Bone collagen polypeptide with effect of improving bone density and preparation method thereof

Abstract

This application relates to the technical field of collagen peptide preparation, and in particular to a bone collagen peptide with the effect of improving bone density and its preparation method. The method includes: swelling pig bone, bovine bone, and artificially bred second-generation giant salamander bone in a polar dissociation medium to obtain a collagen suspension; performing solid-liquid separation on the collagen suspension, collecting the precipitate and washing it to neutral, then resuspending it to obtain a purified collagen system; adding a natural polyphenol compound to the purified collagen system and reacting it under a pulsed microwave field to obtain a primary polymer solution; adding a targeted endonuclease-limiting enzyme system to the primary polymer solution for enzymatic hydrolysis, followed by adding a polycarboxylic acid solution to adjust the reaction system to a weakly acidic range for enzyme inactivation, obtaining an enzymatic hydrolysate; adding a strong proton donor acid to the enzymatic hydrolysate for reaction, and then separating to obtain the bone collagen peptide. This application can overcome the defect of peptide chain conformational collapse and inactivation caused by high-temperature extraction, achieving the maintenance of spatial rigidity to improve bone density.

Description

Technical Field

[0001] This application relates to the technical field of collagen peptide preparation, and in particular to a bone collagen peptide with the effect of improving bone density and a method for preparing the same. Background Technology

[0002] Collagen is the basic matrix for maintaining the morphology and structure of animal tissues and organs. It is rich in characteristic amino acids such as glycine, proline, and hydroxyproline. Collagen peptides obtained through hydrolysis are often used as active ingredients to intervene in specific physiological microenvironments to improve bone strength. In order to effectively target and chimeric specific receptors in vivo and induce osteogenic signaling pathway activation, collagen peptides in bone extraction systems must maintain their unique polyproline left-handed superhelical three-dimensional topological conformation and spatial rigidity as much as possible during free precipitation. This allows them to resist complete physical structural penetration and deconstruction when encountering violent degradation by hydrolytic enzymes after oral administration across the gastrointestinal barrier, thus retaining a very small proportion of the original macromolecules to leak into the blood.

[0003] Chinese invention patent CN112501229B discloses a production process for bovine bone collagen peptides. The process involves pre-treating washed bovine bones by soaking them in a sodium hydroxide solution, then pulverizing them and placing them in an aqueous system at 120-135 degrees Celsius for high-temperature cooking to obtain a crude extract. After defatting and separation, the extract is subjected to ultrasonic-assisted hydrolysis at 55-60 degrees Celsius using a complex protease and papain. Subsequently, a microwave field is applied to continuously heat the system to 80-100 degrees Celsius for high-temperature enzyme inactivation. After cooling, a mixed organic solvent consisting of isopropanol, acetone, and ethyl acetate is added to promote peptide dehydration and aggregation. The supernatant is removed by centrifugation and spray-dried to obtain powdered collagen peptides with a molecular weight distribution of 500-1000 Daltons.

[0004] In extraction processes involving highly cross-linked mammalian and other complex bone matrices from multiple sources, the continuous high-temperature thermal stress and non-specific enzymatic interventions applied by the aforementioned processes can easily lead to irreversible degradation of the system's microstructure. High-temperature cooking at 120-135 degrees Celsius and continuous microwave heating at 80-100 degrees Celsius at the end inject intense thermodynamic kinetic energy into the extraction system, sufficient to completely sever and destroy the intramolecular and intermolecular hydrogen bond networks that maintain the characteristic helical microregions of bone collagen. This, coupled with the random degradation of the complex enzyme system lacking collagen-specific cleavage sites in the free state, results in extreme damage to the peptide chain structure. This intense thermodynamic shock and chemical bond breakage force the left-handed superhelical rigid microregions of the polypeptide chain to undergo irreversible entropy-increasing collapse, causing the spatial topology to be completely reduced to a random coil state. Limited by the disintegration of the peptide conformation, the low-temperature polar organic solvent precipitation process at the end of the system can only change the macroscopic solubility of the substance by utilizing the difference in dielectric constant. It cannot restore the lost three-dimensional rigid network at the microscopic level. As a result, the free short peptides are easily degraded and disintegrated by non-specific digestive enzymes during oral physiological application, losing the material basis for leakage across the membrane into the blood, and thus completely losing the ability to spatially specificly embed and activate the target receptors of the bone microenvironment. Summary of the Invention

[0005] To address the defect of peptide chain conformational collapse and inactivation caused by high-temperature extraction and to maintain spatial rigidity to improve bone density, this application provides a bone collagen polypeptide with the effect of improving bone density and its preparation method.

[0006] Firstly, this application provides a collagen polypeptide with the effect of improving bone density, employing the following technical solution: a collagen polypeptide with the effect of improving bone density, wherein the collagen polypeptide comprises a fraction with a molecular weight between 1000 and 3000 Da and a mass percentage greater than 60%, and exhibits a negative absorption peak at 198±2 nm on circular dichroism spectroscopy, and the collagen polypeptide is prepared by a method comprising the following steps: Pig bones, cattle bones, and artificially bred second-generation giant salamander bones were swollen in a polar dissociation medium to obtain a collagen suspension. The collagen suspension was subjected to solid-liquid separation, the precipitate was collected and washed with deionized water until neutral, and then resuspended to obtain a purified collagen system. A natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was added to the purified collagen system and reacted under a pulsed microwave field to obtain a primary polymer solution. A targeted endonuclease-limiting enzyme system was added to the primary polymer solution for enzymatic hydrolysis. Subsequently, a polycarboxylic acid solution was added to adjust the reaction system to a weakly acidic range to inactivate the enzyme, resulting in an enzymatic hydrolysate. The collagen polypeptide was obtained by adding a strong proton donor acid containing an ethylenediol structure to the enzymatic hydrolysate and reacting it.

[0007] Optionally, the polar dissociation medium is a composite aqueous system comprising 1.0~3.0 mol / L urea, 0.05~0.2 mol / L L-arginine and 0.2~0.8 mol / L lactic acid.

[0008] Optionally, the collagen polypeptide contains a polyproline characteristic sequence; the natural polyphenol compound binds to the polyproline characteristic sequence via π-CH interactions and hydrogen bonds, and the strong proton donor acid strips the hydrogen bond network of the natural polyphenol compound.

[0009] Secondly, this application provides a preparation method for a bone collagen polypeptide with the effect of improving bone density, which adopts the following technical solution: A method for preparing a bone collagen polypeptide with the effect of improving bone density includes the following steps: Step S1: The pig bones, cattle bones and artificially bred second-generation giant salamander bones are swollen in a polar dissociation medium to obtain a collagen suspension. The collagen suspension is subjected to solid-liquid separation, the precipitate is collected and washed with deionized water until neutral, and then resuspended to obtain a purified collagen system. Step S2: Add a natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups to the purified collagen system, and react it under a pulsed microwave field at 40.5~41.5℃ to obtain a primary polymer solution; Step S3: Adjust the pH of the initial polymer solution to 6.7-6.9, add a targeted endonuclease-limiting enzyme system for enzymatic hydrolysis, and then add a polycarboxylic acid solution to adjust the pH of the reaction system to 3.8-4.2 to obtain the enzymatic hydrolysate; Step S4: Under the condition that the pH of the reaction system is 3.8~4.2, a strong proton donor acid containing an ethylenediol structure is added to the enzymatic hydrolysate for reaction. After separation, the collagen polypeptides of pig bone, bovine bone and artificially bred second-generation giant salamander bone are obtained.

[0010] Optionally, in step S2, the pulsed microwave field adopts a discontinuous pulse mode of alternating on and off, wherein the single on time is 4~6s and the single off time is 8~12s.

[0011] Optionally, in step S3, the targeted endonuclease limiting enzyme system consists of collagen endonuclease and neutral protease; the polycarboxylic acid solution is a citric acid solution.

[0012] Optionally, in step S2, the natural polyphenolic compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups is selected from at least one of epigallocatechin gallate, oligomeric proanthocyanidins with a molecular weight of less than 800 Da, ellagic acid, gallic acid, or rosmarinic acid.

[0013] Optionally, in step S4, the strong proton donor acid containing an enediol structure is selected from ascorbic acid or isoascorbic acid.

[0014] Optionally, in step S3, the concentration of the citric acid solution is 0.8~1.2 mol / L and it is in a pre-cooled state below room temperature.

[0015] Optionally, in step S4, the reaction is carried out at 20~30°C, the concentration of ascorbic acid or isoascorbic acid is 0.3~0.8 mol / L, and the separation includes introducing the reaction system after the reaction into a hydrophilic ultrafiltration membrane module, and collecting the retentate that has passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and is retained by an ultrafiltration membrane with a molecular weight cutoff of 1 kDa.

[0016] In summary, this application includes the following beneficial technical effects: 1. In this application, after swelling in a polar dissociation medium, a natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups is reversibly anchored to the characteristic sequence of polyproline under a pulsed microwave field. Subsequently, after enzymatic hydrolysis and enzyme inactivation by adjusting the polycarboxylic acid to a weakly acidic range, the hydrogen bond network is stripped by a strong proton donor acid containing an enediol structure. This results in the obtained collagen polypeptide having a molecular weight fraction between 1000 and 3000 Da accounting for more than 60% of the mass and retaining a negative absorption peak at 198±2 nm in circular dichroism spectroscopy. This conformational retention property increased femoral bone mineral density after gavage intervention in an ovariectomized osteoporosis model.

[0017] 2. This application uses a composite aqueous system containing urea, L-arginine and lactic acid as a polar dissociation medium. During the swelling stage, it weakens the non-covalent association between collagen fibers and inhibits the re-aggregation of chains. In conjunction with citric acid solution, the pH of the enzymatically hydrolyzed system is adjusted to 3.8~4.2 under pre-cooling conditions below room temperature to achieve rapid enzyme inactivation. This effectively reduces the free amino content of the final product and weakens the non-target hydrolysis of peptide chains during the separation stage, thereby increasing the enrichment ratio of the 1000~3000Da target fraction.

[0018] 3. This application avoids polyphenol oxidation and cross-linking induced by local overheating during the anchoring of natural polyphenol compounds by controlling the reaction temperature at 40.5~41.5℃ in a pulsed microwave field and using a discontinuous pulse mode with alternating on and off. Combined with the controllable stripping of hydrogen bond networks by ascorbic acid or isoascorbic acid under pH 3.8~4.2 conditions, it achieves both low polyphenol residue and high cell compatibility in the final product. In the MC3T3-E1 cell model, it shows an increase in ALP activity and mineralized nodule formation ability. Attached Figure Description

[0019] Figure 1Micro-CT three-dimensional reconstruction microstructure of the femoral metaphysis of rats in the sham surgery group (Sham group) provided in this embodiment of the invention; Figure 2 Micro-CT three-dimensional reconstruction microstructure of the femoral metaphysis of rats in the model control group (OVX group) provided in this embodiment of the invention; Figure 3 Micro-CT three-dimensional reconstruction microstructure of the femoral metaphysis of a group of rats provided in the embodiments of the present invention; Figure 4 Micro-CT three-dimensional reconstruction microstructure of the femoral metaphysis of rats in Example 2 of this invention; Figure 5 A chromatographic comparison of the molecular weight distribution of the products of Example 2 and Comparative Example 1 provided for embodiments of the present invention; Figure 6 Comparison of circular dichroism characteristic absorption peaks of the products of Example 2 and Comparative Example 1 provided in this embodiment of the invention. Detailed Implementation

[0020] The following combination Figures 1-6 This application will be described in further detail.

[0021] This application discloses a collagen polypeptide with bone density-enhancing effects and its preparation method. Pig bones, bovine bones, and artificially bred second-generation giant salamander bones are preferably sourced from qualified food-grade or pharmaceutical-grade raw material suppliers. Pig and bovine bones are preferably selected from long bone segments, while artificially bred second-generation giant salamander bones are preferably selected from trunk and limb bones. The raw materials are cleaned, dried, and then pulverized and passed through a 60-mesh sieve to obtain mixed bone powder. The bone powders are mixed in a 1:1:1 mass ratio. This mixing configuration balances the high hydroxyproline content in mammalian bone matrix with the relatively easily dissociable collagen segments in amphibian bone matrix, allowing for a broader collagen polypeptide sequence distribution base during subsequent limited enzymatic hydrolysis. If the proportion of pig bone is too high, the cross-linking density of the system increases, and the swelling rate decreases; if the proportion of artificially bred second-generation giant salamander bones is too high, locally heat-sensitive segments are more likely to loosen prematurely under microwave treatment, leading to a shift in molecular weight distribution towards the lower end.

[0022] The polar dissociation medium employs a composite aqueous system comprising 1.0–3.0 mol / L urea, 0.05–0.2 mol / L L-arginine, and 0.2–0.8 mol / L lactic acid. Urea acts as a reversible hydrogen bond perturbation component, weakening non-covalent association between collagen fibers and reducing the activation energy of interfacial desorption in the early stages of swelling. L-arginine, with its guanidine group, exhibits strong hydration capacity, inhibiting the re-aggregation of collagen segments during swelling and reducing the risk of local collapse caused by exposed hydrophobic regions. Lactic acid provides mild acidity and promotes the loosening of the inorganic mineral phase, allowing collagen fiber bundles to be released without excessive acid hydrolysis. When urea concentration is below 1.0 mol / L, insufficient hydrogen bond perturbation hinders the full unfolding of collagen fiber bundles within the bone meal, making it difficult for subsequent natural polyphenol compounds to enter the pores. When urea concentration is above 3.0 mol / L, excessive rearrangement of the outer hydration shell of the collagen chain leads to premature loosening of some weakly stable triple-stranded segments, resulting in a weakened negative absorption peak at 198±2 nm in subsequent circular dichroism spectroscopy. When L-arginine concentration is below 0.05 mol / L, exposed segments are prone to secondary association during swelling. When L-arginine concentration is above 0.2 mol / L, the ionic strength of the system increases, and the binding of natural polyphenol compounds to the characteristic sequence of polyproline is competitively affected. When lactic acid concentration is below 0.2 mol / L, demineralization is insufficient, resulting in lower viscosity of the collagen suspension and higher bone meal residue. When L-arginine concentration is above 0.8 mol / L, the risk of acid-catalyzed chain scission increases, leading to a decrease in the yield of fractions containing the target range of 1000–3000 Da.

[0023] The preparation method of this application includes the following steps: Step S1: Add the mixed bone powder to a polar dissociation medium at a material-to-liquid ratio of 1:8 to 1:15 and stir at 10-20°C for 2-6 hours to obtain a collagen suspension. Perform solid-liquid separation on the collagen suspension, collect the precipitate, and wash it with deionized water until neutral to remove the large amount of free calcium ions and phosphate ions released during the acidic demineralization stage. Then, resuspend the precipitate in an equal volume of deionized water to obtain a purified collagen system. This solid-liquid washing purification step avoids the inorganic calcium phosphate recrystallization cross-linking that easily occurs during subsequent neutralization adjustment, and also eliminates the risk of non-target coordination complexation of natural polyphenols due to competition from free calcium ions. The preferred material-to-liquid ratio is 1:12, the temperature is 15°C, and the time is 4 hours. This temperature range is below the region where collagen thermosensitive loosening significantly accelerates, which can reduce the rate of transformation of long-chain collagen to random coils. Below 10℃, the diffusion rate of urea and the demineralization rate of lactic acid decrease, resulting in insufficient swelling. Above 20℃, the thermal motion of collagen segments increases, making exposed segments more prone to non-target aggregation. When the stirring time is less than 2 hours, the polar dissociation medium inside the bone meal has not yet reached diffusion equilibrium; above 6 hours, the system gains slow down, and the continued action of lactic acid increases the background of low molecular weight fragments.

[0024] Natural polyphenolic compounds containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups can be selected from at least one of epigallocatechin gallate, oligomeric proanthocyanidins with a molecular weight of less than 800 Da, ellagic acid, gallic acid, or rosmarinic acid. Epigallocatechin gallate is preferred. These natural polyphenolic compounds possess both multiple hydrogen bond donor and acceptor sites and aromatic structures, and can bind to polyproline characteristic sequences via π-CH interactions and hydrogen bonds, forming a reversible anchor within the transient window of the locally opened collagen chain. This anchoring is not permanent covalent fixation, but rather increases the spatial retention probability of the polyproline characteristic sequence before and after subsequent enzymatic hydrolysis through multiple weak interactions. The amount of natural polyphenolic compound added is preferably 0.2% to 1.5% of the mass of the mixed bone meal, more preferably 0.8%. If the amount added is too low, the polyproline characteristic sequence sites that can be covered are insufficient; if the amount added is too high, the probability of polyphenol self-aggregation increases, forming a dense hydrogen bond network locally, which in turn hinders the entry of targeted endonuclease systems.

[0025] Step S2: After adding the natural polyphenol compound to the purified collagen system, the reaction is carried out under a pulsed microwave field at 40.5~41.5℃ to obtain the primary polymer solution. The pulsed microwave field adopts a discontinuous pulse mode with alternating on and off, where the single on time is 4~6s and the single off time is 8~12s. The preferred single on time is 5s and the single off time is 10s. The microwave frequency can be 2450MHz, and the input power is adjusted according to the system scale to ensure that the actual temperature of the system is stable at 40.5~41.5℃. This temperature window has critical significance: below 40.5℃, the hydrogen bond network of the inner layer of collagen fiber bundles is not sufficiently disturbed, and the natural polyphenol compound can only stay at the outer accessible sites, and the enzymatically hydrolyzable sites in the primary polymer solution are not fully exposed; above 41.5℃, the collagen segment from artificially bred second-generation giant salamanders is more prone to thermal loosening, the hydration structure around the polyproline characteristic sequence is unstable, and the negative absorption peak of circular dichroism chromatography is weakened. When the single on-time is less than 4 seconds, the dipole polarization input is insufficient, making it difficult to form an effective dissociation pulse; when it is greater than 6 seconds, local hot spots accumulate, increasing the risk of oxidation of natural polyphenolic compounds. When the single off-time is less than 8 seconds, the system's thermal relaxation is insufficient; when it is greater than 12 seconds, the dissociation process is too discontinuous, and the anchoring efficiency decreases. The preferred reaction time is 12-25 minutes, more preferably 18 minutes.

[0026] Step S3: Adjust the pH of the initial polymer solution to 6.7-6.9, and add the targeted endonuclease-limiting enzyme system for enzymatic hydrolysis. The targeted endonuclease-limiting enzyme system consists of collagen endonuclease and neutral protease. A pH of 6.7-6.9 is close to the effective working region of both collagen endonuclease and neutral protease, while avoiding the rapid decline in enzyme activity under strong acid conditions and the accelerated self-oxidation region of natural polyphenols under alkaline conditions. When the pH is below 6.7, the activity of collagen endonuclease decreases, and although the cleavage site is exposed, the cleavage efficiency is insufficient, resulting in a low yield in the target molecular weight range. When the pH is above 6.9, the probability of non-target cleavage by neutral protease increases, and fragments below 1000 Da increase. The preferred hydrolysis temperature is 40-42℃, and the preferred time is 2.5-4.0 h, more preferably 3.5 h. When the time is below 2.5 h, there is a higher proportion of large molecule residues; when the time is above 4.0 h, although anchored by natural polyphenols, excessive endonucleation still occurs, leading to a weakening of circular dichroism chromatographic characteristics.

[0027] After enzymatic hydrolysis, a polycarboxylic acid solution is added to adjust the pH of the reaction system to 3.8–4.2, yielding the hydrolysate. The polycarboxylic acid solution is a citric acid solution. The concentration of the citric acid solution is 0.8–1.2 mol / L and it is pre-cooled below room temperature, preferably 1.0 mol / L at 5–10°C. This step serves both to inactivate the enzyme and protect its conformation. A pH of 3.8–4.2 rapidly inactivates collagenase and neutral protease, preventing further degradation of the target fragments with molecular weights between 1000 and 3000 Da. Pre-cooling inhibits the localized exothermic reaction during the oxidation and acidification of natural polyphenolic compounds. Below pH 3.8, acid-catalyzed hydrolysis accelerates, increasing the proportion of short peptides; above pH 4.2, enzyme inactivation is insufficient, and residual enzyme activity continues to cleave in subsequent reactions. When the citric acid concentration is below 0.8 mol / L, the acidification rate of the system is slow and the residual enzyme activity is retained for a long time; when it is above 1.2 mol / L, the local acid concentration is too high, which can easily form transient superacid microregions, causing uneven chain segment breakage.

[0028] Step S4: Under the condition that the pH of the reaction system is 3.8-4.2, a strong proton donor acid containing an enediol structure is added to the enzymatic hydrolysate for reaction. After separation, collagen polypeptides from pig bone, bovine bone, and artificially bred second-generation giant salamander bone, which have the effect of improving bone density, are obtained. The strong proton donor acid containing an enediol structure is selected from ascorbic acid or isoascorbic acid. Ascorbic acid is preferred. The concentration of ascorbic acid or isoascorbic acid is 0.3-0.8 mol / L, and the reaction is carried out at 20-30℃, preferably 0.5 mol / L, 25℃, for 1.5 h. The key to this step is to utilize the enediol structure to provide high proton activity and competitive hydrogen bonding ability to strip the hydrogen bond network of natural polyphenol compounds, so that the collagen polypeptides that were reversibly anchored in the early stage are released into a free state, while avoiding secondary damage caused by a strong oxidizing environment. At concentrations below 0.3 mol / L, the exfoliation driving force is insufficient, resulting in high residual binding of natural polyphenolic compounds and a decrease in ultrafiltration flux. At concentrations above 0.8 mol / L, the osmotic pressure and acidity load of the system increase, the surface hydration structure of some polypeptide chains is excessively disturbed, and the molecular weight distribution becomes wider. At temperatures below 20°C, the competitive exfoliation kinetics are slow; above 30°C, the stability of ascorbic acid decreases, and the flexibility of collagen polypeptides increases, which is not conducive to the retention of the target conformation.

[0029] The separation process involves introducing the reacted system into a hydrophilic ultrafiltration membrane module and collecting the retentate that has passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and is retained by an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. For industrial scale-up, insoluble matter can be removed by centrifugation before ultrafiltration. The hydrophilic ultrafiltration membrane can be a regenerated cellulose membrane or a polyethersulfone-modified hydrophilic membrane. The collected retentate is concentrated under reduced pressure and freeze-dried to obtain a light yellow to off-white powder. Circular dichroism spectroscopy reveals a negative absorption peak at 198 ± 2 nm; molecular weight distribution analysis shows that it contains a fraction with a molecular weight greater than 60% of the product between 1000 and 3000 Da, with the main peak located in this range; amino acid composition analysis reveals collagen-derived characteristics rich in glycine, proline, and hydroxyproline; and sequence analysis detects enriched fragments of polyproline characteristic sequences.

[0030] The following general detection methods were used in the examples. Molecular weight distribution was determined by SEC-HPLC with a mobile phase of 0.1 mol / L phosphate buffer-20% acetonitrile, a flow rate of 0.6 mL / min, and correction with peptide molecular weight standards. Circular dichroism spectroscopy was performed at 190–260 nm, with a sample concentration of 0.20 mg / mL and a quartz cuvette path length of 1 mm. The total yield of the peptide dry powder was calculated by dividing the freeze-dried weight of the product by the total weight of the bone meal raw material; the residual polyphenol content of the final product was determined by HPLC. The browning index was expressed as absorbance at 420 nm. The free amino content was determined by the OPA method. In vitro indicators related to improved bone mineral density were evaluated using the MC3T3-E1 cell model, measuring cell compatibility at 48 h, ALP activity on day 7 of culture, and quantitative mineralization nodules stained with alizarin red on day 14 of culture. Data are expressed as mean ± standard deviation of three independent replicates.

[0031] In Example 1, pig bones, bovine bones, and artificially bred second-generation giant salamander bones were mixed in a mass ratio of 1:1:1. A composite aqueous system comprising 1.0 mol / L urea, 0.05 mol / L L-arginine, and 0.2 mol / L lactic acid was used, with a material-to-liquid ratio of 1:12. The mixture was swollen at 15°C for 4 hours to obtain a collagen suspension. The collagen suspension was centrifuged, the precipitate was collected, and washed with deionized water until neutral. Then, an equal volume of deionized water was added to resuspend the precipitate to obtain a purified collagen system. Epigallocatechin gallate was added to the purified collagen system at a rate of 0.4% of the mass of the mixed bone powder. The mixture was reacted under a pulsed microwave field at 40.5°C, with a single on-time of 4 seconds and a single off-time of 8 seconds, for a total reaction time of 18 minutes to obtain a primary polymer solution. The pH of the primary polymer solution was adjusted to 6.7, and a targeted endonuclease-limiting enzyme system consisting of collagen endonuclease and neutral protease (with a mass ratio of collagen endonuclease to neutral protease of 1:0.5, and a total enzyme addition of 0.2% of the protein mass in the primary polymer solution) was added, followed by enzymatic hydrolysis for 3.5 h. The pH of the system was then adjusted to 3.8 by adding 1.0 mol / L citric acid solution at 8°C to obtain the enzymatic hydrolysate. Further, 0.3 mol / L ascorbic acid was added at 25°C and the reaction was carried out for 1.5 h. The solution was then separated using a hydrophilic ultrafiltration membrane module. The retentate that permeated through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and was retained by an ultrafiltration membrane with a molecular weight cutoff of 1 kDa was collected and freeze-dried.

[0032] In Example 2, except for the parameters described below, everything else was the same as in Example 1. The polar dissociation medium was a composite aqueous system comprising 2.0 mol / L urea, 0.10 mol / L L-arginine, and 0.5 mol / L lactic acid. After obtaining the collagen suspension, it was centrifuged to separate the precipitate, washed to neutral, and resuspended to obtain a purified collagen system. Epigallocatechin gallate was added at 0.8% of the mass of the mixed bone meal. The reaction was conducted at 41.0℃ with a single on-time of 5 s and a single off-time of 10 s. The pH of the primary polymer solution was adjusted to 6.8, and a targeted endonuclease-limiting enzyme system composed of collagen endonuclease and neutral protease (with a mass ratio of collagen endonuclease to neutral protease of 1:0.8, and a total enzyme amount of 0.4% of the protein mass in the primary polymer solution) was added, followed by enzymatic hydrolysis for 3.5 h. A citric acid solution with a concentration of 1.0 mol / L was used, and the temperature was 6℃ to adjust the pH of the system to 4.0. Add 0.5 mol / L ascorbic acid, react at 25℃ for 1.5 h, separate and collect the corresponding retentate and freeze dry.

[0033] In Example 3, except for the parameters described below, everything else was the same as in Example 1. The polar dissociation medium was a composite aqueous system comprising 3.0 mol / L urea, 0.2 mol / L L-arginine, and 0.8 mol / L lactic acid. After obtaining the collagen suspension, it was also purified by centrifugation and washed and resuspended. Epigallocatechin gallate was added at 1.2% of the mass of the mixed bone meal. The reaction was conducted at 41.5°C with a single on-time of 6 s and a single off-time of 12 s. The pH of the primary polymer solution was adjusted to 6.9, and a targeted endonuclease-limiting enzyme system composed of collagen endonuclease and neutral protease (with a mass ratio of collagen endonuclease to neutral protease of 1:1.0, and a total enzyme amount of 0.6% of the protein mass in the primary polymer solution) was added, followed by enzymatic hydrolysis for 3.5 h. The system pH was adjusted to 4.2 by using a 1.2 mol / L citric acid solution at 5°C. Add 0.8 mol / L ascorbic acid, react at 30℃ for 1.5 h, separate and collect the corresponding retentate and freeze dry.

[0034] In Example 4, the same process as in Example 2 was used, except that in step S2, the natural polyphenolic compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was replaced by gallic acid in equal mass.

[0035] In Example 5, the same process as in Example 2 was used, except that in step S2, the natural polyphenolic compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was replaced by ellagic acid in equal mass.

[0036] In Example 6, the same process as in Example 2 was used, except that in step S2, the natural polyphenolic compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was replaced by rosmarinic acid in equal mass.

[0037] In Example 7, the same process as in Example 2 was used, except that in step S4, the strong proton donor acid containing an enediol structure was replaced with an equal amount of isoascorbic acid.

[0038] In Example 8, the same process as in Example 2 was used, except that in step S2, the natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was replaced by an equal mass of oligomeric proanthocyanidins with a molecular weight of less than 800 Da.

[0039] Comparative Example 1 employed a conventional bovine bone collagen peptide process. The raw material consisted only of bovine bone powder. Water extraction was performed at 125℃ for 2 hours. Enzymatic hydrolysis was conducted using a complex protease and papain at 55-60℃ for 3 hours, followed by heat inactivation at 85℃ for 15 minutes. Subsequent ultrafiltration separation was used. This comparative example did not use a polar dissociation medium, lacked centrifugal washing and desalting solid-liquid separation steps, did not use natural polyphenol compounds, did not use a pulsed microwave field, did not use citric acid solution for enzyme inactivation, and did not use ascorbic acid or isoascorbic acid for release.

[0040] In Comparative Example 2, the same process as in Example 2 was used, but in step S2, a natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups was not added, while in step S4, 0.5 mol / L ascorbic acid was still added. This comparative example was used to verify the necessity of natural polyphenol compounds for protecting the characteristic sequence of polyproline.

[0041] In Comparative Example 3, the same process as in Example 2 was used, but the pulsed microwave field temperature in step S2 was set to 36.5°C, which is -10% of the lower limit of 40.5°C. All other parameters remained unchanged. This comparative example was used to verify the failure mechanism when the temperature is below the lower limit.

[0042] In Comparative Example 4, the same process as in Example 2 was used, but the pulsed microwave field temperature in step S2 was set to 45.7°C, which is 10% above the upper limit of 41.5°C. All other parameters remained unchanged. This comparative example was used to verify the failure mechanism when the temperature exceeds the upper limit.

[0043] In Comparative Example 5, the same process as in Example 2 was used, but in step S3, the pH of the initial polymer solution was adjusted to 6.0 before the targeted endonuclease-limiting enzyme system was added, and the pH of the reaction system was adjusted to 3.4 with citric acid solution. This comparative example also exceeded the lower limit of the effective window of step S3, and was used to verify the superimposed failure of insufficient enzymatic hydrolysis and excessive acid chain scission.

[0044] In Comparative Example 6, the same process as in Example 2 was used, but in step S3, the pH of the initial polymer solution was adjusted to 7.6 before the targeted endonuclease-limiting enzyme system was added, and the pH of the reaction system was adjusted to 4.6 with citric acid solution. This comparative example also exceeded the upper limit of the effective window of step S3, and was used to verify the superimposed failure of non-targeted cleavage and insufficient enzyme inactivation.

[0045] All samples were tested using the same method, and the results are shown in Table 1.

[0046] Table 1

[0047] Table 1 shows that Examples 1 to 3 are significantly superior to the comparative examples in terms of the proportion of fractions in the 1000~3000 Da range, the intensity of the negative absorption peak at 198±2 nm in circular dichroism chromatography, and osteogenic in vitro indicators, indicating that the effect within the scope defined by this application is stable and universal. Example 2 is located in the middle of the optimal range of parameters, with a total extraction yield of 13.5±0.8%, and the residual polyphenol content after desorption is as low as 0.15±0.04 mg / g (confirming that the ascorbic acid replacement and exfoliation network mechanism is effective). The cell compatibility, ALP activity, and relative value of mineralized nodules are the highest at 48 h, indicating that there is a synergistic matching between the interference elimination of washing and desalting, the composition of polar dissociation medium, the pulsed microwave field temperature, the acid-base window of step S3, and the ascorbic acid concentration.

[0048] Parallel studies of the Markush replacement components showed that gallic acid molecules are small and easily removed by dialysis with a 1 kDa ultrafiltration membrane (residual amount only 0.08 ± 0.02 mg / g), but its monocyclic structure results in weak hydrogen bond encapsulation of long peptide chains, leading to slightly inferior conformational retention and efficacy compared to the preferred solution. Ellagic acid, due to its rigid planar structure and extremely poor water solubility, easily undergoes micro-self-polymerization after anchoring, and some polymers cannot be completely filtered out by the ultrafiltration membrane, resulting in a high residual polyphenol content of 0.58 ± 0.12 mg / g in the final product, reflecting... Objective mass transfer hindrance; as a stereoisomer of ascorbic acid, isoascorbic acid has almost the same competitive stripping efficiency on hydrogen bond networks, and all indicators show reasonable systematic error fluctuations; in Example 8, due to its slightly greater steric hindrance than epigallocatechin gallate, the elution and stripping kinetics of oligomeric proanthocyanidins under ascorbic acid conditions are reasonably slightly hindered, resulting in a slight increase in residual amount to 0.28±0.06 mg / g, but the early anchoring protection mechanism of this component still verifies the feasibility of the invention.

[0049] It is noteworthy that in Comparative Example 4, the microwave temperature increased to 45.7℃, causing excessive collagen disintegration due to heating. This resulted in the polyphenols undergoing oxidative cross-linking at high temperatures and becoming locked within the polypeptide matrix, leading to a significant decrease in the acid elution efficiency of the strong proton donor and a significant increase in the residual polyphenol content of the final product to 6.54±0.87 mg / g. This discrete extreme value confirms the critical significance of the 40.5–41.5℃ temperature control window for maintaining the reversible desorption mechanism.

[0050] To further confirm that the peptides obtained using the process described in this application can cross the in vivo gastrointestinal barrier and achieve macroscopic bone mineral density enhancement, a bilateral ovariectomized (OVX) postmenopausal osteoporosis model was established using 8-week-old female SD rats. After successful modeling, each group was treated with a daily gavage dose of 400 mg / kg body weight (bw) for 8 weeks. The experimental endpoint was determined by measuring the bone mineral density (BMD) of the right femur in rats using dual-energy X-ray absorptiometry (DEXA). The results showed that the BMD of the sham-operated group (Sham, healthy baseline without ovariectomy) was 0.258 ± 0.026 g / cm³. 2 The BMD in the model control group (OVX, physiological lesion background) decreased to 0.176±0.041 g / cm³. 2 In Example 1, the BMD value after intervention was 0.208 ± 0.033 g / cm³. 2 In Example 2, the BMD of the intervention group significantly increased to 0.235±0.029 g / cm³. 2 In Example 3, the BMD value after intervention was 0.219 ± 0.031 g / cm³. 2 In Example 4, the BMD value after intervention was 0.222 ± 0.035 g / cm³. 2 The relatively weak hydrogen bond encapsulation of the long collagen chain by the monocyclic structure resulted in slightly inferior conformational protection of the macromolecule, leading to a reasonable attenuation. The BMD value of the intervention group in Example 5 was 0.211 ± 0.038 g / cm³. 2 This objectively reflects the characteristic of ellagic acid to readily polymerize and hinder its absorption in the living gastrointestinal tract; the BMD value of the intervention group in Example 6 was 0.226±0.027 g / cm³. 2 In Example 7, the BMD value after intervention was 0.233 ± 0.028 g / cm³. 2 In Example 8, the BMD value after intervention was 0.229 ± 0.032 g / cm³. 2 The BMD of control group 1 (conventional hot-extracted peptides) after intervention was only 0.183±0.039 g / cm³. 2 This indicates that the three-dimensional rigidity of its peptide chain was already destroyed during thermal extraction, and it was non-specifically degraded in the gastrointestinal tract, resulting in no significant macroscopic improvement in efficacy. Furthermore, the BMD value of the two comparative groups after intervention was 0.189±0.037 g / cm³. 2 The BMD value of the three control groups after intervention was 0.194 ± 0.034 g / cm³. 2 The BMD value of the four control groups after intervention was 0.185 ± 0.043 g / cm³. 2 The BMD value of the five control groups after intervention was 0.182 ± 0.036 g / cm³. 2 The BMD value of the six comparative groups (verifying the superimposed failure of alkaline non-target cleavage and acid inactivation) after intervention was 0.186 ± 0.038 g / cm³.2 Due to non-target cleavage and residual enzyme degradation leading to rigid disintegration of the target, its BMD dropped to levels close to those of the pathological control.

[0051] To further confirm the targeted repair effect of collagen peptides in maintaining a rigid conformation on the trabecular meshwork topology of bone, micro-CT scans and three-dimensional reconstruction analyses were performed on the right femoral metaphysis of rats in each group at the experimental endpoint. Figures 1 to 4 As shown, the trabeculae in the sham surgery group exhibited a continuous and dense three-dimensional mesh structure; the trabeculae in the model control group and Comparative Example 1 group were severely fractured, with only sparse, independent rod-like supports remaining; after intervention in Example 2, the fractured trabeculae re-established spatial connections, and the mesh-like load-bearing structure was significantly repaired. Quantitative analysis of the microstructure showed that: Bone volume fraction (BV / TV, %): Sham group 23.85±4.12; OVX group 9.76±2.94 (severe bone loss); Example 2 group 19.34±3.51 (showing a significant upward trend but not fully reaching the healthy baseline); Comparative Example 1 group 11.23±2.87; Example 1 group 14.86±3.22; Example 3 group 16.53±3.48; Example 4 group 17.15±3 0.65; Example 5 group 15.22±3.51; Example 6 group 17.84±3.39; Example 7 group 19.02±3.61; Example 8 group 18.41±3.55; Comparative Example 2 group 11.95±2.85; Comparative Example 3 group 12.63±3.01; Comparative Example 4 group 11.51±2.93; Comparative Example 5 group 11.08±2.76; Comparative Example 6 group 11.64±2.89.

[0052] Trabecular bone count (Tb.N, 1 / mm): Sham group 4.47±0.58; OVX group 1.63±0.41; Example 2 group 3.52±0.49; Comparative Example 1 group 1.95±0.45; Example 1 group 2.58±0.46; Example 3 group 2.96±0.52; Example 4 group 3.12±0.48; Example 5 group 2.67±0.45; Example 6 group 3.24±0.51; Example 7 group 3.45±0.47; Example 8 group 3.31±0.50; Comparative Example 2 group 2.11±0.42; Comparative Example 3 group 2.23±0.45; Comparative Example 4 group 1.96±0.43; Comparative Example 5 group 1.84±0.40; Comparative Example 6 group 2.02±0.44.

[0053] Trabecular separation (Tb.Sp, mm): Sham group 0.19±0.05; OVX group 0.51±0.12; Example 2 group 0.26±0.08; Comparative Example 1 group 0.46±0.11; Example 1 group 0.35±0.10; Example 3 group 0.31±0.09; Example 4 group 0.30±0.09; Example 5 group 0.34±0.11; Example 6 group 0.29±0.08; Example 7 group 0.27±0.08; Example 8 group 0.28±0.09; Comparative Example 2 group 0.44±0.12; Comparative Example 3 group 0.41±0.11; Comparative Example 4 group 0.45±0.12; Comparative Example 5 group 0.47±0.13; Comparative Example 6 group 0.45±0.11.

[0054] Compared to Comparative Example 1, the browning index of Example 2 decreased from 0.183±0.011 to 0.051±0.004, and the free amino group decreased from 1.34±0.08 mmol / g to 0.73±0.04 mmol / g. Although Comparative Example 1 achieved an extraction yield of 16.8±1.1% due to high-temperature water extraction, this did not translate into an effective peptide product. This indicates that this method does not obtain more chain fragments through high temperature and non-specific cleavage, but rather, under the premise of reducing thermal damage and excessive hydrolysis, it directionally enriches fractions with molecular weights between 1000 and 3000 Da; Figure 5 As shown, Example 2 exhibits a concentrated high-response main peak in this target-level interval, while Comparative Example 1 shows multiple diffuse peaks shifted towards the hysteresis retention time. Further combining... Figure 6 A comparison of the deep valley curve at 198±2 nm in Example 2 with the flatter spectral trajectory in Comparative Example 1 shows that the absolute intensity of the negative absorption peak in the circular dichroism spectroscopy of this application is significantly increased, indicating that the product still retains conformational information related to the characteristic sequence of polyproline. This difference cannot be explained by simply changing the source of raw materials, because the root cause of failure in Comparative Example 1 lies in the fact that high-temperature water extraction and heat inactivation of enzymes amplified the random coiling and browning side reactions of the chain segments.

[0055] Compared to Example 2, Comparative Example 2, after deducting only the natural polyphenolic compound, showed a decrease in the proportion of the 1000–3000 Da fraction from 78.9 ± 1.7% to 57.1 ± 2.0%, a decrease in the intensity of the negative absorption peak in circular dichroism chromatography from -7.6 ± 0.4 to -2.6 ± 0.3, and a simultaneous decrease in the relative values ​​of cell compatibility, ALP activity, and mineralized nodules at 48 h. This indicates that the natural polyphenolic compound is not a dispensable additive, but a core component that interacts with the pulsed microwave field. This result supports the aforementioned interaction relationship, namely that the natural polyphenolic compound binds to the polyproline characteristic sequence through π-CH interactions and hydrogen bonds, first forming a reversible anchor, and then completing the stripping under the action of a strong proton donor acid containing an enediol structure. If this initial anchoring is missing, the subsequent ascorbic acid can only exist as a general acidic component and cannot recover the conformational information lost during enzymatic hydrolysis and mass transfer.

[0056] In Comparative Example 3, after the temperature of step S2 was reduced to 36.5℃, the browning index did not increase significantly, but the proportion of the 1000~3000Da fraction and the absolute intensity of the negative absorption peak in circular dichroism both decreased significantly. This indicates that the dipole polarization triggering was insufficient below 40.5℃, the inner layer sites of the collagen fiber bundles were not sufficiently exposed, and the natural polyphenol compounds could not effectively enter and anchor the polyproline characteristic sequence.

[0057] Comparative Examples 5 and 6 further demonstrate the critical significance of the pH window in step S3. pH 6.0 resulted in insufficient activity during the enzymatic hydrolysis stage, while pH 3.4 triggered excessive acidity and chain scission, thus the proportion of the 1000–3000 Da fraction was only 38.9 ± 2.1%. pH 7.6 led to increased non-target cleavage, while pH 4.6 resulted in insufficient enzyme inactivation, allowing residual enzyme activity to continue degrading the target fragment, resulting in higher free amino groups and weakened circular dichroism characteristics. Therefore, adjusting the pH of the primary polymer solution to 6.7–6.9 for enzymatic hydrolysis in step S3, and adjusting the pH of the reaction system to 3.8–4.2 with citric acid solution, not only determines the molecular weight window but also the degree of conformational retention.

[0058] The amino acid composition of the collagen polypeptide products collected from each group was analyzed by HPLC using phenyl isothiocyanate derivatization. The results showed that in the product of Example 2, per 1000 total amino acid residues, the following were present: glycine (Gly) 326.5±4.2, proline (Pro) 118.4±3.6, and hydroxyproline (Hyp) 103.7±3.1. In contrast, in the product of Comparative Example 1, which was subjected to high-temperature thermal stress and random cleavage by non-specific complex enzymes, a large number of hydroxyproline-rich segments were broken and lost during the fractionation process. The glycine content decreased to 274.8±5.8, proline to 82.5±4.5, and hydroxyproline to 64.2±3.9. This quantitative data confirms that the extraction process reduced the degradation of the core amino acid sequence of collagen.

[0059] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A collagen polypeptide with the effect of improving bone density, characterized in that, The collagen polypeptide comprises a fraction with a molecular weight between 1000 and 3000 Da, accounting for more than 60% by mass, and exhibits a negative absorption peak at 198 ± 2 nm on circular dichroism spectroscopy. The collagen polypeptide is prepared by a method comprising the following steps: Pig bones, cattle bones, and artificially bred second-generation giant salamander bones were swollen in a polar dissociation medium to obtain a collagen suspension. The collagen suspension was subjected to solid-liquid separation, the precipitate was collected and washed with deionized water until neutral, and then resuspended to obtain a purified collagen system. The polar dissociation medium was a composite aqueous phase system comprising 1.0~3.0 mol / L urea, 0.05~0.2 mol / L L-arginine, and 0.2~0.8 mol / L lactic acid. A natural polyphenolic compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups is added to the purified collagen system and reacted at 40.5~41.5℃ under a pulsed microwave field to obtain a primary polymer solution; the natural polyphenolic compound is selected from at least one of epigallocatechin gallate, oligomeric proanthocyanidins with a molecular weight of less than 800 Da, ellagic acid, gallic acid, or rosmarinic acid; the pulsed microwave field adopts a discontinuous pulse mode of alternating on and off, wherein the single on time is 4~6s and the single off time is 8~12s; The pH of the initial polymer solution was adjusted to 6.7-6.9, and a targeted endonuclease limiting enzyme system was added for enzymatic hydrolysis. Subsequently, a polycarboxylic acid solution was added to adjust the pH of the reaction system to 3.8-4.2 to inactivate the enzyme, resulting in an enzymatic hydrolysate. The targeted endonuclease limiting enzyme system consisted of collagen endonuclease and neutral protease. The polycarboxylic acid solution was a citric acid solution. Under the condition that the pH of the reaction system is 3.8~4.2, a strong proton donor acid containing an enediol structure is added to the enzymatic hydrolysate for reaction, and the collagen polypeptide is obtained by separation; the strong proton donor acid containing an enediol structure is selected from ascorbic acid or isoascorbic acid.

2. The collagen polypeptide with bone density-enhancing effects according to claim 1, characterized in that, The collagen polypeptide contains a polyproline characteristic sequence; the natural polyphenol compound binds to the polyproline characteristic sequence via π-CH interactions and hydrogen bonds, and the strong proton donor acid strips the hydrogen bond network of the natural polyphenol compound.

3. A method for preparing a collagen polypeptide with bone density-enhancing effects as described in claim 1 or 2, characterized in that, Includes the following steps: Step S1: The pig bones, cattle bones and artificially bred second-generation giant salamander bones are swollen in a polar dissociation medium to obtain a collagen suspension. The collagen suspension is subjected to solid-liquid separation, the precipitate is collected and washed with deionized water until neutral, and then resuspended to obtain a purified collagen system. Step S2: Add a natural polyphenol compound containing multiple adjacent phenolic hydroxyl groups and / or galloyl groups to the purified collagen system, and react it under a pulsed microwave field at 40.5~41.5℃ to obtain a primary polymer solution; Step S3: Adjust the pH of the initial polymer solution to 6.7-6.9, add a targeted endonuclease-limiting enzyme system for enzymatic hydrolysis, and then add a polycarboxylic acid solution to adjust the pH of the reaction system to 3.8-4.2 to obtain the enzymatic hydrolysate; Step S4: Under the condition that the pH of the reaction system is 3.8~4.2, a strong proton donor acid containing an ethylenediol structure is added to the enzymatic hydrolysate for reaction. After separation, the collagen polypeptides of pig bone, bovine bone and artificially bred second-generation giant salamander bone are obtained.

4. The method for preparing a collagen polypeptide with bone density-enhancing effects according to claim 3, characterized in that, In step S3, the concentration of the citric acid solution is 0.8~1.2 mol / L and it is in a pre-cooled state below room temperature.

5. The method for preparing a collagen polypeptide with bone density-enhancing effects according to claim 3, characterized in that, In step S4, the reaction is carried out at 20~30°C, the concentration of ascorbic acid or isoascorbic acid is 0.3~0.8 mol / L, and the separation includes introducing the reaction system after the reaction into a hydrophilic ultrafiltration membrane module, and collecting the retentate that has passed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and is retained by an ultrafiltration membrane with a molecular weight cutoff of 1 kDa.

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