A fish gelatin polypeptide nanomaterial prepared based on self-condensation reaction and its application

Abstract

The present invention belongs to the technical field of new materials, and particularly relates to a fish gelatin polypeptide nanomaterial prepared based on a self-condensation reaction and its application. The fish gelatin polypeptide nanomaterial has a core-shell structure, wherein: the core layer is hydrolyzed tannin, and the shell layer is calcium ions and fish gelatin polypeptide; the hydrolyzed tannin is connected to the calcium ions through a coordination bond, and the hydrolyzed tannin is connected to the fish gelatin polypeptide through a hydrogen bond. The present invention adopts a one-step method to cause a self-condensation reaction of fish gelatin polypeptide, hydrolyzed tannin and calcium salt in a polar organic solvent, forming a "core-shell" structure with calcium ion-polypeptide on the outside as the shell and hydrolyzed tannin on the inside as the core. The fish gelatin polypeptide nanomaterial prepared by the present invention has a particle size of 80-110 nm and a hydroxyproline content of 1.5-2.7 wt%; when used as a calcium supplement preparation, it has good ability to supplement bone collagen, improves the strength of bone tissue, and will not cause abnormal development of internal organs.

Description

Technical Field

[0001] The present invention belongs to the technical field of new materials, and particularly relates to a pharmaceutical preparation, specifically to a fish gelatin polypeptide nanomaterial prepared based on a self-condensation reaction and its application. Background Art

[0002] Calcium deficiency can cause a series of physiological and health diseases. For example, children have a relatively strong demand for calcium during the growth period. Long-term calcium deficiency will slow down children's growth and development, retard their intellectual development, cause poor bone calcification reaction, abnormal bone structure or even deformity, resulting in short stature and even rickets; calcium deficiency in young people is likely to lead to irritability, cramps, and poor bone calcification, thus triggering pathological fractures; calcium deficiency in middle-aged and elderly people as well as postmenopausal women is likely to cause symptoms such as osteoporosis and osteoproliferation, leading to physiological fractures, and there is also a risk of diseases such as hypertension, obesity, and colorectal cancer; calcium deficiency in pregnant women is not only unfavorable for the growth of the fetus but also affects the physical health of the pregnant woman herself. Therefore, ensuring an adequate calcium intake is of great significance to the human body.

[0003] In view of the problems of generally insufficient calcium intake and low calcium absorption rate in dietary calcium, in addition to reasonably arranging diet and adjusting the dietary structure in daily life and consuming more calcium-rich foods, for some specific populations, some calcium supplements should also be appropriately used to improve the calcium absorption rate. Currently, the calcium supplements circulating on the market can be mainly divided into three categories: inorganic calcium, organic acid calcium, and organic calcium. Among them: inorganic calcium salts mainly include calcium carbonate, calcium hydrogen phosphate, calcium hydroxide, calcium chloride, etc. This type of calcium supplement has a strong stimulating effect on people with low gastric acid secretion or gastric acid secretion disorders. Long-term use is likely to cause discomfort symptoms such as constipation and stones. Therefore, this type of calcium supplement cannot be blindly used and needs to be selected according to specific populations. Organic acid calcium salts are products prepared by a series of reactions between organic acids and inorganic calcium salts, such as calcium gluconate, calcium lactate, calcium pyruvate, calcium citrate, calcium acetate, etc. The disadvantage of this type of calcium supplement is that the calcium content is relatively low, and a large amount needs to be taken to achieve a certain effect of improving bone density. Organic calcium salts are also called chelated calcium, which refers to the chelation reaction of amino acids or polypeptides with calcium ions through ionic bonds, coordination bonds, or adsorption to generate amino acid chelated calcium or polypeptide chelated calcium. Although the activity of polypeptides is beneficial to promoting calcium absorption, there is also a defect that the polypeptides that can be chelated on the calcium surface are limited, resulting in an insignificant effect on promoting calcium absorption. In the case of the same calcium content, a relatively large dose of chelated calcium still needs to be taken.

[0004] Therefore, it is necessary to develop a fourth type of new calcium supplement material with high calcium content, high biological utilization rate, and no toxic side effects, which can combine the high calcium content of inorganic calcium and the active polypeptides of organic calcium, and increase the content of hydroxyproline in the material to improve the ability to supplement bone collagen and the strength of bone tissue. Summary of the Invention

[0005] The present invention provides a fish gelatin polypeptide nanomaterial prepared based on self-condensation reaction and its applications, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial option or create conditions.

[0006] To overcome the above technical problems, a first aspect of the present invention provides a fish gelatin polypeptide nanomaterial.

[0007] Specifically, a fish gelatin polypeptide nanomaterial has a core-shell structure, wherein: the core layer is hydrolyzed tannin, and the shell layer is calcium ions and fish gelatin polypeptide; the hydrolyzed tannin is connected to the calcium ions through a coordination bond, and the hydrolyzed tannin is connected to the fish gelatin polypeptide through a hydrogen bond.

[0008] As a highly respected tonic in traditional Chinese medicine health preservation, fish gelatin has a history of use in coastal areas of China for many years. Its production process is relatively simple. After the fish swim bladder is taken down, the blood film and fat layer on the swim bladder are cleaned, and it becomes raw fish gelatin after drying or sun drying. Fish gelatin contains a large amount of collagen polypeptide, which can effectively promote the absorption of calcium in animals. However, for the polypeptide chelated calcium directly prepared from fish gelatin polypeptide and calcium salt, the bonding effect between the polypeptide and calcium is not ideal, and the activity of the polypeptide cannot be fully exerted.

[0009] Tannin is a secondary metabolite secreted in plants. There are a large number of phenolic hydroxyl groups in tannin molecules, which enable it to undergo a coordination reaction with metal ions and also a hydrogen bond binding reaction with polypeptides or proteins.

[0010] During the research process, the applicant analyzed the molecular structure of valonea tannin using nuclear magnetic resonance carbon spectrum, and its 13 CNMR spectrum and its molecular structure are as Figure 1 shown. It can be Figure 1 seen that valonea tannin belongs to polyphenols with ellagic acid (HHDP) and gallic acid as extension units and open-ring glucose as the parent nucleus. From its molecular structure characteristics, valonea tannin belongs to typical hydrolyzed tannin. (Jiaman Liu, et al. Valonea Tannin: Tyrosinase Inhibition Activity, Structural Elucidation and Insights into the Inhibition Mechanism, Molecules, 26, 2747)

[0011] Meanwhile, the applicant analyzed the molecular structure of myrica tannin using nuclear magnetic resonance carbon spectrum, and its 13 CNMR spectrum and its molecular structure are as Figure 2 shown. It can beFigure 2 It can be known that myricetin belongs to polyphenols with flavan-3-ol as the monomer, and both the extension unit and the terminal unit are (epi)gallocatechin. From the characteristics of its molecular structure, it can be seen that myricetin belongs to typical condensed tannins. (BoTeng, et al. Comparison of Polyflavonoids in Bayberry Tanning Effluent and Commercial Bayberry Tannin: Prerequisite Information for Vegetable Tanning Effluent Recycling, Journal of Cleaner Production, 112, 972-979)

[0012] In addition, the applicant also used gel permeation chromatography (GPC) to measure the hydrodynamic radius of oak gall tannin and myricetin molecules under the conditions of different solvents (methanol-water, acetonitrile-water, acetone-water, and ethanol-water solution) as the mobile phase. In order to avoid the phenomenon that the phenolic hydroxyl group of tannin forms hydrogen bonds with the active groups on the GPC chromatographic column, resulting in distortion of the retention time of the results, during the measurement of the hydrodynamic radius, the content of organic solvent in the mobile phase is at least 50%, and the selected organic solvents are all polar organic solvents (methanol, ethanol, acetone, acetonitrile) that can play a role in hydrogen bond disruption. The hydrodynamic volume is as Figure 3 shown, where Figure 3 -A is the hydrodynamic volume of oak gall tannin in methanol-water solutions with different ratios; Figure 3 -B is the hydrodynamic volume of oak gall tannin in acetonitrile-water solutions with different ratios; Figure 3 -C is the hydrodynamic volume of oak gall tannin in acetone-water solutions with different ratios; Figure 3 -D is the hydrodynamic volume of oak gall tannin in ethanol-water solutions with different ratios; Figure 3 -E is the hydrodynamic volume of myricetin in methanol-water solutions with different ratios; Figure 3 -F is the hydrodynamic volume of myricetin in acetonitrile-water solutions with different ratios; Figure 3 -G is the hydrodynamic volume of myricetin in acetone-water solutions with different ratios; Figure 3 -H is the hydrodynamic volume of myricetin in ethanol-water solutions with different ratios.

[0013] From Figure 3It can be known that in an aqueous methanol solution, the hydrodynamic volume of hydrolyzable tannins represented by oak gall tannin is closely related to the ratio of methanol to water. The higher the relative content of methanol in the mobile phase, the smaller the GPC retention time of oak gall tannin. When the ratio of methanol to water in the mobile phase is 5:0, the retention time of oak gall tannin is 11 - 16 min; when the ratio is 5:1, the retention time is 10 - 15 min; when it is 5:3, it is 8 - 14 min; and when it is 5:5, it is 7.5 - 12.5 min. GPC belongs to size exclusion chromatography with a molecular sieve function. The shorter the retention time in the GPC chromatogram, the larger the hydrodynamic volume. Therefore, the above results indicate that the larger the proportion of methanol in the mobile phase, the smaller its molecular volume. This phenomenon not only occurs in methanol - aqueous solutions, but also in ethanol - aqueous solutions, acetonitrile - aqueous solutions, and acetone - aqueous solutions for oak gall tannin.

[0014] It should be noted that when the same experiment was carried out with condensed tannins represented by myricetin tannin, completely different results were observed. In the elution system with methanol - water as the mobile phase, when changing the water concentration, although the peak shape of tannin on GPC changed, in all the experiments involved, the retention time of myricetin tannin was 12.5 - 17.5 min. This indicates that the hydrodynamic radius of myricetin tannin did not change significantly. Similarly, in ethanol - aqueous solutions, acetonitrile - aqueous solutions, and acetone - aqueous solutions, the proportion of organic solvents had little effect on the hydrodynamic radius of oak gall tannin.

[0015] Zanchi et al. from the Institut de Physique Théorique et Physique des Hautes Energies in France used small - angle X - ray diffraction (SAXS) combined with model analysis and observed that there were certain differences in the molecular lengths of catechin hexamers (belonging to condensed tannins) in ethanol and in water. And this phenomenon was explained as follows: The molecules of tannins have a certain "molecular flexibility", and their molecular volume can change with the solution environment. (Zanchi et al. Rigidity, conformation and solvation of native and oxidized tannin macromolecules in water - ethanol solution, The Journal of Chemical Physics, 130, 245103) During the applicant's research process, it was found that there were significant differences in the degree to which the hydrodynamic radii of hydrolyzable tannins and condensed tannins were affected by solvents (as Figure 3 shown). The difference in the structures between condensed tannins and hydrolyzable tannins is the fundamental reason for this phenomenon.

[0016] Condensed tannins have flavan-3-ol as their structural units, which belong to planar structures, and the molecular skeleton is formed by connecting two benzene rings with a heterocyclic ring. The structural units are connected by C-C bonds. Due to the large steric hindrance between the structural units, the spatial structure of tannin molecules has a certain "rigidity". Therefore, in different solvent environments, the molecular volume of condensed tannins does not change much. The structural units of hydrolyzable tannins only contain 1 (gallic acid) or 2 (ellagic acid) benzene rings, and the structural units are connected by ester bonds. Therefore, the steric hindrance between the structural units is small, and the "flexibility" of the molecular structure is relatively significant. Increasing the proportion of organic solvents in the solvent environment forces the hydrophobic groups (benzene rings) in tannin molecules to approach and aggregate with each other, while exposing the hydrophilic groups (phenolic hydroxyl groups) fully outside. And this process of exposing phenolic hydroxyl groups driven by organic solvents requires the stereochemical structure of the molecule to change accordingly. The molecules of condensed tannins have strong "rigidity", and this change in stereochemical structure is not very significant. The molecules of hydrolyzable tannins have strong "flexibility" characteristics. Therefore, when the solvent environment is changed, the hydrodynamic radius of hydrolyzable tannins also changes significantly.

[0017] Based on this physicochemical phenomenon, the present invention utilizes the physicochemical characteristics of hydrolyzable tannins, namely "phenolic hydroxyl groups are exposed outside and benzene rings are aggregated inside", in a polar organic solvent environment to carry out the coordinated complexation reaction of hydrolyzable tannins with calcium ions and fish gelatin polypeptides. At this time, the complexation reaction and the hydrogen bond binding reaction between fish gelatin polypeptides and tannins mainly occur on the phenolic hydroxyl groups exposed on the surface, forming a "core-shell" structure with calcium ion-polypeptide as the "shell" outside and hydrolyzable tannins as the core inside. It is found that under the hydrogen bond action of hydrolyzable tannins, the binding ability of the material to fish gelatin polypeptides can be effectively improved, the content of hydroxyproline can be increased, thereby further promoting the absorption effect of calcium ions, improving the bone tissue strength, and at the same time improving the replenishing ability of bone collagen.

[0018] As a further improvement of the above solution, the average particle size of the fish gelatin polypeptide nanomaterial is 80-110 nm. The fish gelatin polypeptide nanomaterial with a small particle size is more conducive to the absorption of calcium ions and collagen.

[0019] As a further improvement of the above solution, the Zeta potential of the fish gelatin polypeptide nanomaterial is -6.70 mV to -8.2 mV.

[0020] The average potential of hydrolyzed tannins (such as oak gall tannin) in methanol solution is -28 mV. This is because oak gall tannin aggregates to form micelles in methanol solution, exposing phenolic hydroxyl groups on the surface of the micelles, and after the surface phenolic hydroxyl groups dissociate, the surface is negatively charged. The Zeta potential of polypeptides (such as fish gelatin polypeptide) is about +5 mV, which is caused by the dissociation of amino groups in the polypeptide. Calcium salts do not form colloids in solution, so almost no Zeta potential peak can be seen. However, after forming the fish gelatin polypeptide nanomaterial of the present invention, the Zeta potential of the nanoparticles is -6.70 mV to -8.2 mV. This potential is significantly different from that of fish gelatin polypeptide or oak gall tannin, further indicating the formation of the "core-shell" structure of the present invention.

[0021] As a further improvement of the above scheme, the content of hydroxyproline in the fish gelatin polypeptide nanomaterial is 1.5 - 2.7 wt%. The high content of hydroxyproline in the fish gelatin polypeptide nanomaterial of the present invention indicates that the content of the polypeptide combined with hydrolyzed tannins in the present invention is high, which is more conducive to promoting calcium absorption, supplementing bone collagen, and improving bone tissue strength.

[0022] As a further improvement of the above scheme, the hydrolyzed tannins are selected from at least one of oak gall tannin, Chinese gallnut tannin, and tannic acid. These hydrolyzed tannins all have structures and properties similar to those of the above oak gall tannin.

[0023] As a further improvement of the above scheme, the fish gelatin polypeptide is selected from at least one of the fish gelatin polypeptides of Boeseman's croaker, Miichthys miiuy, Pseudotolithus elongatus, and Larimichthys crocea. The molecular weights of these fish gelatin polypeptides are all relatively small, being small molecular peptides, and having a good effect of promoting calcium absorption. The molecular weights of these fish gelatin polypeptides are all relatively small, being small molecular peptides, and having a good effect of promoting calcium absorption.

[0024] The second aspect of the present invention provides a preparation method of a fish gelatin polypeptide nanomaterial.

[0025] Specifically, a preparation method of a fish gelatin polypeptide nanomaterial, the preparation method is used to prepare the fish gelatin polypeptide nanomaterial of the present invention, and the solvent used in the preparation method is a polar organic solvent, including the following steps:

[0026] Dissolve the hydrolyzed tannins in the solvent, perform primary stirring, and dropwise add a calcium salt solution, a surfactant, and a fish gelatin polypeptide during the stirring. After the dropping is completed, perform secondary stirring; then dropwise add an alkali solution, perform tertiary stirring, and then centrifuge and collect the precipitate; after washing and drying, obtain the fish gelatin polypeptide nanomaterial.

[0027] The present invention adopts a one-step method, in which fish glue polypeptide, hydrolyzed tannin and calcium salt undergo a self-condensation reaction in a polar organic solvent. Among them: the hydrolyzed tannin and the calcium salt undergo a coordination reaction to form a coordination chemical bond; at the same time, a hydrogen bond binding reaction occurs between the hydrolyzed tannin and the fish glue polypeptide. The coordination reaction and the hydrogen bond binding reaction mainly occur on the phenolic hydroxyl groups exposed on the surface of the tannin, forming a core-shell structure with calcium ion-polypeptide on the outside and hydrolyzed tannin on the inside.

[0028] As a further improvement of the above solution, the mass ratio of the hydrolyzed tannin, the calcium salt solution, the surfactant and the polypeptide is 1:(0.02 - 4):(0.03 - 0.64):(0.1 - 3.5).

[0029] As a further improvement of the above solution, the preparation method of the fish glue polypeptide nanomaterial includes the following steps:

[0030] Dissolve 1 part by mass of hydrolyzed tannin in 50 - 1200 parts by mass of solvent, and perform primary stirring treatment for 5 - 15 min using a high-speed stirrer with a rotation speed of 8000 - 15000 rpm. During stirring, add 0.02 - 4 parts by mass of calcium salt solution, 0.03 - 0.64 parts by mass of surfactant, and 0.1 - 3.5 parts by mass of fish glue polypeptide solution dropwise. After the dropwise addition is completed, perform secondary stirring treatment for 3 - 15 min, then add 2.3 - 10 parts by mass of alkali solution dropwise and perform tertiary stirring treatment for 10 - 30 min. Subsequently, centrifuge the solution and collect the precipitate, wash the precipitate with solvent, and dry it to obtain the fish glue polypeptide nanomaterial.

[0031] Preferably, the polar organic solvent is selected from at least one of methanol, ethanol, acetone, and acetonitrile.

[0032] Preferably, the calcium salt solution is selected from at least one of calcium chloride solution, calcium sulfate, calcium chlorate, and calcium perchlorate solution; more preferably, the concentration of the calcium salt solution is 3 - 5 wt%.

[0033] Preferably, the surfactant is selected from at least one of fatty alcohol polyoxyethylene ether sulfate (abbreviation: AES), higher fatty alcohol sulfate (abbreviation: AS), and dodecyl trimethyl ammonium chloride (abbreviation: anti-sticking agent DT).

[0034] Preferably, the alkali solution is selected from at least one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, and potassium bicarbonate.

[0035] The third aspect of the present invention provides an application of a fish glue polypeptide nanomaterial.

[0036] Specifically, a calcium supplement preparation contains the fish glue polypeptide nanomaterial of the present invention.

[0037] The above technical solution of the present invention has at least the following technical effects or advantages compared with the prior art:

[0038] The fish gelatin polypeptide nanomaterial of the present invention has a core-shell structure, wherein: the core layer is hydrolyzed tannin, and the shell layer is calcium ions and polypeptides. During preparation, a one-step method is adopted to cause a self-condensation reaction of fish gelatin polypeptide, hydrolyzed tannin and calcium salt in a polar organic solvent. The hydrolyzed tannin and calcium salt undergo a coordination reaction to form a coordination chemical bond; at the same time, a hydrogen bond binding reaction between the hydrolyzed tannin and fish gelatin polypeptide occurs. The coordination reaction and hydrogen bond binding reaction mainly occur on the phenolic hydroxyl groups exposed on the tannin surface, forming a "core-shell" structure with calcium ion-polypeptide on the outside as the shell and hydrolyzed tannin on the inside as the core. The fish gelatin polypeptide nanomaterial prepared by the present invention has a particle size of 80-110 nm and a hydroxyproline content of 1.5-2.7 wt%; when used as a calcium supplement preparation, it has good bone collagen supplement ability, improves bone tissue strength, and will not cause abnormal development of internal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 For the 13 CNMR spectrum and its molecular structure of valonea tannin;

[0040] Figure 2 For the 13 CNMR spectrum and its molecular structure of myrica tannin;

[0041] Figure 3 For the hydrodynamic volume of valonea tannin and myrica tannin in solvents with different ratios;

[0042] Figure 4 For the schematic diagram of the "core-shell" structure formed by tannin and metal ions and the SEM images of nanoparticles formed by valonea tannin and myrica tannin and calcium ions;

[0043] Figure 5 For the DSC diagram of fish gelatin polypeptide nanomaterial particles and their reactants;

[0044] Figure 6 For the Zeta potential diagram of fish gelatin polypeptide nanomaterial particles and their reactants;

[0045] Figure 7 For the FT-IR diagram of fish gelatin polypeptide nanomaterial particles and their reactants;

[0046] Figure 8 For the size comparison diagram of the tibias of rats taking the samples of Example 1 and Comparative Examples 1-4. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will be specifically described below in conjunction with embodiments to facilitate the understanding of those skilled in the art. It is necessary to specifically point out here that the embodiments are only used to further illustrate the present invention and cannot be construed as a limitation on the protection scope of the present invention. Those skilled in the art who make non-essential improvements and adjustments to the present invention based on the above-mentioned invention content should still fall within the protection scope of the present invention. At the same time, for the raw materials not specifically described below, they are all commercially available products; the process steps or preparation methods not specifically mentioned are all process steps or preparation methods well-known to those skilled in the art.

[0048] Example 1

[0049] A fish glue polypeptide nanomaterial has a core-shell structure, wherein: the core layer is valonea tannin, and the shell layer is calcium ions and small yellow croaker fish glue polypeptide; valonea tannin is connected to calcium ions through a coordination bond, and valonea tannin is connected to small yellow croaker fish glue polypeptide through a hydrogen bond.

[0050] A preparation method of a fish glue polypeptide nanomaterial includes the following steps:

[0051] Dissolve 1 part by mass of valonea tannin in 50 parts by mass of methanol, initially stir for 10 min on a high-speed stirrer with a rotation speed of 8000 rpm, and dropwise add 0.02 part by mass of a calcium chloride solution with a concentration of 4 wt%, 0.03 part by mass of fatty alcohol polyoxyethylene ether sulfate (AES), and 0.1 part by mass of small yellow croaker fish glue polypeptide solution during stirring. After the dropping is completed, continue secondary stirring for 3 min, then dropwise add 2.3 parts by mass of sodium hydroxide solution, perform tertiary stirring for 10 min, then centrifuge the solution and collect the precipitate, wash the precipitate with methanol, and dry it to obtain the fish glue polypeptide nanomaterial sample of this example.

[0052] Examples 2-10

[0053] The differences between Examples 2-10 and Example 1 lie in: the preparation process parameters of the fish glue polypeptide nanomaterial, the specific selection and dosage of each raw material, as shown in Table 1 specifically.

[0054] Table 1: Preparation conditions of the fish glue polypeptide nanomaterial in Examples 2-10

[0055]

[0056] Comparative Example 1

[0057] The difference between Comparative Example 1 and Example 1 is only that: in Comparative Example 1, myrica tannin is used to replace valonea tannin, and the types, addition amounts of other raw materials and the preparation method are the same as those in Example 1.

[0058] Comparative Example 2

[0059] A preparation method of a fish glue polypeptide nanomaterial, comprising the following steps:

[0060] Mix 0.02 parts by mass of a calcium chloride solution with a concentration of 4 wt%, 0.03 parts by mass of fatty alcohol polyoxyethylene ether sulfate (AES), and 0.1 parts by mass of a corvina fish glue polypeptide solution, stir for 3 min, then add 2.3 parts by mass of sodium hydroxide solution dropwise, stir for 10 min, then centrifuge the solution and collect the precipitate, wash the precipitate with methanol, and dry to obtain the fish glue polypeptide nanomaterial sample of this comparative example.

[0061] Comparative Example 3

[0062] Comparative Example 3 is commercially available calcium carbonate.

[0063] Comparative Example 4

[0064] Comparative Example 4 is a blank group of water.

[0065] Performance test

[0066] 1. Microstructural analysis

[0067] Figure 4 It is the schematic diagram of the core-shell structure and the SEM image of the samples prepared in Example 1 and Comparative Example 1. Among them Figure 4 -A is the schematic diagram of the "core-shell" structure formed by valonea tannin, fish glue polypeptide and metal calcium ions; Figure 4 -B is the SEM image of the sample prepared in Example 1; Figure 4 -C is the SEM image of the sample prepared in Comparative Example 1. It can be seen from Figure 4 that: Since the three-dimensional configuration of hydrolyzable tannins (valonea) is easily regulated by the solvent system, the coordination reaction is carried out in methanol, and the particle size of the calcium-tannin nanoparticles formed is about 100 nm ( Figure 4 -B), but the three-dimensional configuration of condensed tannins is not easily regulated by the solvent system, and the particle size of the particles formed in methanol is about 3 μm ( Figure 4 -C). It can be seen from this that in a polar solvent environment, forming nanoparticles with polypeptide-metal ions is unique to hydrolyzable tannins, while the molecular structure of condensed tannins is relatively rigid, and the formed particles are too large to obtain nanoparticles, thus affecting the calcium supplement effect.

[0068] 2. Differential scanning calorimetry (DSC) analysis

[0069] Figure 5 It is the DSC diagram of the fish glue nano-polypeptide sample prepared in Example 1 and its reactants. It can be seen from Figure 5It can be seen that: at 119.15 °C, an endothermic peak appeared in the fish gelatin polypeptide nanoparticles. There is no such endothermic peak in CaCl2, fish gelatin polypeptide, and oak gall tannin used for synthesizing the nanoparticles, indicating that the endothermic phenomenon is caused by the destruction of the nanoparticle structure.

[0070] 3. Zeta potential analysis

[0071] Figure 6 It is the Zeta potential diagram of the fish gelatin nanopolypeptide sample prepared in Example 1 and its reactants. From Figure 6 It can be seen that: the average potential of oak gall tannin in methanol solution is -28 mV. This is because oak gall tannin aggregates to form micelles in methanol solution, exposing phenolic hydroxyl groups on the surface of the micelles, and a large number of negatively charged groups are carried on the surface after the dissociation of surface phenolic hydroxyl groups. The Zeta potential of fish gelatin polypeptide is about +5 mV, which is caused by the dissociation of amino groups in the polypeptide; calcium chloride does not form a colloid in solution, so the Zeta potential peak can hardly be seen. However, after the formation of fish gelatin polypeptide nanoparticles, the Zeta potential of the particles is about -6.7 mV. This potential is significantly different from that of fish gelatin polypeptide or oak gall tannin, indicating the formation of a "core-shell" structure.

[0072] 4. Fourier transform infrared spectroscopy (FT-IR) analysis

[0073] Figure 7 It is the FT-IR diagram of the fish gelatin nanopolypeptide sample prepared in Example 1 and its reactants. From Figure 7 It can be seen that: the hydroxyl stretching vibration peaks of oak gall tannin and fish gelatin polypeptide are located at 3212 cm -1 and 3281 cm -1 , while the hydroxyl stretching vibration of the nanoparticles has a significant red shift of about 120 cm -1 (2071 cm -1 ). This result means that the hydroxyl group is the main reaction site of the nanoparticles. In addition, the carbonyl peak of the nanoparticles (1698 cm -1 ) and the carbonyl peak of oak gall tannin (1698 cm -1 ) appear at the same position, indicating that the carbonyl group mainly comes from oak gall tannin and does not participate in the coordination reaction. The absorption peaks at 1396 cm -1 and 881 cm -1 mainly come from the coordination bonds formed by calcium ions (1392 cm -1 , 870 cm -1 ) and oak gall tannin. In addition, the absorption peak at 1238 cm -1 in fish gelatin polypeptide appears in the fish gelatin nanopolypeptide particles, indicating that fish gelatin polypeptide exists in the nanoparticles.

[0074] 5. Material properties

[0075] For the samples prepared in Examples 1-10 and Comparative Examples 1 and 2, the positions of their heat absorption peaks and average potentials were tested using DSC and a Zeta potential analyzer respectively; the average particle size was tested using dynamic light scattering analysis (DLS); after the samples were degraded by the thermal-acid degradation method, an amino acid analyzer was used to test the hydroxyproline content in each product, and the test results are shown in Table 2.

[0076] Table 2: Performance parameter table of the samples in Examples 1-10

[0077] Sample DSC Absorption Peak (°C) Average Potential (mv) Average Particle Size (nm) Hydroxyproline Content (%) Example 1 115 -6.70 80 1.8 Example 2 114 -7.13 83 1.6 Example 3 113 -7.66 86 1.5 Example 4 118 -8.20 91 1.9 Example 5 120 -8.00 95 2.1 Example 6 116 -8.11 110 2.3 Example 7 110 -7.42 105 2.7 Example 8 113 -7.49 92 1.9 Example 9 116 -6.92 87 1.8 Example 10 115 -6.83 107 1.6 Comparative Example 1 —— —— 3000 —— Comparative Example 2 62 No Peak Appeared No Peak Appeared ——

[0078] Calcium supplementation effect experiment:

[0079] Using the samples prepared in Example 1 and Comparative Examples 1-4 as calcium supplementation preparations, SD rats were gavaged for 8 weeks, with a gavage volume of 300 mg / kg body weight / day, once a day. After the gavage, the tibias of the SD rats were taken, their sizes were observed and their lengths were measured. The results are as Figure 8 shown. It can be Figure 8 seen that for the SD rats gavaged with the sample of Example 1, both the femoral diameter and the length of the tibia are significantly larger than those of Comparative Examples 1-4.

[0080] At the same time, the bone calcium and bone hydroxyproline contents in the tibias of the SD rats gavaged for 8 weeks were tested, and a universal testing machine was used to test the maximum load, flexural modulus and flexural strength of the tibias. The test results are shown in Table 3.

[0081] Table 3: Performance comparison table of the tibias of SD rats gavaged with different samples

[0082]

[0083]

[0084] It can be seen from Table 3 that for the SD rats fed with the sample of Example 1, the hydroxyproline content in their tibias is significantly higher than that of Comparative Examples 1-4, indicating that Example 1 has a better ability to supplement bone collagen. In addition, the maximum load, flexural modulus and flexural strength of the tibias of the SD rats are significantly higher than those of Comparative Examples 1-4, indicating that Example 1 can make the tibias stronger and tougher, and has a better ability to improve the strength of bone tissue.

[0085] In addition, the relative weights of the organs and the body weight gain of the SD rats gavaged for 8 weeks were also tested, and the test results are shown in Table 4.

[0086] Table 4: Table of relative organ weights and body weight growth rates of SD rats gavaged with different samples

[0087] Grouping Heart (%) Lung (%) Liver (%) Spleen (%) Kidney (%) Body Weight Growth Rate (%) Example 1 <![CDATA[0.36±0.06 a > <![CDATA[0.73±0.16 a > <![CDATA[3.34±0.67 a > <![CDATA[0.26±0.02 a > <![CDATA[0.71±0.04 a > <![CDATA[30.23±1.61 a > Comparative Example 1 0.35±0.06 0.76±0.11 3.26±0.77 0.28±0.06 0.73±0.09 27.41±3.66 Comparative Example 2 0.35±0.05a <![CDATA[0.75±0.07 a > <![CDATA[2.92±0.29 a > <![CDATA[0.27±0.02 a > <![CDATA[0.67±0.0a a > <![CDATA[25.38±4.39 a > Comparative Example 3 0.36±0.05a <![CDATA[0.78±0.09 a > <![CDATA[2.79±0.28 a > <![CDATA[0.21±0.03 a > <![CDATA[0.69±0.04 a > <![CDATA[28.98±6.43 a a]]> Comparative Example 4 0.34±0.05a <![CDATA[0.85±0.04 a > <![CDATA[3.12±0.39 a > <![CDATA[0.25±0.05 a > <![CDATA[0.74±0.08 a > <![CDATA[27.63±5.34 a >

[0088] As can be seen from Table 4, for the SD rats fed with the samples of Example 1, there were no significant differences in the relative weights of their internal organs compared with Comparative Example 4, and there were also no statistical differences in the body weight growth rates of the rats compared with Comparative Examples 1-4, indicating that Example 1 would not cause abnormal development of the internal organs of SD rats.

[0089] For those of ordinary skill in the art to which the present invention pertains, several simple deductions or substitutions can be made without departing from the concept of the present invention, without the need for creative labor. Therefore, any simple improvements made by those skilled in the art based on the disclosure of the present invention should fall within the protection scope of the present invention. The above embodiments are the preferred embodiments of the present invention, and all processes similar to the present invention and equivalent changes made thereto should fall within the protection scope of the present invention.

Claims

1. A fish gelatin polypeptide nanomaterial, characterized in that, The fish gelatin polypeptide nanomaterial has a core-shell structure, wherein: the core layer is hydrolyzed tannin, and the shell layer is calcium ions and fish gelatin polypeptide; the hydrolyzed tannin is connected to the calcium ions through a coordination bond, and the hydrolyzed tannin is connected to the fish gelatin polypeptide through a hydrogen bond; The hydrolyzed tannin is selected from at least one of oak gall tannin, gallnut tannin, and tannic acid; The fish gelatin polypeptide is selected from at least one of the fish gelatin polypeptides of Boeseman's croaker, Miichthys miiuy, Pseudotolithus elongatus, and Larimichthys crocea. ​ 2. The fish glue polypeptide nanomaterial according to claim 1, wherein The average particle size of the fish gelatin polypeptide nanomaterial is 80-110 nm.

3. The fish gelatin polypeptide nanomaterial according to claim 1, wherein The Zeta potential of the fish gelatin polypeptide nanomaterial is -6.70 mV to -8.2 mV.

4. The fish gelatin polypeptide nanomaterial according to claim 1, wherein The hydroxyproline content in the fish gelatin polypeptide nanomaterial is 1.5-2.7 wt%.

5. A preparation method of fish glue polypeptide nanomaterial, characterized in that, The preparation method is used to prepare the fish gelatin polypeptide nanomaterial according to any one of claims 1 to 4. The solvent used in the preparation method is a polar organic solvent, and it includes the following steps: Dissolve the hydrolyzed tannin in the solvent, perform primary stirring, and dropwise add a calcium salt solution, a surfactant, and fish gelatin polypeptide during the stirring. After the dropping is completed, perform secondary stirring; then dropwise add an alkali solution, perform tertiary stirring, and then centrifuge and collect the precipitate; after washing and drying, obtain the fish gelatin polypeptide nanomaterial.

6. The preparation method of the fish glue polypeptide nanomaterial according to claim 5, characterized in that, The mass ratio of the hydrolyzed tannin, the calcium salt solution, the surfactant, and the fish gelatin polypeptide is 1:(0.02-4):(0.03-0.64):(0.1-3.5).

7. The preparation method of the fish glue polypeptide nanomaterial according to claim 5, wherein The polar organic solvent is selected from at least one of methanol, ethanol, acetone, and acetonitrile; The calcium salt solution is selected from at least one of calcium chloride solution, calcium sulfate, calcium chlorate, and calcium perchlorate solution; The surfactant is selected from at least one of fatty alcohol polyoxyethylene ether sulfate, higher fatty alcohol sulfate, and dodecyl trimethyl ammonium chloride; The alkali solution is selected from at least one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, and potassium bicarbonate.

8. A calcium supplement, characterized in that, The calcium supplement contains the fish gelatin polypeptide nanomaterial according to any one of claims 1 to 4.

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