness of the preparation method of this application and the uniformity and significantly improved calcium loading capacity of the prepared products.

[0090] Test Example 2: DPPH Free Radical Scavenging Ability Detection Procedure: Take 2 mL of bird's nest peptide-calcium complex sample solutions of different concentrations, add 2 mL of DPPH solution, mix well, let stand at room temperature for 30 min, centrifuge at 5000 r / min for 10 min, and take the supernatant to measure the absorbance at 517 nm.

[0091] Preparation of test solution: The products obtained in Examples 1-11 and Comparative Examples 1-9 were mixed with ultrapure water to prepare a 1 mg / mL sample solution.

[0092] DPPH free radical scavenging rate is calculated using the following formula:

[0093] Where A0 represents the absorbance value of 2 mL anhydrous ethanol + 2 mL DPPH radical solution; A1 represents the absorbance value of 2 mL sample solution + 2 mL DPPH radical solution; and A2 represents the absorbance value of 2 mL sample solution + 2 mL anhydrous ethanol.

[0094] The DPPH radical scavenging rates of the products prepared in Examples 1-11 and Comparative Examples 1-9 were tested. The results are shown in Table 3 (in Table 3, # indicates a highly significant statistical difference compared to Example 1, showing a highly significant decrease; ns indicates no statistical difference compared to Example 1).

[0095] Table 3

[0096] Conclusion: The products prepared in the embodiments of this application have good antioxidant effects. The products prepared without calcium chloride (Comparative Examples 1-3), without high-voltage pulsed electric field treatment (Comparative Examples 8-9), and with high-voltage pulsed electric field treatment, where any one of the electric field strength, pulse width, pulse frequency, and treatment time is outside the parameter range proposed in this application (Comparative Examples 1-7), exhibit significantly lower DPPH free radical scavenging ability than the products prepared by the method of this application. This demonstrates the effectiveness of the preparation method of this application and the uniformity and significantly enhanced antioxidant activity of the prepared products.

[0097] Test Example 3: Alkaline phosphatase (ALP) Activity Detection in Osteoblasts. Alkaline phosphatase (ALP) is a key functional marker enzyme in osteoblast differentiation and bone formation. Its activity level can directly reflect the degree of osteoblast differentiation and maturity and the functional status of bone formation. Elevated ALP activity indicates active osteoblast differentiation, enhanced transformation of cells into mature osteoblast phenotypes, vigorous bone matrix mineralization-related metabolism, and improved new bone formation capacity. Therefore, it is often used as an important basis for effective osteogenic induction and enhanced bone regeneration function.

[0098] MC3T3-E1 cells in the logarithmic growth phase were harvested, cell counts were performed, and cell concentration was adjusted to 1×10⁻⁶ cells. 5 Cells were seeded per well into 12-well plates and cultured in a 5% CO2, 37°C incubator until adherence. The control group was supplemented with DMEM medium and cultured with mouse bone marrow mesenchymal stem cell osteogenic differentiation-inducing medium containing 1 mg / mL of the products prepared in Examples 1-11 and Comparative Examples 1-9. The medium was changed every 3-4 days. After 7 days of culture, the culture medium was collected, centrifuged at 1500 r / min for 10 min, and the supernatant was used to determine the relative ALP activity over 7 days (the relative ALP activity of the control group cultured in differentiation-inducing medium without any products prepared in the examples or comparative examples is used as the unit).

[0099] The results are shown in Table 4 (in Table 4, # indicates a highly significant statistical difference compared with Example 1, showing a highly significant decrease; ns indicates no statistical difference compared with Example 1).

[0100] Table 4

[0101] Conclusion: The products prepared in the embodiments of this application have good osteoblast ALP activity and promote osteoblast differentiation. The products prepared without calcium chloride (Comparative Examples 1-3), without high-voltage pulsed electric field treatment (Comparative Examples 8-9), and with high-voltage pulsed electric field treatment, where any one of the electric field strength, pulse width, pulse frequency, and treatment time was outside the parameter range proposed in this application (Comparative Examples 1-7), showed significantly lower osteoblast ALP activity than the products prepared by the method of this application. This demonstrates the effectiveness of the preparation method of this application and the uniformity and significantly enhanced osteoblast differentiation-promoting activity of the prepared products.

[0102] Example 1. Ultraviolet Spectroscopy Analysis: Ultraviolet absorption spectroscopy is one of the methods for detecting changes in the tertiary structure of proteins, specifically reflected by changes in the microenvironment of amino acid residues. Second-guided ultraviolet spectroscopy has proven to be an effective analytical tool for detecting protein structural changes involving the microenvironment of aromatic amino acids, mainly because it has the unique ability to resolve the spectral bands of each aromatic amino acid. The presence of organic coordination and metal ion complexes can cause significant changes in absorption peaks.

[0103] The bird's nest peptide and the bird's nest peptide-calcium complex prepared in Example 1 were respectively prepared into 1 mg / mL solutions with ultrapure water, and the ultraviolet scanning wavelength range was 200-400 nm.

[0104] Results: This application investigated the formation of the bird's nest peptide-calcium complex using UV-Vis spectroscopy. As shown in Figure 2, bird's nest peptide exhibits a maximum absorbance at 284 nm; the maximum absorption peak of the bird's nest peptide-calcium complex is at 283 nm, shifting towards blue compared to the bird's nest peptide sample. Simultaneously, the peak shape and intensity show significant changes, attributed to the charge transfer effect resulting from the coordination of calcium ions with carboxyl or amino groups. This demonstrates a significant difference between the bird's nest peptide-calcium complex and bird's nest peptide products.

[0105] 2. Fluorescence Spectroscopy Analysis: Fluorescence spectroscopy can reflect the oxidation level of proteins and changes in tryptophan residues in the microenvironment, and is one of the important techniques for detecting the tertiary conformation of proteins. At specific excitation wavelengths, aromatic amino acids such as tryptophan, tyrosine, and phenylalanine can produce endogenous fluorescence. The endogenous fluorescence in proteins mainly originates from the indole group of tryptophan. When the excitation wavelength is 295 nm, the absorption peak of the endogenous fluorescence produced by tryptophan is around 350 nm. At this point, polypeptide folding can lead to a decrease in fluorescence intensity. Therefore, in fluorescence spectra, changes in absorption peaks and fluorescence intensity can reflect the interaction between organic ligands and metal ions in proteins.

[0106] The bird's nest peptide and the bird's nest peptide-calcium complex prepared in Example 1 were respectively prepared into 1 mg / mL solutions using ultrapure water. The effects of calcium on the intrinsic fluorescence and conformation of the peptides were determined using a multifunctional microplate reader. The measurement conditions were: excitation wavelength 280 nm, emission wavelength 300-500 nm, and cleft width 5 nm.

[0107] Results: As shown in Figure 3, all components of the bird's nest peptide and the bird's nest peptide-calcium complex exhibited fluorescence absorption, with the maximum absorption peak around 350 nm. The fluorescence spectrum of the bird's nest peptide-calcium complex showed a significant change compared to that of the bird's nest peptide, with a marked decrease in fluorescence intensity. This phenomenon is attributed to the fluorescence quenching effect of calcium ion modification on the bird's nest peptide, and the folding effect caused by calcium ions binding to the bird's nest peptide, leading to the migration of the indole group of tryptophan from the molecular surface to the interior of the molecule. This demonstrates a significant difference between the bird's nest peptide-calcium complex and the bird's nest peptide product.

[0108] 3. Fourier Transform Infrared Spectroscopy (FTIR) is one of the methods for investigating peptide structure and the reaction mechanism of metal ion modification. Changes in absorption peaks can reflect the coordinated reaction between metal ions and organic groups.

[0109] 1 mg of bird's nest peptide and the bird's nest peptide-calcium complex prepared in Example 1 were mixed and ground with 100 mg of dry KBr (potassium bromide) powder. Using KBr as a blank background, the mixtures were pressed into thin sheets. Fourier transform infrared spectroscopy was used to analyze the mixture from 4000 to 400 cm⁻¹. –1 Infrared spectral scanning was performed within the wavenumber range, with a cumulative total of 32 scans for each sample.

[0110] Results: As shown in Figure 4, the infrared spectrum of the bird's nest peptide-calcium complex is significantly different from that of bird's nest peptide. This is because when calcium ions bind with bird's nest peptide, the structure of some functional groups of bird's nest peptide changes, causing changes in the absorption peaks. This proves that there is a significant difference between the bird's nest peptide-calcium complex and bird's nest peptide products.

[0111] 4. Zeta potential analysis: Zeta potential is a physicochemical indicator that reflects the surface charge state of a substance. Partial ionization of amino acid residues will generate charge on the protein surface.

[0112] The zeta potentials of bird's nest peptides and the bird's nest peptide-calcium complex prepared in Example 1 were analyzed using a Zetasizer Nano ZS90 nanoparticle size and zeta potential analyzer. Both samples were prepared into 1 mg / mL solutions with ultrapure water and added to a U-shaped cell, then equilibrated at 25 °C for 60 s. All measurements were performed at 25 °C and repeated 12 times.

[0113] Results: As shown in Figure 5, the average potential of the bird's nest peptide was -16.9 mV, and the electronegativity of the bird's nest peptide-calcium complex was significantly reduced, with an average potential of -5.12 mV, indicating that Ca... 2+ The formation of shared electron pairs with negatively charged binding sites such as carboxyl groups in bird's nest peptides leads to a reduction in the surface charge of bird's nest peptides, demonstrating a significant difference between bird's nest peptide-calcium complex and bird's nest peptide products.

[0114] 5. Scanning Electron Microscopy and Energy Dispersive Spectroscopy (EDS) Analysis: Two samples, bird's nest peptide and the bird's nest peptide-calcium complex prepared in Example 1, were uniformly coated onto a scanning electron microscope (SEM) sample column and subjected to gold sputtering. After applying a certain voltage, scanning images were acquired at a set magnification. The scanning conditions were: accelerating voltage 15.0 kV, beam current 6.9 × 10⁻⁶ kV. -2 The samples were observed at mA and a working distance of 6.7 mm, and images were acquired at 500x magnification. The elemental composition and content of the samples were determined using energy-dispersive spectroscopy.

[0115] Results: Bird's nest peptides (Figure 6A) exhibit a smooth, sheet-like structure interspersed with small fragments. Scanning electron microscopy (SEM) of the bird's nest peptide-calcium complex (Figure 6B) reveals a rough surface with numerous dense granular substances and crystalline aggregates. This change may be due to the aggregation of carboxyl and amino groups in the peptide chain caused by hydrogen bonds between water molecules, and further aggregation in different directions due to anisotropic intermolecular forces and surface tension. Alternatively, it may be caused by the retention of calcium by the porous structure of the bird's nest peptides. Furthermore, the surface of the bird's nest peptide-calcium complex contains a small number of crystalline structures, possibly calcium salt crystals adsorbed on the peptide surface. The SEM images show that the bird's nest peptide-calcium complex, formed after the binding of bird's nest peptides and calcium, is a novel substance different from bird's nest peptides.

[0116] Energy dispersive spectroscopy (EDS) analysis showed that bird's nest peptides (Figure 7A) contain a small amount of calcium, indicating that they contain a small amount of mineral elements; while the bird's nest peptide-calcium complex (Figure 7B) contains a large amount of calcium. Quantitative analysis of the elements (see Table 5) showed that the normalized mass percentage of calcium in the bird's nest peptide-calcium complex increased from 1.81% to 12.40%. This demonstrates a significant increase in the bound calcium content in the bird's nest peptide-calcium complex, which has become a dense aggregate.

[0117] Table 5

[0118] 6. Effect of CCK8 assay on osteoblast proliferation rate of bird's nest peptide-calcium complex. The proliferation stage is the first stage in which osteoblasts participate in the bone formation process. At this time, the precursor osteoblasts proliferate rapidly and form multilayer cells. Various intracellular organelles work together to synthesize, process and deliver collagen to the extracellular space to form the organic structural framework of bone tissue.

[0119] Logarithmic growth phase MC3T3-E1 cells were harvested, cell counts were performed, and cell concentration was adjusted to 6 × 10⁻⁶ cells. 3Cells were seeded per well in 96-well plates and cultured in a 5% CO2 incubator at 37°C until adherence. Culture media containing different concentrations of the bird's nest peptide-calcium complex prepared in Example 1 were used, with groups including 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, and 2000 μg / mL. The control group used DMEM medium without the bird's nest peptide-calcium complex. After culturing for 7 days, the supernatant was discarded, and the cells were washed once with PBS. 100 μL of medium containing 10% CCK8 was added to each well, and the cells were incubated in a 5% CO2 incubator at 37°C for 2 hours. The absorbance at 450 nm was measured.

[0120] Results: The CCK-8 assay was used to determine the proliferation rate of MC3T3-E1 osteoblasts cultured for 7 days with different concentrations of bird's nest peptide-calcium complex. As shown in Figure 8, ns indicates no significant difference between the two groups (P > 0.05). During the 7-day culture period, the effect of bird's nest peptide-calcium complex on the proliferation of MC3T3-E1 cells showed a clear concentration-dependent effect. All groups showed significant differences compared to the control group, but there were no significant differences among the groups other than the control group. Specifically, when the concentration was in the range of 100 μg / mL to 2000 μg / mL, the relative cell viability significantly increased and showed a dose-dependent enhancement, with the 2000 μg / mL concentration group reaching its peak relative cell viability. In conclusion, the bird's nest peptide-calcium complex has good osteoblast proliferation-promoting activity. Based on this, subsequent experiments selected 100 μg / mL and 400 μg / mL bird's nest peptide-calcium complex for subsequent cell-related studies.

[0121] 7. The effect of bird's nest peptide-calcium complex on ALP staining in MC3T3-E1 cell supernatant. Cell differentiation is the second stage in which MC3T3-E1 cells exert their bone-forming function. Alkaline phosphatase (ALP) is one of the important markers in this cell differentiation process, mainly expressed in the early stage of differentiation, and is also an essential enzyme in the mineralization process of MC3T3-E1 cells. The secretion of ALP varies with the stage of differentiation. When it is released from the intracellular to the extracellular space, it marks the maturation of the extracellular matrix. Extracellular ALP can increase the concentration of calcium and phosphorus ions near the bone collagen fiber network, thus providing favorable conditions for osteoblasts to exert their mineralization function. The higher the ALP activity, the more conducive it is to bone formation and repair. Osteoblast differentiation is mainly manifested in the synthesis and precipitation of bone matrix proteins, that is, the production of extracellular matrix and the initiation of matrix mineralization.

[0122] MC3T3-E1 cells in the logarithmic growth phase were harvested, cell counts were performed, and cell concentration was adjusted to 1×10⁻⁶ cells. 5Cells were seeded per well in 12-well plates and cultured in a 5% CO2 incubator at 37°C until adherence. A blank control group was supplemented with DMEM medium. Mouse bone marrow mesenchymal stem cells were cultured in osteogenic induction differentiation-inducing medium containing 0 μg / mL (model group), 100 μg / mL, and 400 μg / mL of the bird's nest peptide-calcium complex prepared in Example 1, respectively. Culture was continued for 7 and 14 days. The culture dishes were removed, the medium was discarded, and the cells were fixed with 4% paraformaldehyde. After fixation, the cells were washed once with PBS, and then ALP staining solution was prepared according to the instructions. Staining was performed for approximately 30 minutes, followed by washing with PBS 1-2 times. The cells were then photographed under a microscope.

[0123] Results: As shown in Figure 9, the left images are staining results after 7 days of culture, and the right images are staining results after 14 days of culture. ALP enzyme activity was stained in MC3T3-E1 cells; the darker the color, the higher the ALP activity. The staining results clearly show that the ALP staining in the 100 μg / mL bird's nest peptide-calcium complex group and the 400 μg / mL bird's nest peptide-calcium complex group was significantly darker than that in the model group, indicating that the bird's nest peptide-calcium complex can significantly promote the increase of ALP activity in MC3T3-E1 cells.

[0124] 8. Determination of Mineralization Levels in MC3T3-E1 Cells by the Effect of Bird's Nest Peptide-Calcium Complex: The formation of mineralized nodules in the extracellular matrix is ​​a marker of osteoblast maturation. Mineralization is the final step in osteoblast differentiation. Calcium ions in the mineralized nodules, after complexing with alizarin red, turn a deep red color, which can visually reflect the degree of bone formation and has long been used as the ultimate marker of bone formation. Furthermore, matrix mineralization is also the most direct evidence of calcium deposition capacity.

[0125] MC3T3-E1 cells in the logarithmic growth phase were harvested, cell counts were performed, and cell concentration was adjusted to 1×10⁻⁶ cells. 5 Cells were seeded per well in 12-well plates and cultured in a 5% CO2 incubator at 37°C until adherence. A blank control group was supplemented with DMEM medium. Mouse bone marrow mesenchymal stem cells were cultured in osteogenic induction differentiation-inducing medium containing 0 μg / mL (model group), 100 μg / mL, and 400 μg / mL of the bird's nest peptide-calcium complex prepared in Example 1, respectively. After 21 days of culture, the medium was discarded, cells were washed once with PBS, fixed with 4% paraformaldehyde for 20 min, and washed three times with PBS. A suitable amount of Alizarin Red S staining solution was added to evenly cover the cells, and staining was performed at room temperature for 30 minutes. Cells were thoroughly washed with distilled water and then observed and photographed under a microscope. Finally, the absorbance of each well was measured at 562 nm, with three replicates for each sample group.

[0126] Results: Therefore, this application used Alizarin Red staining to detect the effect of the bird's nest peptide-calcium complex on bone mineralization of MC3T3-E1 cells, and measured the absorbance of the staining products at 562 nm (absorbance is proportional to the amount of calcium deposited). As shown in Figure 10, after culturing cells with different concentrations of bird's nest peptide-calcium complex for 21 days, dense opaque clumps appeared between osteoblasts. Compared with the control group and the model group, the MC3T3-E1 cells in the 100 μg / mL bird's nest peptide-calcium complex group and the 400 μg / mL bird's nest peptide-calcium complex group had a larger mineralization staining area, with visible patchy red staining, and more and larger mineralized nodules, indicating that both 100 μg / mL and 400 μg / mL bird's nest peptide-calcium complex could significantly promote the formation of mineralization products.

[0127] To quantify the binding amount of Alizarin Red, the absorbance of stained cells at 562 nm was measured after destaining. Referring to Figure 11, ns indicates no significant difference between the two groups (P > 0.05). The OD values ​​of the blank control group and the model group were 0.24 and 0.76, respectively. Compared with the model group, the 100 μg / mL bird's nest peptide-calcium complex and 400 μg / mL bird's nest peptide-calcium complex treatment groups significantly increased the binding amount of Alizarin Red (P < 0.001), by 1.47 and 1.27, respectively. The 100 μg / mL bird's nest peptide-calcium complex group was higher than the 400 μg / mL group, but there was no significant difference between the two groups (P > 0.05). These results indicate that the bird's nest peptide-calcium complex can induce MC3T3-E1 cells to form more mineralized nodules, effectively promoting their mineralization capacity.

[0128] 9. The effect of the bird's nest peptide-calcium complex on the mRNA expression level of MC3T3-E1 cell-related factors. Osteoblasts are key cells involved in the dynamic process of bone formation and the regulation of bone remodeling. They not only secrete bone matrix but also synthesize collagen and new bone tissue through calcification mechanisms. Osteoblasts can secrete cytokines such as ALP, Runx2, CoLI, and OCN. Among them, ALP and Runx2 are related to early osteogenic differentiation, while OCN and CoLI are related to late osteogenic differentiation. These cytokines are the main phenotypic markers of osteoblast precursor differentiation during bone formation. In addition, Osterix and CoLI are also the main phenotypic markers of pre-osteoblast proliferation and differentiation during bone formation. Therefore, osteogenic differentiation-related genes such as ALP, CoLI, and OCN can be used to monitor the differentiation process of pre-osteoblasts into fully mature osteoblasts and serve as markers of osteoblast differentiation and bone formation, indirectly reflecting the degree of osteoblast differentiation and maturation.

[0129] Cell proliferation and mineralization are two independently regulated biological stages. The aforementioned results indicate that there was no significant difference in cell proliferation between the 100 μg / mL and 400 μg / mL groups, suggesting that the 100 μg / mL bird's nest peptide-calcium complex has reached a saturated proliferative effect. Therefore, considering both economic efficiency and bioavailability, the 100 μg / mL bird's nest peptide-calcium complex will be selected for further research.

[0130] MC3T3-E1 cells in the logarithmic growth phase were harvested, cell counts were performed, and cell concentration was adjusted to 1×10⁻⁶ cells. 5 Cells were seeded per well in 12-well plates and cultured in a 5% CO2, 37°C incubator until adherence. A blank control group was supplemented with DMEM medium. Cells were cultured in mouse bone marrow mesenchymal stem cell osteogenic induction differentiation medium containing 0 μg / mL (model group) and 100 μg / mL bird's nest peptide-calcium complex, respectively, for 14 days. The culture dishes were then removed, the medium was discarded, and total RNA was extracted from MC3T3-E1 cells using the Trizol method. The total RNA was adjusted to a uniform concentration, and cDNA was synthesized according to the reverse transcription kit instructions. The cDNA was then diluted to a uniform concentration and amplified using a real-time PCR reaction system prepared according to the kit instructions. β-actin was used as an internal control gene, and a 2... -△△Ct The relative mRNA expression level of the target gene was calculated using a method with three replicates per group. Primer sequences are shown in Table 6.

[0131] Table 6

[0132] Results: The aforementioned experimental results have shown that the bird's nest peptide-calcium complex significantly promotes osteoblast proliferation, differentiation, and mineralization. Based on this, this application further investigated the effect of the bird's nest peptide-calcium complex on the relative expression levels of osteoblast differentiation-related genes ALP, COLI, and OCN mRNA. As shown in Figure 12, after treatment with 100 μg / mL bird's nest peptide-calcium complex for 14 days, compared with the model group, the mRNA expression levels of ALP, COLI, and OCN in MC3T3-E1 cells were significantly increased (P<0.01), being 3.40, 2.07, and 2.16 times that of the model group, respectively. The results indicate that the bird's nest peptide-calcium complex can further promote osteoblast differentiation and maturation by significantly upregulating the expression of osteoblast differentiation-related genes.

[0133] 10. Calcium Retention Rate of Bird's Nest Peptide-Calcium Complex in a Simulated Gastrointestinal Environment. Dietary nutrients typically need to be digested in the stomach before being absorbed by the body in the intestines. The stability of calcium supplements in the gastrointestinal environment directly affects the body's absorption and utilization of calcium. In the gastrointestinal environment, H... +Factors such as proteases can induce the release of calcium from chelates, leading to the formation of insoluble precipitates and Ca(OH)2, thus reducing calcium bioavailability. Therefore, evaluating the stability of the bird's nest peptide-calcium complex in a simulated gastrointestinal environment is of great significance for clarifying its practical application value.

[0134] The preparation method for simulated gastrointestinal fluid is based on the method in the Pharmacopoeia of the People's Republic of China: The preparation method for simulated gastric fluid SGF is as follows: Take 0.384 mL of concentrated hydrochloric acid and add it to 80 mL of deionized water. After mixing, add 1 g of pepsin and dissolve it completely. Then add water to make up to 100 mL to obtain simulated human gastrointestinal fluid. At this time, the pH value of simulated gastric fluid is about 2.0.

[0135] Preparation method of simulated intestinal fluid SIF: Dissolve 0.68g KH2PO4 in 50 mL of water and adjust the pH to 6.8 with NaOH; dissolve 1g trypsin in a small amount of water, mix the two solutions and add water to 100 mL.

[0136] (1) To prepare a 10 mg / mL bird's nest peptide-calcium complex solution prepared in Example 1, 20 mL of the above solution was placed in a 100 mL Erlenmeyer flask. The pH was adjusted to 2.0 with 1 mol / L HCl solution. 20 mL of simulated gastric juice SGF was added. The mixture was shaken in a 37°C water bath (0 min, 15 min, 30 min, 60 min, 120 min). After shaking, the mixture was placed in boiling water for 5 min to inactivate the enzyme. After cooling the sample to room temperature, it was centrifuged at 8000 rpm for 10 min using a refrigerated centrifuge. The supernatant was placed in a digestion tube, and the calcium content was determined by inductively coupled plasma mass spectrometry to determine the free calcium content. A control group of 10 mg / mL bird's nest peptide-calcium complex solution was set up.

[0137]

[0138] (2) Simulated intestinal digestion: After the bird's nest peptide-calcium complex solution was digested with simulated gastric juice for 120 min, the enzyme was inactivated by boiling water bath for 5 min. The pH was adjusted to 7.6 with 0.5 mol / L NaOH solution. 5 mL of intestinal fluid SIF was added, and the mixture was shaken in a 37℃ water bath for (0 min, 15 min, 30 min, 60 min, 120 min). Then, the mixture was placed in boiling water for 5 min to inactivate the enzyme. After the sample was cooled to room temperature, it was centrifuged at 8000 rpm for 10 min using a refrigerated centrifuge. The supernatant was placed in a digestion tube, and the calcium content was determined by inductively coupled plasma mass spectrometry to determine the free calcium content. The control group was a bird's nest peptide-calcium complex solution with a concentration of 10 mg / mL.

[0139]

[0140] Results: As shown in Figure 13, letters a, b, c, and d indicate statistically significant differences; different letters represent significant differences, while the same letter represents no significant difference. During simulated gastrointestinal digestion, the calcium retention rate of the bird's nest peptide-calcium complex exhibited a digestion stage-dependent characteristic. In the simulated gastric digestion stage (acidic environment), the calcium retention rate of the bird's nest peptide-calcium complex remained relatively stable within 0–120 min, maintaining 45%–48%, indicating good stability in the gastric environment. Entering the simulated intestinal digestion stage (neutral environment), the calcium retention rate significantly increased with prolonged digestion time, rising from an initial 69% to 85% at 120 min, and the calcium retention rate at each time point was significantly higher than that of the gastric digestion group at the same time point (P<0.05). These results indicate that the bird's nest peptide-calcium complex prepared in this invention can maintain high calcium binding stability in the gastrointestinal tract, especially with a significantly improved calcium retention rate in the neutral intestinal environment. This suggests that calcium is less prone to dissociation and formation of insoluble precipitates, thus increasing the proportion of calcium absorbable in the intestine and making it more beneficial for human absorption and utilization.

[0141] In summary, the ion-modified bird's nest peptide complex of this application possesses both antioxidant activity and osteoblast differentiation-promoting bioactivity, exhibits good tolerance to the gastrointestinal digestive environment, and is beneficial for the human body's absorption and utilization of its nutrients. The method for preparing the ion-modified bird's nest peptide complex of this application can efficiently and stably prepare the product, with batch-to-batch consistency, simple operation, and suitability for large-scale application.

[0142] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are 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.

[0143] 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. An ion-modified bird's nest peptide complex that enhances bone density and promotes growth and development, characterized in that, It includes bird's nest peptides modified with metal ions; wherein the metal ion loading of the ion-modified bird's nest peptide complex is 15% to 25%; and the metal ion is calcium ion.

2. The ion-modified bird's nest peptide complex according to claim 1, characterized in that, The bird's nest peptides include polypeptides with a molecular weight of 500 Da to 3000 Da.

3. A method for preparing the ion-modified bird's nest peptide complex according to any one of claims 1-2, characterized in that, include: Bird's nest is pre-processed to obtain bird's nest peptides; The bird's nest peptide is modified with a metal salt to obtain a metal ion-modified bird's nest peptide complex; wherein, the modification treatment includes pulsed electric field treatment; and the metal salt is calcium chloride.

4. The method according to claim 3, characterized in that, The pretreatment includes stewing, enzymatic hydrolysis, and ultrafiltration.

5. The method according to claim 4, characterized in that, The enzymatic hydrolysis treatment uses a compound enzyme, which includes trypsin and flavor protease; the ratio of trypsin to flavor protease is (1~5):1; the amount of compound enzyme added is 4000 U / g~8000 U / g; the pH value of the enzymatic hydrolysis treatment is 7.0~8.0; the temperature of the enzymatic hydrolysis treatment is 50 ℃~55 ℃; and the time of the enzymatic hydrolysis treatment is 4 h~6 h.

6. The method according to claim 3, characterized in that, The molar ratio of the bird's nest peptide to the metal ion of the metal salt is (1~5):1; before the modification treatment, the pH value of the mixture of the bird's nest peptide and the metal salt is 6.5~8.

5.

7. The method according to claim 3, characterized in that, The electric field strength of the pulsed electric field treatment is 5 kV / cm to 30 kV / cm; the pulse frequency of the pulsed electric field treatment is 50 Hz to 200 Hz; the pulse width of the pulsed electric field treatment is 2 μs to 10 μs; and the processing time of the pulsed electric field treatment is 4 s to 10 s.

8. The method according to claim 3, characterized in that, Further includes: The modified solution is purified to obtain the metal ion-modified bird's nest peptide complex; the purification process includes ultrafiltration and drying.

9. A food or biological material, characterized in that, Includes the ion-modified bird's nest peptide complex according to any one of claims 1-2 or the ion-modified bird's nest peptide complex prepared according to any one of claims 3-8.

10. The use of the ion-modified bird's nest peptide complex according to any one of claims 1-2 or the ion-modified bird's nest peptide complex prepared according to any one of claims 3-8 in the preparation of food or biomaterials that promote bone health.

Images