A peptide composition for promoting hematopoiesis based on donkey-hide gelatin peptide and a preparation method thereof

Abstract

The present application belongs to the technical field of medical preparations in biomedical disciplines, and relates to a peptide composition for promoting hematopoiesis based on donkey-hide gelatin peptide and a preparation method thereof. The composition is composed of a nano-scale peptide iron complex formed by self-assembly of donkey-hide gelatin oligopeptide, plant source iron targeting transport oligopeptide and ferrous salt. The donkey-hide gelatin oligopeptide is obtained by multi-stage ultrafiltration retention after limited enzyme hydrolysis of complex protease on donkey-hide gelatin. The plant source iron targeting transport oligopeptide is a short peptide with a total mole ratio of histidine and glutamic acid residues greater than 40%. The above two oligopeptides wrap ferrous ions through coordination chelation, avoid the generation of iron ion precipitate in the weak alkaline environment of the intestinal tract, and simultaneously deliver the iron ions to the absorption site by using the affinity of oligopeptides to intestinal epithelial cells, so as to improve the transmembrane transport amount of iron elements.

Description

Technical Field

[0001] This invention belongs to the field of medical preparation technology in biomedicine, and relates to a peptide composition based on donkey-hide gelatin peptide that promotes hematopoiesis and its preparation method. It is used to prepare a peptide composition based on donkey-hide gelatin peptide that promotes hematopoiesis, which can improve the transmembrane transport of iron. Background Technology

[0002] Currently, donkey-hide gelatin peptides are a commonly used raw material in the development of hematopoietic biochemical agents. Conventional processes involve hydrolyzing donkey-hide gelatin substrates with proteases, followed by ultrafiltration to retain a mixture of peptides within a specific molecular weight range as the effective hematopoietic-promoting component. In intervention programs, to accelerate hemoglobin synthesis, these donkey-hide gelatin peptides are often physically mixed with conventional iron supplements and taken orally. This conventional approach relies on donkey-hide gelatin peptides providing nutritional support for the hematopoietic microenvironment, while also depending on free iron ions as a core trace element for hemoglobin synthesis. After entering the digestive tract, the physically mixed donkey-hide gelatin peptides and iron supplements are dispersed by gastrointestinal fluids, allowing them to separate. The donkey-hide gelatin peptides are absorbed through amino acid and peptide transport channels in intestinal epithelial cells, while iron ions are transported across the membrane via divalent metal ion transport proteins in intestinal epithelial cells.

[0003] The aforementioned conventional physical mixing schemes present a core technical problem in actual intestinal absorption. Because the intestinal environment is weakly alkaline, free ferrous ions from conventional physical mixing readily combine with hydroxide ions in the intestine, forming insoluble hydroxide precipitates, thus causing the iron ions to lose their ability to be absorbed across the membrane. Existing donkey-hide gelatin peptides only possess passive diffusion or transport absorption characteristics; their molecular structure lacks the structural basis for specific binding to iron ions and active mediation of iron ion transmembrane transport. Therefore, they cannot target and protect iron ions at the intestinal absorption site, resulting in extremely low actual bioavailability of iron in the physical mixture. Donkey-hide gelatin peptides and iron cannot be delivered synchronously during the intestinal absorption stage.

[0004] In the prior art, CN115724911B and CN115974979B disclose specific sequence donkey-hide gelatin oligopeptides and their iron chelates, which can improve anemia and enhance immunity; CN114886116B discloses a conditioning composition for women of childbearing age containing donkey-hide gelatin peptides. However, the above patents all use a single donkey-hide gelatin peptide to directly chelate with iron, without introducing plant-derived iron-targeting transport oligopeptides, and cannot form a core-shell structured nanocomposite. The problems of iron easily precipitating in the weakly alkaline environment of the intestine, the inability of donkey-hide gelatin peptides and iron to be delivered synchronously, and low iron bioavailability still exist. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and solve the technical problems that, after physical mixing of donkey-hide gelatin peptides and iron, free ferrous ions easily form insoluble hydroxide precipitates in the weakly alkaline environment of the intestine, resulting in extremely low bioavailability of iron and the inability of donkey-hide gelatin peptides and iron to be delivered synchronously during the intestinal absorption stage. This invention provides a peptide composition based on donkey-hide gelatin peptides that promotes hematopoiesis and its preparation method.

[0006] To achieve the above-mentioned objectives, this invention provides a hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides. The peptide composition comprises a nanoscale peptide-iron complex formed by the self-assembly of donkey-hide gelatin oligopeptides, plant-derived iron-targeting transport oligopeptides, and ferrous salts in a chelation reaction system. Based on the total mass percentage of the peptide composition, the donkey-hide gelatin oligopeptides account for 60%-80% of the total mass, the plant-derived iron-targeting transport oligopeptides account for 15%-30% of the total mass, and the ferrous salts account for 5%-10% of the total mass based on iron content. The donkey-hide gelatin oligopeptides are oligopeptide components with a molecular weight of 500 Da-2000 Da obtained by restrictive enzymatic hydrolysis of donkey-hide gelatin substrates with specific endopeptides and retention through a multi-stage ultrafiltration membrane assembly. The plant-derived iron-targeting transport oligopeptides are a mixture of short peptides with a molecular weight less than 500 Da and a total molar percentage of histidine and glutamic acid residues greater than 40% in the amino acid sequence, obtained by enzymatic hydrolysis of plant proteins.

[0007] The high proportion of histidine imidazole ring and glutamic acid side chain carboxyl groups in plant-derived iron-targeting transport oligopeptides can serve as coordination sites, specifically coordinating and chelating with ferrous ions to form a stable iron-peptide chelated core. Donkey-hide gelatin oligopeptides self-assemble with iron-chelated plant-derived oligopeptides through intermolecular hydrogen bonds and hydrophobic interactions to form a core-shell structured nanoscale peptide-iron complex. Among them, donkey-hide gelatin oligopeptides constitute the outer hydrophilic shell of the complex, improving the dispersion stability of the complex in an aqueous environment. After entering the weakly alkaline environment of the intestine, the outer oligopeptide shell of this complex can shield ferrous ions from direct contact with hydroxide ions in the intestine, avoiding the formation of insoluble hydroxide precipitates. At the same time, by leveraging the affinity of plant-derived short peptides for the intestinal epithelial cell membrane, the complex as a whole is mediated to achieve transmembrane absorption through peptide transport channels, breaking the limitation of the separate transport of donkey-hide gelatin peptides and iron ions in traditional physical mixing systems, and achieving the simultaneous delivery of the two. Donkey-hide gelatin oligopeptides can directly provide amino acid nutritional support for the proliferation and differentiation of hematopoietic cells, and work synergistically with ferrous ions to provide core raw materials for hemoglobin synthesis, thereby achieving a synergistic enhancement of hematopoietic promotion efficacy.

[0008] The plant protein described in this invention is any one of soy protein isolate, pea protein, or wheat gluten. The plant-derived iron-targeting transport oligopeptide is a short peptide component whose elution volume is within a specific peak range after the plant protein is hydrolyzed by a complex protease and separated by gel filtration chromatography.

[0009] In practice, soy protein isolate, pea protein, and wheat gluten are all high-quality plant protein raw materials rich in histidine and glutamate residues. They can be enzymatically hydrolyzed to generate a large number of short peptides containing histidine and glutamate coordination sites. Gel filtration chromatography can accurately separate short peptide components with a molecular weight of less than 500 Da based on the molecular exclusion effect. By collecting specific peak ranges, targeted short peptides with a total molar ratio of histidine and glutamate residues greater than 40% can be enriched, ensuring that the obtained oligopeptides have sufficient iron ion chelating sites and intestinal targeted transport activity.

[0010] The ferrous salt described in this invention is any one of ferrous sulfate, ferrous chloride, ferrous lactate, or ferrous citrate. Before being added to the chelation reaction system, the ferrous salt is dissolved in deoxygenated deionized water containing 0.1%-0.5% ascorbic acid to form a ferrous salt precursor solution.

[0011] In practice, the aforementioned ferrous salts are all water-soluble ferrous iron supplements, which can provide sufficient ferrous ions to participate in the chelation reaction; deoxygenated deionized water can remove dissolved oxygen in the water, and together with the reducing properties of ascorbic acid, it can effectively inhibit the oxidation of ferrous ions to ferric ions during solution preparation and subsequent chelation reaction, ensuring that the iron ions participating in the chelation reaction remain in the ferrous active state, while improving the valence stability of iron in the final composition during storage.

[0012] The dynamic light scattering particle size of the nanoscale peptide-iron complex of the present invention is 80nm-150nm. In the chelation reaction system, the ratio of the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide to the molar number of ferrous ions in the ferrous salt is 2:1 to 4:1. The initial pH value of the chelation reaction system is adjusted to 5.5-6.5.

[0013] In practice, nanocomposites within this particle size range can effectively circumvent clearance by the intestinal reticuloendothelial system while exhibiting excellent transmembrane transport efficiency. This molar ratio ensures that the coordination sites of ferrous ions are fully saturated by the histidine imidazole ring and the carboxyl group of the glutamate side chain, forming a stable chelate structure and preventing the presence of free ferrous ions. The weakly acidic environment of pH 5.5-6.5 can prevent the hydrolysis and precipitation of ferrous ions during the reaction process, while providing the optimal protonation environment for the coordination and chelation of histidine and glutamate residues, ensuring the efficient conduct of the chelation reaction and improving the formation rate and structural stability of the peptide-iron complex.

[0014] This invention also provides a method for preparing a hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides. The preparation method includes the following steps: Step S1, adding donkey-hide gelatin substrate to water to prepare a donkey-hide gelatin solution, adding a specific endopeptide protease to perform a restricted enzymatic hydrolysis reaction to obtain a donkey-hide gelatin hydrolysate; Step S2, passing the donkey-hide gelatin hydrolysate sequentially through a first-stage ultrafiltration membrane and a second-stage ultrafiltration membrane for multi-stage separation, collecting the retentate and freeze-drying it to obtain donkey-hide gelatin oligopeptides; Step S3, separating the plant protein hydrolysate by chromatography, collecting the eluent rich in histidine and glutamic acid residues, and freeze-drying it to obtain plant-derived iron-targeting transport oligopeptides; Step S4, dissolving the donkey-hide gelatin oligopeptides and the plant-derived iron-targeting transport oligopeptides in water to form a mixed peptide solution, adding a ferrous salt solution dropwise to the mixed peptide solution, performing a self-assembly reaction under light-protected conditions, and freeze-drying the mixture after the reaction to obtain the peptide composition.

[0015] This preparation method involves the stepwise preparation of high-purity donkey-hide gelatin oligopeptides and plant-derived iron-targeting transport oligopeptides, followed by liquid-phase dropwise addition to achieve simultaneous chelation and self-assembly of ferrous ions with the two oligopeptides. The restriction enzymatic hydrolysis and multi-stage ultrafiltration steps allow for the targeted preparation of donkey-hide gelatin oligopeptides within the target molecular weight range, ensuring their hematopoietic activity and shell-forming ability. The chromatographic separation step enriches plant-derived short peptides with high-abundance iron-chelating sites, ensuring their iron-targeting binding and transport capabilities. The dropwise self-assembly reaction under light-protected conditions achieves uniform chelation of ferrous ions, preventing aggregation and precipitation caused by excessively high local iron concentrations. Furthermore, freeze-drying yields a solid composite product without damaging the peptide-iron complex structure, ensuring its storage stability and bioactivity.

[0016] In step S1 of this invention, the specific endopeptide is a complex protease composed of flavor protease and alkaline protease at an enzyme activity ratio of 1:2 to 1:4. The conditions for the limiting enzymatic hydrolysis reaction are as follows: the substrate mass concentration of the donkey-hide gelatin solution is 5%-10%, the amount of the complex protease added is 1%-3% of the donkey-hide gelatin substrate mass, the enzymatic hydrolysis temperature is 45℃-55℃, the pH value of the enzymatic hydrolysis system is 7.5-8.5, the enzymatic hydrolysis reaction time is 4h-6h, and after the enzymatic hydrolysis reaction is completed, the temperature is raised to 85℃-95℃ and kept at 10min-15min to inactivate the enzyme.

[0017] In practice, the ratio of the complex protease and the enzymatic hydrolysis conditions can ensure the targeted and restricted enzymatic hydrolysis of the donkey-hide gelatin substrate, obtaining a high yield of target molecular weight oligopeptides; the high-temperature enzyme inactivation step after enzymatic hydrolysis can completely inactivate the complex protease, terminate the enzymatic hydrolysis reaction, avoid excessive enzymatic hydrolysis leading to the degradation of the target oligopeptide components, and ensure the stability of the molecular weight distribution of oligopeptide components in the donkey-hide gelatin enzymatic hydrolysate.

[0018] In step S2 of this invention, the molecular weight cutoff of the first-stage ultrafiltration membrane is 3000 Da-5000 Da, and the molecular weight cutoff of the second-stage ultrafiltration membrane is 500 Da. The enzymatic hydrolysate of donkey-hide gelatin is first separated by the first-stage ultrafiltration membrane to remove the large molecular weight protein retentate, and the first-stage permeate is collected. Then, the first-stage permeate is pumped into the second-stage ultrafiltration membrane, and the retentate of the second-stage ultrafiltration membrane is collected. The retentate is freeze-dried at a temperature of -40°C to -30°C and a vacuum degree of less than 20 Pa.

[0019] In practice, the separation sequence and molecular weight cutoff settings of the two-stage ultrafiltration can accurately remove large ineffective components and small free amino acids from the enzymatic hydrolysate, obtaining high-purity 500Da-2000Da donkey-hide gelatin oligopeptides. The freeze-drying conditions allow the water in the retentate to sublimate directly under low-temperature vacuum conditions, avoiding the structural damage and activity loss of oligopeptides caused by high-temperature drying, thus ensuring the bioactivity and structural integrity of the obtained donkey-hide gelatin oligopeptides.

[0020] In step S4 of this invention, the ferrous salt solution is prepared by dissolving ferrous salt in deoxygenated deionized water containing 0.1%-0.5% ascorbic acid by mass. The pH of the mixed peptide solution is adjusted to 5.5-6.5. In the mixed peptide solution, the ratio of the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide to the molar number of ferrous ions in the ferrous salt solution is 2:1 to 4:1. The ferrous salt solution is added dropwise to the mixed peptide solution at a dropping rate of 10 ml / min-20 ml / min, and the reaction is carried out under light-protected stirring conditions at a speed of 200 rpm-400 rpm for 2 h-4 h.

[0021] In practice, the ferrous salt solution is prepared using deoxygenated deionized water containing ascorbic acid, which can inhibit the oxidation of ferrous ions throughout the process and ensure the effectiveness of the chelation reaction. The pH value and coordination molar ratio provide the optimal reaction environment for the chelation of ferrous ions, ensuring the full progress of the chelation reaction. The drop acceleration rate and stirring speed can avoid the hydrolysis precipitation and complex aggregation of ferrous ions caused by excessively high local concentrations when the ferrous salt solution is added, ensuring that the generated peptide-iron complex has uniform particle size and stable structure. The light-protected conditions can further prevent the photo-oxidation of ferrous ions and improve the conversion rate of the target product.

[0022] Compared with the prior art, the beneficial effects of this invention are as follows: Firstly, by combining donkey-hide gelatin oligopeptides with specific molecular weight ranges with plant-derived iron-targeting transport oligopeptides rich in histidine and glutamate residues, and then self-assembling them with ferrous salts under specific pH conditions to form nanoscale peptide-iron complexes, the problem of free iron precipitation and untargeted delivery in the intestinal environment is solved. The high molar ratio of histidine and glutamate residues in the plant-derived iron-targeting transport oligopeptides, as defined in the claims, enables them to spatially encapsulate and coordinately chelate divalent iron ions through imidazole rings and side-chain carboxyl groups. When this nanoscale complex enters the weakly alkaline intestinal environment, the externally encapsulated oligopeptides shield iron ions from direct contact with hydroxide ions, avoiding the formation of hydroxide precipitation. This complex utilizes the affinity of plant-derived short peptides for intestinal epithelial cell membranes, changing the passive absorption mode of single donkey-hide gelatin peptides, and simultaneously delivering iron ions to the absorption site in a complex form, thereby increasing the transmembrane transport of iron.

[0023] Secondly, by limiting the specific mass ratio of donkey-hide gelatin oligopeptides and plant-derived iron-targeting transport oligopeptides, as well as the molar ratio of amino acid residues to iron ions in both, the uniformity of particle size and structural stability of the self-assembled complex were ensured. Restrictive enzymatic hydrolysis was performed using a complex enzyme composed of flavor protease and alkaline protease, combined with multi-stage ultrafiltration membranes to retain donkey-hide gelatin oligopeptides of specific molecular weights, eliminating interference from large, ineffective proteins and increasing the drug loading density of effective hematopoietic components in the complex. Pre-dissolving ferrous salts in deoxygenated deionized water containing ascorbic acid inhibited the oxidation of iron ions, ensuring that the iron ions participating in the chelation reaction remained in a divalent active state, thus maintaining the iron stability of the final composition during storage. Control of the dropping rate and reaction conditions during preparation limited the occurrence of side reactions and improved the conversion rate of the target peptide-iron complex. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments.

[0025] Example 1: The hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides in this embodiment consists of a nanoscale peptide-iron complex formed by the self-assembly of donkey-hide gelatin oligopeptides, plant-derived iron-targeting transport oligopeptides, and ferrous salts in a chelation reaction system. By mass percentage of the total composition, donkey-hide gelatin oligopeptides account for 70%, plant-derived iron-targeting transport oligopeptides account for 22%, and ferrous salts account for 8% by mass of iron.

[0026] Among them, the donkey-hide gelatin oligopeptide is an oligopeptide component with a molecular weight of 500Da-2000Da obtained by restrictive enzymatic hydrolysis of donkey-hide gelatin substrate by a complex protease composed of flavor protease and alkaline protease, and then retained by a two-stage ultrafiltration membrane assembly; the plant-derived iron-targeting transport oligopeptide is a short peptide mixture with a molecular weight of less than 500Da and a total molar percentage of histidine and glutamic acid residues of 45% in the amino acid sequence obtained by enzymatic hydrolysis of soybean protein isolate; the ferrous salt is ferrous sulfate.

[0027] The preparation method includes the following steps: Step S1: Preparation of donkey-hide gelatin enzymatic hydrolysate The donkey-hide gelatin substrate conforming to the 2025 edition of the Pharmacopoeia of the People's Republic of China was pulverized and passed through an 80-mesh sieve. It was then added to deoxygenated deionized water to prepare a donkey-hide gelatin solution with a substrate concentration of 8%. After stirring evenly, the pH of the system was adjusted to 8.0 with 0.1 mol / L sodium hydroxide solution. A specific complex protease was then added. The complex protease was a mixture of flavor protease (enzyme activity ≥ 50000 U / g) and alkaline protease (enzyme activity ≥ 200000 U / g) at an enzyme activity ratio of 1:3. The total amount of the complex protease added was 2% of the donkey-hide gelatin substrate mass. The system was placed in a constant temperature water bath at 50℃ and subjected to a restricted enzymatic hydrolysis reaction for 5 hours under constant temperature and stirring conditions. After the enzymatic hydrolysis reaction, the system was rapidly heated to 90℃ and kept at this temperature for 12 minutes to inactivate the enzyme. It was then cooled to room temperature and centrifuged at 4000 rpm for 15 minutes. The supernatant was collected to obtain the donkey-hide gelatin enzymatic hydrolysate.

[0028] Step S2: Preparation of donkey-hide gelatin oligopeptides A two-stage ultrafiltration membrane module made of polyethersulfone was used to fractionate the enzymatic hydrolysate of donkey-hide gelatin. The molecular weight cutoff of the first-stage ultrafiltration membrane was 3000 Da, and that of the second-stage ultrafiltration membrane was 500 Da. First, the enzymatic hydrolysate was pumped into the first-stage ultrafiltration membrane system, with the operating pressure controlled at 0.25 MPa and the feed temperature at 25°C. Circulation and filtration continued until the permeate volume reached 90% of the feed volume. The first-stage permeate was collected, and the high-molecular-weight cutoff was discarded. Then, the first-stage permeate was pumped into the second-stage ultrafiltration membrane system, with the operating pressure controlled at 0.3 MPa and the feed temperature at 25°C. Circulation and filtration continued until the cutoff volume reached 10% of the feed volume. The cutoff of the second-stage ultrafiltration membrane was collected, and the low-molecular-weight permeate was discarded. The resulting cutoff was placed in a vacuum freeze dryer and freeze-dried at -35°C and a vacuum of 10 Pa for 24 hours to obtain donkey-hide gelatin oligopeptides. Gel permeation chromatography determined that the molecular weight distribution range was 500 Da–2000 Da.

[0029] Step S3: Preparation of plant-derived iron-targeting transport oligopeptides Soy protein isolate was prepared into a 10% (w / w) protein solution by adding deionized water and adjusting the pH to 8.0. A complex protease consisting of alkaline protease and neutral protease was added at a concentration of 2% of the substrate mass. The solution was hydrolyzed at 55°C for 4 hours. After enzyme inactivation and centrifugation, the plant protein hydrolysate was obtained. The hydrolysate was separated using a Sephadex G-10 gel filtration chromatography column with 0.02 mol / L phosphate buffer (pH 6.0) as the mobile phase, a flow rate of 1.0 mL / min, and a detection wavelength of 220 nm. Elution peaks with elution volumes in the 12-18 min range were collected based on the elution curve. The molecular weight of these peaks was determined to be less than 500 Da by high performance liquid chromatography, and the total molar percentage of histidine and glutamic acid residues was 45% by an automated amino acid analyzer. The collected eluent was desalted and then freeze-dried at -35°C and 10 Pa for 24 hours to obtain plant-derived iron-targeting transport oligopeptides.

[0030] Step S4: Self-assembly preparation of peptide-iron complex According to the above mass ratio, take the donkey-hide gelatin oligopeptide obtained in step S2 and the plant-derived iron-targeting transport oligopeptide obtained in step S3, add them to deoxygenated deionized water, and stir until completely dissolved to form a mixed peptide solution with a total peptide mass concentration of 5%; adjust the pH of the mixed peptide solution to 6.0 with 0.1 mol / L hydrochloric acid solution, and stir continuously under light-protected conditions at a stirring speed of 300 rpm; take ferrous sulfate, dissolve it in deoxygenated deionized water containing 0.3% ascorbic acid, and prepare a ferrous salt precursor solution with an iron ion concentration of 0.5 mol / L, wherein the plant-derived iron target The ratio of the total molar number of histidine and glutamic acid residues in the transport oligopeptide to the molar number of ferrous ions in the ferrous salt was 3:1. The ferrous salt precursor solution was slowly added dropwise to the mixed peptide solution at a constant dropping rate of 15 mL / min. After the addition was complete, the reaction was continued to be stirred in the dark at a constant temperature for 3 h, and the reaction temperature was controlled at 25 °C. After the reaction was completed, the reaction solution was filtered through a 0.22 μm microporous membrane for sterilization. The filtrate was freeze-dried at -35 °C and a vacuum of 10 Pa for 24 h to obtain nano-sized peptide-iron complex powder, which is the hematopoietic peptide composition based on donkey-hide gelatin peptide.

[0031] Dynamic light scattering analysis showed that the average particle size of the peptide-iron complex prepared in this example was 102 nm, and the particle size distribution coefficient (PDI) was 0.128.

[0032] Example 2: In this embodiment, the hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides comprises, by weight percentage, 60% donkey-hide gelatin oligopeptides, 30% plant-derived iron-targeting transport oligopeptides, and 10% ferrous salts by weight of iron.

[0033] The remaining formulation, raw material sources, and preparation methods are completely consistent with those in Example 1.

[0034] Example 3: In this embodiment, the hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides comprises, by weight percentage, 80% donkey-hide gelatin oligopeptides, 15% plant-derived iron-targeting transport oligopeptides, and 5% ferrous salts by weight of iron.

[0035] The remaining formulation, raw material sources, and preparation methods are completely consistent with those in Example 1.

[0036] Example 4: The hematopoietic peptide composition based on donkey-hide gelatin peptides in this embodiment uses pea protein as the raw material for preparing plant-derived iron-targeting transport oligopeptides. The short peptide mixture obtained by enzymatic hydrolysis has a molecular weight of less than 500 Da and a total molar percentage of histidine and glutamic acid residues of 43%.

[0037] The remaining formulation, raw material dosage, and preparation method steps are completely consistent with those in Example 1.

[0038] Example 5: The hematopoietic peptide composition based on donkey-hide gelatin peptides in this embodiment uses wheat gluten as the raw material for preparing plant-derived iron-targeting transport oligopeptides. The short peptide mixture obtained by enzymatic hydrolysis has a molecular weight of less than 500 Da and a total molar ratio of histidine and glutamic acid residues of 42%.

[0039] The remaining formulation, raw material dosage, and preparation method steps are completely consistent with those in Example 1.

[0040] Example 6: The hematopoietic peptide composition based on donkey-hide gelatin peptide in this embodiment is ferrous chloride as the ferrous salt, and the rest of the formulation, raw material dosage and preparation method are completely consistent with those in Example 1.

[0041] Example 7: The hematopoietic peptide composition based on donkey-hide gelatin peptide in this embodiment is ferrous lactate salt, and the rest of the formulation, raw material dosage and preparation method are completely consistent with those in Example 1.

[0042] Example 8: The hematopoietic peptide composition based on donkey-hide gelatin peptides in this embodiment is ferrous citrate salt, and the remaining formulation, raw material dosage and preparation method are completely consistent with those in Example 1.

[0043] Example 9: In this embodiment, the hematopoietic peptide composition based on donkey-hide gelatin peptide has a ratio of 2:1 between the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide and the molar number of ferrous ions in the ferrous salt. The remaining formulation, raw material dosage, and preparation method steps are completely consistent with those in Example 1.

[0044] Example 10: The hematopoietic peptide composition based on donkey-hide gelatin peptides in this embodiment has the same formulation and raw material dosage as in Example 1. In the preparation method, the limiting enzymatic hydrolysis reaction parameters in step S1 are adjusted as follows: the complex protease is a mixture of flavor protease and alkaline protease at an enzyme activity ratio of 1:2, the substrate mass concentration of donkey-hide gelatin solution is 5%, the amount of complex protease added is 1% of the substrate mass of donkey-hide gelatin, the enzymatic hydrolysis temperature is 45℃, the pH value of the enzymatic hydrolysis system is 7.5, the enzymatic hydrolysis reaction time is 4h, and the enzyme inactivation condition is 85℃ for 15min.

[0045] The remaining preparation methods and steps are completely consistent with those in Example 1.

[0046] Example 11: The hematopoietic peptide composition based on donkey-hide gelatin peptides in this embodiment has the same formulation and raw material dosage as in Example 1. In the preparation method, the limiting enzymatic hydrolysis reaction parameters in step S1 are adjusted as follows: the complex protease is a mixture of flavor protease and alkaline protease at an enzyme activity ratio of 1:4, the substrate mass concentration of donkey-hide gelatin solution is 10%, the amount of complex protease added is 3% of the substrate mass of donkey-hide gelatin, the enzymatic hydrolysis temperature is 55℃, the pH value of the enzymatic hydrolysis system is 8.5, the enzymatic hydrolysis reaction time is 6h, and the enzyme inactivation condition is 95℃ for 10min.

[0047] The remaining preparation methods and steps are completely consistent with those in Example 1.

[0048] Example 12: The hematopoietic peptide composition based on donkey-hide gelatin peptide in this embodiment has the same formulation and raw material dosage as in Example 1. In the preparation method, the self-assembly reaction parameters in step S4 are adjusted as follows: the initial pH value of the mixed peptide solution is adjusted to 5.5, the dropping rate of the ferrous salt precursor solution is 10 mL / min, the stirring speed is 200 rpm, and the reaction time in the dark is 2 h.

[0049] The remaining preparation methods and steps are completely consistent with those in Example 1.

[0050] Comparative Example 1: In this comparative example, the plant-derived iron-targeting transport oligopeptide component was removed from the formulation. The missing component was made up by the mass percentage of donkey-hide gelatin oligopeptide. That is, based on the total mass percentage of the composition, the mass percentage of donkey-hide gelatin oligopeptide was 92%, and based on the mass of iron, the mass percentage of ferrous salt was 8%.

[0051] The sources of other raw materials and the preparation methods and steps are completely consistent with those in Example 1.

[0052] Comparative Example 2: This comparative example uses the conventional scheme of the prior art described in the background. The composition is a physical mixture of donkey-hide gelatin oligopeptide and ferrous salt. By mass percentage of the total composition, the donkey-hide gelatin oligopeptide accounts for 70%, the soybean protein isolate hydrolysate (without gel filtration chromatography enrichment, the total molar percentage of histidine and glutamic acid residues is 18%) accounts for 22%, and the ferrous sulfate accounts for 8% by mass of iron. The preparation method is as follows: the above raw materials are added to a three-dimensional mixer according to the ratio and mixed for 30 minutes until uniform to obtain the composition powder. No chelation self-assembly reaction is performed.

[0053] The sources of the remaining raw materials are exactly the same as in Example 1.

[0054] Comparative Example 3: In this comparative example, the ratio of the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide to the molar number of ferrous ions in the ferrous salt in the chelation reaction system is 1:1. The remaining formulation, raw material amounts, and preparation methods are completely consistent with those in Example 1.

[0055] Comparative Example 4: In this comparative example, the multi-stage ultrafiltration separation step in step S2 is omitted from the preparation method. The product obtained by directly freeze-drying the enzyme-inactivated hydrolysate of donkey-hide gelatin obtained in step S1 is used as donkey-hide gelatin oligopeptide. The remaining formulation, raw material dosage and preparation method steps are completely consistent with those in Example 1.

[0056] Test method: Average particle size determination: A dynamic light scattering particle size analyzer (Malvin Zetasizer Nano ZS90) was used. The sample to be tested was diluted with degassed ultrapure water to a peptide mass concentration of 1 mg / mL. After vortex mixing, the average hydration particle size and particle size distribution coefficient (PDI) of the sample were determined at 25℃. Each sample was measured in parallel 3 times and the arithmetic mean was taken.

[0057] Ferrous ion chelation rate determination: The o-phenanthroline spectrophotometric method was used. The sample to be tested was accurately weighed and dissolved in ultrapure water to prepare a sample solution with an iron concentration of 0.1 mg / mL. 10 mL of the sample solution was taken, and an equal volume of 10% (w / v) trichloroacetic acid solution was added. After vortexing and mixing, the mixture was allowed to stand for 30 min, centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The content of free ferrous ions in the supernatant was determined using the o-phenanthroline method. An equal volume of the sample solution was also taken, and after adding 10% nitric acid solution and heating for digestion, the total ferrous ion content in the sample was determined. The ferrous ion chelation rate was calculated using the following formula: Ferrous ion chelation rate (%) = (Total ferrous ion content - Free ferrous ion content) / Total ferrous ion content × 100% Each sample was measured in triplicate, and the arithmetic mean was taken.

[0058] Storage stability test: The sample to be tested was sealed in an aluminum foil bag and stored in a constant temperature and humidity chamber at 25℃ and 60% relative humidity for 6 months in the dark. The ferrous ion content in the sample before and after storage was measured, and the ferrous ion retention rate was calculated according to the following formula: Ferrous ion retention rate (%) = Ferrous ion content in the sample after 6 months of storage / Ferrous ion content in the sample before storage × 100% Each sample was measured in triplicate, and the arithmetic mean was taken.

[0059] Determination of iron precipitation rate in simulated intestinal environment: A phosphate-buffered saline (PBS) solution with pH 7.4 was prepared to simulate the weakly alkaline environment of the human intestine. The sample to be tested was diluted with the PBS buffer to an iron concentration of 100 μg / mL. After vortexing, the sample was incubated in a 37°C water bath in the dark for 2 hours. After incubation, the sample was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to determine the soluble iron content. The iron precipitation rate was calculated using the following formula: Iron precipitation rate (%) = (Total iron content of sample - Soluble iron content of supernatant) / Total iron content of sample × 100% Each sample was measured in triplicate, and the arithmetic mean was taken.

[0060] Determination of transmembrane transport efficiency in Caco-2 cells: The transmembrane transport efficiency of iron was evaluated using a Caco-2 cell monolayer model. Caco-2 cells were seeded at a density of 1×10^5 cells / well in Transwell cell culture chambers (0.4 μm pore size, 1.12 cm² area) and cultured at 37℃ in a 5% CO2 incubator for 21 days, with the culture medium changed every 2 days. After culture, the transepithelial resistance (TEER) was measured to be ≥500 Ω·cm², confirming the formation of a dense monolayer. Before the experiment, the cell monolayer was washed three times with preheated HBSS buffer. 2 mL of HBSS buffer containing the sample to be tested (iron concentration 50 μg / mL) was added to the supply chamber, and 2 mL of blank HBSS buffer was added to the receiving chamber. The chambers were incubated at 37℃ in a 5% CO2 incubator for 2 h. After incubation, the solution from the receiving chamber was collected, and the iron content was determined using inductively coupled plasma mass spectrometry (ICP-MS). The transmembrane transport efficiency was calculated using the following formula: Transmembrane transport efficiency (%) = (Total mass of iron in the receiving chamber / Total mass of iron in the supply chamber) × 100% Three replicates were set up for each sample in parallel, and the arithmetic mean was taken.

[0061] Determination of hematopoietic function enhancement rate in mice: SPF-grade male Kunming mice, weighing 18-22g, were acclimatized for 3 days. A hemorrhagic anemia model was established using orbital bloodletting, with 0.3mL of blood drawn from the orbital cavity each time for 3 consecutive days. Successful modeling was confirmed when the hemoglobin (Hb) content was ≤90g / L. Mice with successful modeling were randomly divided into an experimental group and a model control group, with 10 mice in each group. The experimental group mice were administered the test sample by gavage at a dose of 2mg / kg·d (calculated as iron). The model control group mice were administered an equal volume of physiological saline by gavage for 14 consecutive days. 24 hours after the last administration, blood was collected from the orbital cavity, and the hemoglobin content in whole blood was measured using an automated hematology analyzer. The hemoglobin enhancement rate was calculated using the following formula: Hemoglobin increase rate (%) = (Average hemoglobin content in the experimental group - Average hemoglobin content in the model control group) / Average hemoglobin content in the model control group × 100% Test results: Table 1. Results of Composition Performance and Efficacy Tests

[0062] Results analysis: The test results of Examples 1-12 show that the formulation and preparation method specified in this embodiment can stably form nanoscale peptide-iron complexes with particle sizes of 80nm-150nm within the parameter range. The ferrous ion chelation rate is ≥88%, the iron precipitation rate under simulated weakly alkaline intestinal environment is ≤6.5%, the transmembrane transport efficiency of Caco-2 cells is ≥24%, the increase in mouse hemoglobin is ≥28%, and the ferrous ion retention rate is ≥86% after 6 months of storage. This fully demonstrates that the technical solution of this embodiment has excellent robustness within the specified parameter range and can stably achieve the expected technical effect. Among them, Example 1 is the optimal solution, with all performance indicators reaching the best level, verifying the optimality of this parameter combination.

[0063] Comparative Example 1 removed the core component, plant-derived iron-targeting transport oligopeptide, and only retained the donkey-hide gelatin oligopeptide to react with ferrous salt. The results showed that the ferrous chelation rate was only 35.2%, a decrease of 61.9% compared with Example 1. The iron precipitation rate in the simulated intestinal environment was as high as 42.6%. The transmembrane transport efficiency and hemoglobin enhancement rate were both less than half of those in Example 1. This fully demonstrates that plant-derived iron-targeting transport oligopeptide is the core material basis for achieving stable chelation of ferrous ions, anti-precipitation in the intestinal environment, and targeted transmembrane transport. Its rich histidine and glutamic acid residues provide sufficient coordination chelation sites for ferrous ions. Without this component, a stable nanoscale peptide-iron complex cannot be formed, and the core problems of easy precipitation and low bioavailability of free iron in the background technology cannot be solved.

[0064] Comparative Example 2 uses the conventional physical mixing scheme described in the background section, which does not involve chelation self-assembly. Ferrous ions exist entirely in free form, resulting in a ferrous chelation rate of 0%. Under simulated weakly alkaline intestinal conditions, the iron precipitation rate is as high as 58.7%, and the transmembrane transport efficiency is only 4.2%, leading to extremely poor hematopoietic promotion effects. This directly demonstrates the core defects of the existing technology. In contrast, this invention, through chelation self-assembly to form a nanoscale peptide-iron complex, completely solves the problem of free iron easily forming hydroxide precipitates in the intestinal environment. It achieves simultaneous transmembrane delivery of donkey-hide gelatin oligopeptides and iron, achieving unexpected technical effects compared to existing technologies, and possessing outstanding substantive features and significant progress.

[0065] Comparative Example 3 adjusted the molar ratio of histidine to glutamic acid residues and ferrous ions to 1:1, which exceeded the 2:1-4:1 range specified in this embodiment. This resulted in a severe shortage of coordination chelation sites, failing to fully saturate the coordination bonds of ferrous ions. Consequently, the ferrous chelation rate dropped significantly to 52.1%, and a large number of free ferrous ions precipitated in the intestinal environment, increasing the iron precipitation rate to 28.9%. The transmembrane transport efficiency and hematopoietic promotion effect decreased significantly. This fully demonstrates that the coordination molar ratio range specified in this embodiment is a key parameter for forming a stable chelation structure and achieving anti-precipitation and targeted transport effects. Deviating from this range will prevent the achievement of the expected technical effects.

[0066] Comparative Example 4 omitted the multi-stage ultrafiltration separation step. The donkey-hide gelatin oligopeptide used was a direct enzymatic hydrolysis freeze-dried product with a wide molecular weight distribution. It contained a large number of macromolecular proteins and small free amino acids, which could not form a uniform and stable nanoscale complex with plant-derived oligopeptides and ferrous ions. The average particle size was as high as 280 nm. The chelation rate, storage stability, anti-precipitation performance and transport efficiency were significantly reduced. This fully demonstrates that the restricted enzymatic hydrolysis combined with the multi-stage ultrafiltration fractionation step in this embodiment can accurately obtain donkey-hide gelatin oligopeptides in the target molecular weight range. This is a key preparation step to ensure the uniformity, stability and bioactivity of the complex structure. Without this step, the expected technical effect cannot be achieved.

[0067] In summary, this embodiment combines donkey-hide gelatin oligopeptides with plant-derived iron-targeting transport oligopeptides rich in histidine and glutamic acid within a specific molecular weight range, and then self-assembles them with ferrous salts to form nanoscale peptide-iron complexes. This completely solves the core technical problems in the prior art, such as easy precipitation of free iron, low iron bioavailability, and the inability to deliver donkey-hide gelatin peptides and iron simultaneously. The performance and efficacy of each embodiment are significantly better than the comparative example, and they have excellent stability and hematopoietic activity, fully demonstrating the inventiveness and beneficial effects of this technical solution.

Claims

1. A peptide composition based on donkey-hide gelatin peptides to promote hematopoiesis, characterized in that, The peptide composition consists of a nanoscale peptide-iron complex formed by the self-assembly of donkey-hide gelatin oligopeptide, plant-derived iron-targeting transport oligopeptide, and ferrous salt in a chelation reaction system. Based on the total mass percentage of the peptide composition, the donkey-hide gelatin oligopeptide accounts for 60%-80% of the mass, the plant-derived iron-targeting transport oligopeptide accounts for 15%-30% of the mass, and the ferrous salt accounts for 5%-10% of the mass based on the mass of iron. The donkey-hide gelatin oligopeptides are oligopeptide components with a molecular weight of 500 Da-2000 Da obtained by restrictive enzymatic hydrolysis of donkey-hide gelatin substrate by specific endopeptide protease and then retention by a multi-stage ultrafiltration membrane assembly. The plant-derived iron-targeting transport oligopeptide is a mixture of short peptides with a molecular weight of less than 500 Da and a total molar percentage of histidine and glutamic acid residues greater than 40% obtained by enzymatic hydrolysis of plant protein.

2. The hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 1, characterized in that, The plant protein is any one of soy protein isolate, pea protein, or wheat gluten. The plant-derived iron-targeting transport oligopeptide is a short peptide component whose elution volume is within a specific peak range after the plant protein has been hydrolyzed by a complex protease and then separated and collected by gel filtration chromatography.

3. The hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 1, characterized in that, The ferrous salt is any one of ferrous sulfate, ferrous chloride, ferrous lactate, or ferrous citrate. Before being added to the chelation reaction system, the ferrous salt is dissolved in deoxygenated deionized water containing 0.1%-0.5% ascorbic acid to form a ferrous salt precursor solution.

4. The hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 1, characterized in that, The dynamic light scattering particle size of the nanoscale peptide-iron complex is 80nm-150nm. In the chelation reaction system, the ratio of the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide to the molar number of ferrous ions in the ferrous salt is 2:1 to 4:

1. The initial pH of the chelation reaction system is adjusted to 5.5-6.

5.

5. A method for preparing a hematopoietic-promoting peptide composition based on donkey-hide gelatin peptides according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: Step S1: Add the donkey-hide gelatin substrate to water to prepare a donkey-hide gelatin solution, add a specific endopeptidase to carry out a restriction enzymatic hydrolysis reaction, and obtain donkey-hide gelatin hydrolysate; Step S2: The enzymatic hydrolysate of donkey-hide gelatin is sequentially passed through a first-stage ultrafiltration membrane and a second-stage ultrafiltration membrane for multi-stage separation. The retentate is collected and freeze-dried to obtain donkey-hide gelatin oligopeptides. Step S3: The plant protein hydrolysate is separated by chromatography, and the eluent rich in histidine and glutamic acid residues is collected and freeze-dried to obtain plant-derived iron-targeting transport oligopeptides. Step S4: Dissolve the donkey-hide gelatin oligopeptide and the plant-derived iron-targeting transport oligopeptide in water to form a mixed peptide solution. Add ferrous salt solution dropwise to the mixed peptide solution and carry out a self-assembly reaction under light-protected conditions. After the reaction is completed, freeze-dry to obtain the peptide composition.

6. The method for preparing the hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 5, characterized in that, In step S1, the specific endopeptide is a complex protease composed of flavor protease and alkaline protease at an enzyme activity ratio of 1:2 to 1:4, and the conditions for the limiting enzymatic hydrolysis reaction are: The substrate concentration of the donkey-hide gelatin solution is 5%-10%, the amount of compound protease added is 1%-3% of the substrate mass of the donkey-hide gelatin, the enzymatic hydrolysis temperature is 45℃-55℃, the pH value of the enzymatic hydrolysis system is 7.5-8.5, the enzymatic hydrolysis reaction time is 4h-6h, and after the enzymatic hydrolysis reaction is completed, the temperature is raised to 85℃-95℃ and kept at 10min-15min to inactivate the enzyme.

7. The method for preparing the hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 5, characterized in that, In step S2, the molecular weight cutoff of the first-stage ultrafiltration membrane is 3000 Da-5000 Da, and the molecular weight cutoff of the second-stage ultrafiltration membrane is 500 Da. The enzymatic hydrolysate of donkey-hide gelatin is first separated by the first-stage ultrafiltration membrane to remove the large molecular weight protein retentate, and the first-stage permeate is collected. Then, the first-stage permeate is pumped into the second-stage ultrafiltration membrane, and the retentate of the second-stage ultrafiltration membrane is collected. The retentate is freeze-dried at a temperature of -40°C to -30°C and a vacuum degree of less than 20 Pa.

8. The method for preparing the hematopoietic-promoting peptide composition based on donkey-hide gelatin peptide according to claim 5, characterized in that, In step S4, the ferrous salt solution is prepared by dissolving ferrous salt in deoxygenated deionized water containing 0.1%-0.5% ascorbic acid by mass. The pH of the mixed peptide solution is adjusted to 5.5-6.

5. In the mixed peptide solution, the ratio of the total molar number of histidine and glutamic acid residues in the plant-derived iron-targeting transport oligopeptide to the molar number of ferrous ions in the ferrous salt solution is 2:1 to 4:

1. The ferrous salt solution is added dropwise to the mixed peptide solution at a dropping rate of 10 ml / min-20 ml / min, and the reaction is carried out under light-protected stirring conditions at a speed of 200 rpm-400 rpm for 2 h-4 h.

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