Preparation method of deer spleen aminopeptide powder

Abstract

The invention discloses a preparation method of deer spleen aminopeptide powder in the field of bioactive substance extraction and functional material compositing, and the performance of the deer spleen aminopeptide powder is improved by compositing a multi-stage functional modified material and deer spleen aminopeptide. The preparation method comprises the following steps: cleaning, homogenizing and crushing fresh deer spleen, extracting with a phosphate buffer solution, adjusting the pH value, carrying out enzymolysis, inactivating enzyme, and treating with an ultrafiltration membrane and a nanofiltration membrane in a specific molecular weight range to obtain a concentrated solution; and mixing the concentrated solution with a multi-stage functional modified material under proper conditions, sterilizing, pre-freezing, and freeze-drying to obtain the deer spleen aminopeptide powder. Wherein the modified material is constructed through quaternization modification, nanopore loading, enzyme inhibitor coupling and targeting ligand modification, and has the functions of charge repulsion, steric hindrance, enzyme inhibition and targeting recognition. The deer spleen aminopeptide powder prepared by the invention has high stability and good bioavailability, and is suitable for functional food or health care products.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction and functional material composite technology, specifically to a method for preparing deer spleen amino peptide powder. Background Technology

[0002] Deer spleen, a traditional medicinal animal organ, is rich in active ingredients such as polypeptides, amino acids, and immunomodulatory factors. Its hydrolysis product, deer spleen amino peptide, exhibits significant application value in functional foods and health products due to its immunomodulatory, antioxidant, and anti-fatigue bioactivities. However, traditional methods for preparing deer spleen amino peptide powder have significant limitations, restricting its large-scale development and high-value-added applications.

[0003] Traditional preparations of deer spleen amino peptide powder rely heavily on conventional processes such as enzymatic hydrolysis, centrifugation, and membrane separation. However, products obtained using these methods often exhibit poor stability. Small molecule peptides are susceptible to degradation by proteases, oxidation, and pH fluctuations in the environment, leading to a significant decrease in bioactivity over storage time. Furthermore, deer spleen amino peptides have a wide molecular weight distribution (mostly 1-5 kDaltons), resulting in limited intestinal absorption efficiency and low bioavailability. In addition, traditional products have limited functionality, serving only as nutritional supplements and lacking targeted delivery or sustained-release capabilities, failing to meet the demands of modern functional foods for "highly efficient absorption and precise action." While existing technologies have attempted to use single polymers such as sodium alginate and chitosan as protective agents, these methods suffer from low transport efficiency and insufficient compatibility between the materials and bioactive components, failing to effectively address the dual bottlenecks of stability and bioavailability.

[0004] To address the aforementioned pain points, developing a novel material with high binding capacity and multi-level functional modification, combined with an optimized enzymatic hydrolysis process for preparing deer spleen amino peptide powder, is crucial. This invention constructs a four-level gradient modified material, integrating multiple functions such as charge repulsion, steric hindrance, enzyme inhibition, and targeted transport, effectively solving the problems of poor stability and low absorption efficiency in traditional deer spleen amino peptide powder. Simultaneously, optimizing enzymatic hydrolysis conditions and membrane separation processes improves the yield and purity of amino peptides, ultimately producing deer spleen amino peptide powder with high stability and good bioavailability, providing a new path for the high-value-added development of functional foods and health products. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing deer spleen amino peptide powder, which solves the technical problems of poor stability, low bioavailability, and limited function of existing deer spleen amino peptide powder.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] A method for preparing deer spleen amino peptide powder, comprising the following steps:

[0008] S1. Take fresh deer spleen, wash it with physiological saline to remove surface blood, remove fat and connective tissue, cut it into a homogenate, add pre-cooled phosphate buffer solution with pH=6.9-7.1, and break the cells with a high-speed homogenizer to obtain a homogenate.

[0009] S2, centrifuge the homogenate at 3.8-4.2℃ and collect the supernatant; adjust the pH to 7.8-8.2, add alkaline protease, and enzymatically hydrolyze at 50-52℃; after enzymatic hydrolysis, inactivate the enzyme in a water bath at 95-97℃ and cool to room temperature;

[0010] S3, the enzymatic hydrolysate is filtered through a 9-11 kDa ultrafiltration membrane, and the permeate is collected; then it is concentrated through a 2-4 kDa nanofiltration membrane to obtain deer spleen amino peptide concentrate.

[0011] S4. The deer spleen amino peptide concentrate is mixed and shaken with QCS-MSN-STI-RGD modified material at 3-5℃; after sterilization by microporous filter membrane, the composite solution is pre-frozen at -50~-52℃ and then freeze-dried at -50℃.

[0012] According to a preferred embodiment of the present invention, in step S1, the volume of the phosphate buffer is 3-5 times the volume of the homogenate; the rotation speed of the high-speed homogenizer is 10,000-12,000 rpm.

[0013] In step S1 of this invention, fresh deer spleen is used as raw material. Its dense tissue structure encapsulates a large number of active ingredients (such as polypeptides, amino acids, and immunomodulatory factors). Initially, the spleen is washed with physiological saline to remove surface blood, fat, and connective tissue, reducing interference from impurities in subsequent processes. The deer spleen is then cut into small pieces and subjected to high-speed homogenization. The mechanical shearing force of the homogenizer (such as the cutting force generated by the high-speed rotation of the blades) disrupts the integrity of the cell membrane, allowing intracellular substances to be fully released into the solution. Pre-cooled phosphate buffer (pH close to the physiological environment, 6.9-7.1) plays a crucial role in this process: on the one hand, it maintains a stable solution pH, preventing denaturation or degradation of active ingredients due to drastic pH changes; on the other hand, it reduces the heat generated during mechanical shearing through buffering, preventing high temperatures from damaging sensitive components. The homogenization of the homogenate directly affects the efficiency of subsequent enzymatic hydrolysis; therefore, it is necessary to ensure sufficient cell disruption and avoid large tissue fragments that hinder enzyme-substrate contact, providing a homogeneous raw material basis for subsequent enzymatic hydrolysis.

[0014] According to a preferred embodiment of the present invention, in step S2, the centrifugation time is 15-20 min; the enzymatic hydrolysis time is 4-6 h; and the enzyme inactivation time is 15-20 min.

[0015] In step S2 of this invention, after centrifuging the homogenate (3.8-4.2℃, 15-20 min) to remove undisturbed cell debris and large particulate impurities, the pH is adjusted to a weakly alkaline environment (7.8-8.2), and alkaline protease is added to initiate the hydrolysis reaction. The alkaline protease cleaves proteins into smaller peptides by recognizing specific peptide bonds (such as the carboxyl-terminal peptide bonds of arginine and lysine). The reaction temperature is controlled at 50-52℃, at which the enzyme activity is highest, ensuring cleavage efficiency while avoiding enzyme denaturation and inactivation due to high temperatures. As enzymatic hydrolysis proceeds, large protein molecules are gradually broken down into small peptides with a concentrated molecular weight distribution (mainly distributed in the 1-5 kDalton range). After enzymatic hydrolysis, the enzyme is inactivated by high temperature (95-97℃, 15-20 min) to terminate the reaction and prevent further enzyme action that could lead to excessive hydrolysis of the target peptides (such as decomposition into smaller amino acids or dipeptides, reducing biological activity). This step ensures that the generated small peptides retain their immunomodulatory, antioxidant, and other biological activities by precisely controlling the enzymatic hydrolysis conditions (pH, temperature, and time), while also facilitating subsequent separation and purification.

[0016] According to a preferred embodiment of the present invention, in step S3, the permeate contains small molecule peptides with a molecular weight of <10kDa; the nanofiltration membrane concentration pressure is 1.4-1.6MPa, and the nanofiltration membrane concentration temperature is 24-26℃.

[0017] In step S3 of this invention, the enzymatic hydrolysate contains impurities such as unhydrolyzed large molecular weight proteins, peptides, and inorganic salts, in addition to the target small molecule peptides. The ultrafiltration membrane (9-11 kDa pore size) uses physical sieving to allow only small molecule peptides with a molecular weight less than 10 kilodaltons to pass through, while retaining large molecular weight impurities (such as proteins and peptides with a molecular weight greater than 10 kilodaltons), thus initially enriching the target peptide. Subsequently, the filtrate is further concentrated using a nanofiltration membrane (2-4 kDa pore size) under pressure (1.4-1.6 MPa): water and small molecule impurities (such as monosaccharides and inorganic salts) are filtered out, while small molecule peptides are retained due to their larger molecular weight, thereby significantly increasing the peptide concentration (from a lower initial concentration to 9-11 mg / mL). This process, through two-stage membrane separation technology (ultrafiltration and nanofiltration), effectively removes impurities and concentrates the target components, providing high-purity raw materials for subsequent functionalization modifications, while avoiding the impact of impurities on the binding efficiency of the modified materials, ensuring the stability of subsequent compounding steps.

[0018] According to a preferred embodiment of the present invention, in step S4, the concentration of the deer spleen amino peptide concentrate is 9-11 mg / mL; the mass ratio of the deer spleen amino peptide concentrate to the QCS-MSN-STI-RGD modified material is (9-11):1; the mixing and oscillation speed is 150-200 rpm, and the oscillation time is 2-4 h; the pore size of the microporous filter membrane is 0.2-0.24 μm; the pre-freezing time is 4-6 h; the vacuum degree of freeze-drying is 10-12 Pa, and the freeze-drying time is 24-30 h.

[0019] In step S4 of this invention, the compounding of deer spleen amino peptide concentrate with the four-level gradient modified material is a key step in improving product performance. The modified material, through a four-level functional design (charge repulsion, steric hindrance, enzyme inhibition, and targeted transport), forms a synergistic protective system with the deer spleen amino peptide: First, the positively charged groups (such as quaternary ammonium salt groups) on the surface of the modified material adsorb the negatively charged deer spleen amino peptide through electrostatic interactions, forming a stable complex and reducing the contact between the amino peptide and negatively charged proteases in the environment; second, the mesoporous silica nanoparticles (pore size 50-100 nm) inside the material construct a physical barrier, preventing larger molecular weight enzyme molecules (such as trypsin, molecular weight ≈25 kDa) from approaching the amino peptide, thus inhibiting enzymatic hydrolysis; simultaneously, the bovine trypsin inhibitor loaded on the material directly neutralizes the enzymatic hydrolysis capacity by binding to the active sites of residual enzymes, further protecting the stability of the amino peptide; finally, the targeting ligands (such as RGD peptide) on the surface of the material can specifically recognize integrin receptors on the surface of M cells in the Pell's aggregate lymph nodes of the intestine, mediating the active uptake of the material by the cells and improving the transmembrane transport efficiency of the amino peptide. The four-fold synergistic effect makes deer spleen amino peptide less prone to degradation during storage (improved stability), and significantly improves absorption efficiency after entering the intestine (enhanced bioavailability), ultimately producing functional deer spleen amino peptide powder with both high stability and high bioavailability.

[0020] According to a preferred embodiment of the present invention, the preparation steps of the QCS-MSN-STI-RGD modified material include:

[0021] A1. Chitosan was dissolved in acetic acid solution to prepare a chitosan solution; 3-chloro-2-hydroxypropyltrimethylammonium chloride was added and the mixture was magnetically stirred at 40-42℃; after the reaction was completed, the mixture was dialyzed with deionized water until neutral and then freeze-dried to obtain quaternized ammonium salt chitosan.

[0022] A2, a quaternized chitosan solution was mixed with a hexadecyltrimethylammonium bromide aqueous solution, tetraethyl orthosilicate was added, the pH was adjusted to 8.9-9.1, and the reaction was carried out at 58-62℃; after centrifugation and washing, the hexadecyltrimethylammonium bromide template was etched with NaOH solution, and then dialyzed and dried again to obtain a quaternized chitosan-mesoporous silica nanoparticle composite.

[0023] A3. Quaternized chitosan-mesoporous silica nanoparticle complex was dispersed in phosphate buffer at pH 7.2-7.6, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was carried out at room temperature; then bovine trypsin inhibitor was added, and the reaction was carried out at 3.8-4.2℃; unreacted bovine trypsin inhibitor was removed by dialysis to obtain the QCS-MSN-STI complex.

[0024] A4. Dissolve the QCS-MSN-STI complex in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.8-6.2, add RGD peptide, carbodiimide, and N-hydroxysuccinimide, and react at room temperature; centrifuge, wash, and lyophilize.

[0025] According to a preferred embodiment of the present invention, in step A1, the mass ratio of chitosan to 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:(2-4); the magnetic stirring reaction time is 24-30h.

[0026] In step A1 of this invention, chitosan molecules contain a large number of amino groups (-NH2). In acetic acid solution (a weakly acidic environment), the amino groups will be partially protonated to positively charged -NH3. + However, the overall composition remains predominantly neutral. Upon addition of 3-chloro-2-hydroxypropyltrimethylammonium chloride, the bromine atom in its molecule acts as a favorable leaving group. Under mild heating conditions of 40-42℃, the amino group (-NH2) of chitosan acts as a nucleophile, attacking the α-carbon atom of 3-chloro-2-hydroxypropyltrimethylammonium chloride, resulting in a nucleophilic substitution reaction. The bromine atom is removed, and the amino group combines with the propidium group to form a stable quaternary ammonium salt structure (-N). + (CH3)3). In this process, 3-chloro-2-hydroxypropyltrimethylammonium chloride not only provides quaternary ammonium salt groups but also binds to the amino groups of chitosan through electrostatic interactions, increasing the positive charge density of the material. After the reaction is complete, unreacted 3-chloro-2-hydroxypropyltrimethylammonium chloride and byproducts are removed by dialysis with deionized water, and finally freeze-dried to obtain a chitosan material with a surface rich in quaternary ammonium salt groups. Its positive charge characteristics lay the foundation for subsequent functionalization modifications.

[0027] According to a preferred embodiment of the present invention, in step A2, the volume ratio of the quaternized chitosan solution to the hexadecyltrimethylammonium bromide aqueous solution is 1:5; the reaction time is 2-4 h; after centrifugation and washing, the temperature of the NaOH solution etching the CTAB template is 60-62 °C, and the etching time is 3-6 h.

[0028] In step A2 of this invention, after mixing the quaternized chitosan solution with the hexadecyltrimethylammonium bromide aqueous solution, the hydrophobic long chains (hexadecyl) of the hexadecyltrimethylammonium bromide spontaneously assemble into an ordered micelle structure in the solution, with the hydrophilic head (quaternary ammonium cation) facing outward and the hydrophobic tail chain facing inward, forming a nanoscale reaction template. After adding tetraethyl orthosilicate (silicon source), under weakly alkaline conditions (pH 8.9-9.1), the silicon source hydrolyzes to generate silanol (Si-OH). The silanol undergoes a condensation reaction with the hydroxyl groups (from water or chitosan) on the micelle surface, gradually depositing a silica layer on the micelle surface. As the reaction proceeds (isothermal at 58-62℃ to promote hydrolysis and condensation), the silica layer gradually thickens, ultimately forming ordered mesoporous silica nanoparticles with micelles as templates. After the reaction was completed, unreacted silicon source and surfactant were removed by centrifugation and washing. Then, the micelle template was etched with a strong base (NaOH) solution (to destroy its hydrophobic core) to expose the mesoporous structure. The residual base and impurities were removed by dialyzing again, and finally, a mesoporous silica nanoparticle composite with quaternized chitosan loaded on the surface was obtained. The ordered pores inside provide space for subsequent loading of functional molecules.

[0029] According to a preferred embodiment of the present invention, in step A3, the reaction time at room temperature is 30-60 min; the reaction time at 3.8-4.2℃ is 12-14 h.

[0030] In step A3 of this invention, when the quaternized chitosan-mesoporous silica nanoparticle composite is dispersed in phosphate buffer, the quaternary ammonium salt groups (-N) on its surface... + (CH3)3) binds initially to the carboxyl group (-COOH) of the bovine trypsin inhibitor (STI) molecule via electrostatic interaction. Upon addition of N-hydroxysuccinimide (NHS) and carbodiimide (EDC), EDC acts as a dehydrating agent, catalyzing the reaction of the carboxyl group with NHS to generate a highly reactive O-acylisourea intermediate. This intermediate readily undergoes nucleophilic substitution with the amino group (-NH2) to form a stable amide bond (-NH-CO-). Because the complex surface is rich in quaternary ammonium salt groups (positively charged), it can attract negatively charged STI molecules (where the carboxyl group dissociates into -COO). - This process promotes effective contact between the two components, thereby covalently coupling STI to the surface of the complex. A low-temperature environment of 3.8-4.2℃ can slow down side reactions (such as hydrolysis) and improve coupling efficiency. After the reaction is complete, unreacted STI is removed by dialysis, finally yielding composite particles loaded with bovine trypsin inhibitors, which are stably bound by covalent bonds and can effectively inhibit the activity of residual proteases.

[0031] According to a preferred embodiment of the present invention, in step A4, the molar ratio of the QCS-MSN-STI complex to the RGD peptide is 1:5; the reaction time at room temperature is 2-4 h.

[0032] In step A4 of this invention, the quaternary ammonium salt groups on the surface of the QCS-MSN-STI complex are pre-bound to the carboxyl groups (-COOH) in the RGD peptide molecule via electrostatic interactions. In 2-(N-morpholino)ethanesulfonic acid buffer (weakly acidic environment, pH 5.8-6.2), the carboxyl groups dissociate into -COO. - The positive charge on the complex surface further attracts the peptide, enhancing the binding force. Upon addition of NHS and EDC, EDC catalyzes the reaction of the carboxyl group with NHS to generate an O-acylisourea intermediate. This intermediate undergoes nucleophilic substitution with unoccupied amino groups (-NH2, from chitosan or the remaining amino group after STI coupling) on ​​the complex surface, forming an amide bond (-NH-CO-), thereby covalently coupling the RGD peptide to the complex surface. Room temperature reaction (2-4 h) ensures complete coupling. After centrifugation and washing to remove unreacted RGD peptides, lyophilization yields composite particles simultaneously loaded with STI and RGD peptides. The introduction of RGD peptides endows the material with targeted recognition capabilities, enabling it to mediate active uptake through binding to receptors on the surface of intestinal cells, thereby enhancing the absorption efficiency of deer spleen amino peptides.

[0033] The beneficial effects of this invention are as follows:

[0034] This invention significantly improves the stability and bioavailability of deer spleen amino peptide powder through multi-dimensional technological innovation, solving the core pain points of traditional preparation methods. Traditional deer spleen amino peptides, due to their small molecular weight and loose structure, are easily affected by environmental protease degradation, oxidation, and pH fluctuations, leading to rapid loss of active ingredients during storage. This invention uses a four-level gradient functionalized modified material to composite with deer spleen amino peptides. The modified material, through its positively charged surface, repels negatively charged proteases, forming the first "protective net" and effectively inhibiting the enzymatic hydrolysis of trypsin and other enzymes. Its internal nanopores physically prevent larger enzyme molecules from approaching the amino peptides, constructing the second "spatial barrier." Simultaneously, the enzyme inhibitor loaded on the material directly neutralizes residual enzyme activity, forming the third "chemical defense line." This triple protective synergistic effect significantly reduces the degradation rate of deer spleen amino peptides after 18 months of storage at room temperature, improving stability several times compared to traditional methods.

[0035] Improved bioavailability is another core advantage of this invention. Traditional deer spleen amino peptides, due to their wide molecular weight distribution, are difficult to penetrate the intestinal mucosa for efficient absorption, resulting in limited bioavailability. In this invention, the nanopores of the modified material can encapsulate and directionally transport deer spleen amino peptides to the surface of M cells in the Peyer's patch lymph nodes of the intestine. The surface-modified targeting ligands specifically bind to receptors on the M cell surface, mediating active uptake and significantly increasing the transmembrane transport efficiency of the amino peptides. Experiments show that deer spleen amino peptides loaded with this modified material exhibit improved intestinal absorption efficiency compared to traditional methods, effectively solving the problem of "difficult absorption."

[0036] Furthermore, this invention expands the application scenarios of deer spleen amino peptide powder through process optimization and functional integration. The multi-level functional modification of the modified material (charge repulsion, steric hindrance, enzyme inhibition, and targeted transport) achieves an integrated "protection-transportation-targeting" design, making the product not only highly stable and well-absorbed but also possessing active targeted delivery capabilities, precisely acting on intestinal immune-related cells to enhance immunomodulatory activity. Simultaneously, the ultrasound-assisted enzymatic hydrolysis and multi-level membrane separation technologies used in the preparation process effectively improve the yield and purity of amino peptides, ensuring consistent and controllable product quality. The deer spleen amino peptide powder prepared by this invention possesses high stability, high bioavailability, and multifunctionality, and can be widely used in functional foods, health products, and other fields, providing a new pathway for the high-value utilization of deer spleen active ingredients. Detailed Implementation

[0037] The following detailed embodiments are only used to further illustrate this application and should not be construed as limiting the scope of protection of this application. Those skilled in the art can make some non-essential improvements and adjustments to this application based on the above application content.

[0038] I. Main Raw Materials:

[0039] Chitosan was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (model: SH-Y012, degree of deacetylation ≥90%, molecular weight 50,000-100,000).

[0040] Acetic acid solution was purchased from Sinopharm Chemical Reagent Co., Ltd. (model: glacial acetic acid AR, concentration ≥99.5%).

[0041] 3-Chloro-2-hydroxypropyltrimethylammonium chloride was purchased from Aladdin Reagent (Shanghai Jingchun Biochemical Technology Co., Ltd.) (Model: A100205, purity ≥98%).

[0042] The dialysis bags were purchased from Shanghai Xinya Purification Equipment Factory (model: RC membrane, molecular weight cutoff 10kDa).

[0043] Hexadecyltrimethylammonium bromide (CTAB) was purchased from Sinopharm Chemical Reagent Co., Ltd. (model: CTAB-AR, purity ≥99%).

[0044] Tetraethyl orthosilicate (TEOS) was purchased from Aladdin Reagent (Shanghai Jingchun Biochemical Technology Co., Ltd.) (model: T105312, purity ≥99.5%).

[0045] Phosphate buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. (model: PBS buffer).

[0046] N-hydroxysuccinimide (NHS) was purchased from Aladdin Reagent (Shanghai Jingchun Biochemical Technology Co., Ltd.) (Model: N100205, purity ≥99%).

[0047] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from Aladdin Reagent (Shanghai Jingchun Biochemical Technology Co., Ltd.) (Model: E100205, purity ≥99%).

[0048] Bovine trypsin inhibitor (PMSF) was purchased from Beyotime Biotechnology Co., Ltd. (model: ST506, purity ≥98%).

[0049] 2-(N-morpholino)ethanesulfonic acid (MES) buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. (model: MES buffer).

[0050] The RGD peptide was purchased from Shanghai Jier Biochemical Technology Co., Ltd. (model: RGD-001, sequence: Arg-Gly-Asp, purity ≥95%).

[0051] The microporous filter membrane was purchased from Shanghai Xinya Purification Device Factory (model: MCE microporous filter membrane, pore size 0.22μm).

[0052] The alkaline protease was purchased from Weilan Biotechnology (model: Alkaline Protease AP-01).

[0053] The nanofiltration membrane was purchased from Times Wharton Technology Co., Ltd. (model: Vontron NF-2, molecular weight cutoff 2kDa).

[0054] II. Implementation Examples

[0055] Example 1

[0056] Preparation of QCS-MSN-STI-RGD modified material: 10g of chitosan with a molecular weight of 50,000 Da was dissolved in 1% acetic acid solution (100mL) to prepare a 10% (w / v) chitosan solution; 20g of 3-chloro-2-hydroxypropyltrimethylammonium chloride was added, and the mixture was magnetically stirred at 41℃ for 27h (200rpm); after the reaction, the solution was placed in a dialysis bag with a molecular weight cutoff of 10kDa, and dialyzed with deionized water until neutral (pH=7.0) for 48h (water was changed every 12h), and then freeze-dried (-50℃, vacuum degree 10Pa, 24h) to obtain quaternized ammonium salt chitosan. 10 g of quaternized chitosan was dissolved in 100 mL of deionized water and mixed with 50 mL of hexadecyltrimethylammonium bromide (CTAB) aqueous solution (10% by mass). 20 mL of tetraethyl orthosilicate (TEOS) was added, and the pH was adjusted to 9.0 (titrated with 1 mol / L NaOH solution). The reaction was carried out at 59 °C for 3 h (200 rpm). After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was washed three times with deionized water. The CTAB template was then etched with 1 mol / L NaOH solution at 61 °C for 4.5 h. The mixture was dialyzed again to neutral using a dialysis bag with a molecular weight cutoff of 10 kDa, and then freeze-dried (-50 °C, vacuum 10 Pa, 24 h) to obtain the quaternized chitosan-mesoporous silica nanoparticle composite. 10 g of the complex was dispersed in 100 mL of phosphate buffer at pH 7.4, and 5 g of N-hydroxysuccinimide (NHS) and 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added. The mixture was reacted at room temperature for 45 min (25 °C). Subsequently, 2 g of bovine trypsin inhibitor (PMSF) was added, and the mixture was reacted at 3.9 °C for 13 h. After the reaction was completed, unreacted PMSF was removed by dialysis using a dialysis bag with a molecular weight cutoff of 10 kDa (48 h, with water changed every 12 h) to obtain the QCS-MSN-STI complex. 10 g of the QCS-MSN-STI complex was dissolved in 100 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 6.0. 50 g of RGD peptide (sequence Arg-Gly-Asp, molecular weight 1060 Da), 3 g of EDC, and 2 g of NHS were added. The mixture was reacted at room temperature for 3 h (25℃). After centrifugation at 8000 rpm for 10 min, the precipitate was washed three times with deionized water and freeze-dried (-50℃, vacuum 10 Pa, 24 h) to obtain the QCS-MSN-STI-RGD modified material (denoted as modified material A).

[0057] Preparation of deer spleen amino peptide powder: Take 500g of fresh deer spleen (about the spleen of one adult deer), wash off the surface blood stains with 0.9% physiological saline (500mL), remove fat and connective tissue (about 100g), and cut the remaining 400g of deer spleen tissue into 1cm pieces. 3Homogenize the cells; add 1500 mL of pre-cooled phosphate buffer (pH 7.0), and homogenize using a high-speed homogenizer (11000 rpm) for 3 min to obtain a homogenate. Centrifuge the homogenate at 3.9℃ (11000 rpm, 17 min), and collect the supernatant (approximately 1200 mL); adjust the pH to 8.0 with 1 mol / L NaOH solution, add alkaline protease (trypsin, 1 g, enzyme activity 100,000 U / g), and incubate at 51℃ for 5 h (200 rpm); inactivate the enzyme in a 96℃ water bath for 17 min, and cool to room temperature (25℃). Filter the hydrolysate through a 9.5 kDa ultrafiltration membrane (molecular weight cutoff 9.5 kDa, membrane area 0.5 m²). 2 Filter the solution and collect the permeate (approximately 1000 mL); then pass the permeate through a 2.5 kDa nanofiltration membrane (molecular weight cutoff 2.5 kDa, membrane area 0.3 m²). 2 Concentrate (pressure 1.5 MPa, temperature 25℃) to obtain deer spleen amino peptide concentrate (concentration 10 mg / mL, volume approximately 300 mL). Mix the concentrate with modified material A (10 g of modified material A) at 3.5℃ and shake (speed 180 rpm, time 3 h); filter the composite solution through a 0.22 μm microporous membrane (membrane area 0.2 m²). 2 After sterilization, it is pre-frozen at -51℃ for 5 hours; after pre-freezing, it is freeze-dried at -50℃ (vacuum degree 11Pa, time 27 hours) to obtain deer spleen amino peptide powder.

[0058] Example 2

[0059] The specific implementation method is the same as in Example 1, except that the QCS-MSN-STI-RGD modified material is prepared as follows: 10g of chitosan is dissolved in 1% acetic acid solution (100mL), 25g of 3-chloro-2-hydroxypropyltrimethylammonium chloride is added, and the mixture is magnetically stirred at 42℃ for 24h; after dialyzing and lyophilization, quaternized ammonium salt chitosan is obtained. This is mixed with CTAB aqueous solution (50mL), 25mL of TEOS is added, pH=8.9, and the mixture is reacted at 61℃ for 2h; after centrifugation and washing, the CTAB template is etched at 62℃ for 3h, and the mixture is dialyzed and dried to obtain the complex. The complex is dispersed in MES buffer (100mL), 6g of NHS and 4g of EDC are added, and the mixture is reacted at room temperature for 60min; 3g of PMSF is added, and the mixture is reacted at 3.8℃ for 12h; after dialyzing, the mixture is lyophilized to obtain the QCS-MSN-STI complex. The complex was dissolved in MES buffer (100 mL), and 60 g of RGD peptide, 4 g of EDC, and 3 g of NHS were added. The mixture was reacted at room temperature for 2 h. After centrifugation and washing, the mixture was lyophilized to obtain modified material B (enzymatic hydrolysis time 2 h, etching time 3 h).

[0060] Preparation of deer spleen amino peptide powder: 600g of fresh deer spleen was washed with physiological saline and homogenized. Four times the volume of pH 7.1 phosphate buffer (2400mL) was added, and homogenization was performed at 12000rpm. The mixture was centrifuged at 3.8℃ (12000rpm, 15min), and the supernatant was adjusted to pH 8.2. Alkaline protease (1.2g) was added, and enzymatic hydrolysis was performed at 52℃ for 4h. The hydrolysate was then inactivated at 97℃ for 15min. The hydrolysate was filtered through a 10kDa ultrafiltration membrane and concentrated through a 2.5kDa nanofiltration membrane (pressure 1.6MPa, 26℃) to obtain a concentrated solution (concentration 11mg / mL). This solution was then shaken with modified material B (11g) at 3℃ (200rpm, 2h), sterilized using a 0.24μm filter membrane, pre-frozen at -52℃ for 4h, and freeze-dried at -50℃ (vacuum 12Pa, 24h).

[0061] Example 3

[0062] The specific implementation method is the same as in Example 1, except that the QCS-MSN-STI-RGD modified material is prepared as follows: 10g of chitosan is dissolved in 1% acetic acid solution (100mL), 30g of 3-chloro-2-hydroxypropyltrimethylammonium chloride is added, and the mixture is magnetically stirred at 40℃ for 30h; after dialyzing and lyophilization, quaternized ammonium salt chitosan is obtained. This is mixed with CTAB aqueous solution (50mL), 15mL of TEOS is added, pH=9.1, and the mixture is reacted at 58℃ for 4h; after centrifugation and washing, the CTAB template is etched at 58℃ for 6h, and the mixture is dialyzed and dried to obtain the complex. The complex is dispersed in MES buffer (100mL), 4g of NHS and 2g of EDC are added, and the mixture is reacted at room temperature for 30min; 2g of PMSF is added, and the mixture is reacted at 4.2℃ for 14h; after dialyzing, the mixture is lyophilized to obtain the QCS-MSN-STI complex. The complex was dissolved in MES buffer (100 mL), and 40 g of RGD peptide, 2 g of EDC, and 2 g of NHS were added. The mixture was reacted at room temperature for 4 h. After centrifugation and washing, the mixture was lyophilized to obtain modified material C (enzymatic hydrolysis time 6 h, etching time 6 h).

[0063] Preparation of deer spleen amino peptide powder: 400g of fresh deer spleen was washed with physiological saline and homogenized. 3.5 times the volume of pH 6.9 phosphate buffer (1400mL) was added, and homogenization was performed at 10000rpm. The mixture was centrifuged at 4.2℃ (10000rpm, 20min), and the supernatant was adjusted to pH 7.8. Alkaline protease (0.8g) was added, and enzymatic hydrolysis was performed at 4.5℃ for 6h. The hydrolysate was then inactivated at 95℃ for 20min. The hydrolysate was filtered through an 8kDa ultrafiltration membrane and concentrated through a 2kDa nanofiltration membrane (pressure 1.4MPa, 24℃) to obtain a concentrated solution (concentration 9mg / mL). This solution was then shaken with modified material C (11g) at 5℃ (150rpm, 4h), sterilized using a 0.2μm filter membrane, pre-frozen at -50℃ for 6h, and freeze-dried at -50℃ (vacuum 10Pa, 30h).

[0064] Comparative Example 1

[0065] The specific implementation method is the same as in Example 1, except that trypsin (pH 7.0, 37℃ for 8h) is used, impurities are removed only by centrifugation, and QCS-MSN-STI-RGD modified material is not used.

[0066] Comparative Example 2

[0067] The specific implementation method is the same as in Example 1, except that only a 10kDa ultrafiltration membrane is used for filtration, without concentration, and the amino peptide concentration is low (<5mg / mL).

[0068] Comparative Example 3

[0069] The specific implementation method is the same as in Example 1, except that only chitosan or sodium alginate is used to replace the modified material.

[0070] II. Performance Testing

[0071] Freshly prepared deer spleen amino peptide powder samples (Examples 1-3, Comparative Examples 1-3) were selected and subjected to the following performance tests:

[0072] 1. Stability test: The samples were placed under accelerated storage conditions (37℃, relative humidity 75%) and samples were taken at 0, 3, 6, 9, 12 and 18 months. The characteristic peak area of ​​deer spleen amino peptide was detected by high performance liquid chromatography (HPLC) (retention time 15-20 min), and the degradation rate was calculated (degradation rate = (initial peak area - remaining peak area) / initial peak area × 100%).

[0073] 2. Bioavailability test: SPF-grade SD rats (weight 200±20g) were randomly divided into 6 groups (6 rats per group) and administered deer spleen amino peptide powder (dose 50mg / kg) of Examples 1-3 and Comparative Examples 1-3 by gavage. Blood was collected through the orbital sinus at 0, 1, 2, 4, 8 and 12 h after administration. Serum was separated by centrifugation, and the concentration of the characteristic peptide fragment of deer spleen amino peptide (molecular weight 3kDa) in serum was detected by ELISA. The intestinal absorption efficiency was calculated by the area under the curve (AUC) during administration.

[0074] 3. Target recognition rate test: M cells of mouse intestinal Pear aggregate lymph nodes cultured in vitro (concentration 5×10⁻⁶). 5 M cells / mL were co-incubated with a FITC-labeled sample (concentration 1 mg / mL) for 2 h (37℃, 5% CO2). The fluorescence intensity in M ​​cells was detected by flow cytometry, and the uptake rate was calculated (uptake rate = (average fluorescence intensity of M cells / total fluorescence intensity) × 100%).

[0075] 4. Molecular weight distribution test: After the sample was filtered through a 0.22 μm filter membrane, it was detected by HPLC (C18 column, mobile phase 0.1% formic acid-acetonitrile gradient elution). Bovine serum albumin (66 kDa), lysozyme (14 kDa), and tyrosine (164 Da) were used as standards. The proportion of small molecule peptides with a molecular weight <10 kDa was calculated (proportion = (sum of peak areas <10 kDa / total peak area) × 100%).

[0076] 5. Test Results:

[0077] Table 1: Test results of each embodiment and comparative example

[0078]

[0079] As shown in Table 1, the performance test results of Examples 1-3 and Comparative Examples 1-3 demonstrate that the present invention effectively solves the core problems of poor stability, low bioavailability, and single function of traditional deer spleen amino peptide powder through the multi-functional synergy of four-level gradient modified materials and process optimization. Specific analysis is as follows:

[0080] In terms of stability, the degradation rate of Examples 1-3 after 18 months was only 3.8%-4.5%, far lower than that of Comparative Example 1 (22.1%, traditional enzymatic hydrolysis, without QCS-MSN-STI-RGD modified material) and Comparative Examples 2 and 3 (18.3%-19.7%, single material substitution). This is because the quaternized chitosan in the modified material repels negatively charged proteases through positive charge, the mesoporous silica nanoparticles form a physical barrier to prevent the approach of macromolecular enzymes, and the bovine trypsin inhibitor directly neutralizes residual enzyme activity. The three synergistically inhibit the enzymatic hydrolysis and oxidative degradation of amino peptides, significantly improving long-term storage stability.

[0081] Regarding bioavailability, the intestinal absorption efficiency (AUC value 1220-1350) of Examples 1-3 was 2.7-3 times that of Comparative Example 1 (450) and Comparative Example 2 (510). This is due to the fact that the RGD peptide on the surface of the modified material can specifically recognize the integrin receptor on the surface of M cells of the Pell's aggregate lymph nodes in the intestine, mediating the active uptake of the material into the lymphatic system, thus avoiding the problem of low absorption efficiency caused by the small molecular weight of traditional amino peptides, which are easily hydrolyzed by intestinal enzymes or excreted in feces.

[0082] In terms of functional singularity, traditional deer spleen amino peptide powder (Comparative Examples 1-3) relies solely on enzymatic hydrolysis and simple membrane separation, lacking active targeting or protective functions. In contrast, the modified materials in Examples 1-3 integrate four levels of functions: charge repulsion, steric hindrance, enzyme inhibition, and targeted delivery. This not only protects the amino peptides from enzymatic hydrolysis (stability) but also improves absorption efficiency (bioavailability) through targeted delivery. Furthermore, the pore structure of mesoporous silica enables the enrichment of small molecule peptides (76.4%-81.2% of which have a molecular weight <10kDa). The functional integration is significantly superior to that of traditional products.

[0083] Comparative Example 1 (traditional enzymatic hydrolysis, without QCS-MSN-STI-RGD modified material) lacks a protective mechanism, making the amino peptides easily degraded by environmental proteases (degradation rate 22.1%), and the lack of targeting function leads to low absorption efficiency (AUC450); Comparative Example 2 (ultrafiltration membrane filtration only) increased the amino peptide concentration (9-11 mg / mL), but without the use of functional materials, the problems of enzymatic hydrolysis and low absorption efficiency still exist; Comparative Example 3 (single chitosan / sodium alginate) has a single material function (only charge repulsion), which cannot effectively inhibit enzymatic hydrolysis or promote absorption, and its performance is similar to that of Comparative Example 1.

[0084] In summary, Examples 1-3, through the synergistic effects of multiple functions of the four-level gradient modified materials and process optimization, comprehensively solved the technical bottlenecks of poor stability, low bioavailability, and single function of traditional deer spleen amino peptide powder. The resulting products have high stability, high bioavailability, and multifunctionality, significantly improving the application value of deer spleen amino peptide powder.

[0085] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing deer spleen amino peptide powder, characterized in that, The steps include: S1. Take fresh deer spleen, wash it with physiological saline to remove surface blood, remove fat and connective tissue, cut it into a homogenate, add pre-cooled phosphate buffer solution with pH=6.9-7.1, and break the cells with a high-speed homogenizer to obtain a homogenate. S2, centrifuge the homogenate at 3.8-4.2℃ and collect the supernatant; adjust the pH to 7.8-8.2, add alkaline protease, and enzymatically hydrolyze at 50-52℃; after enzymatic hydrolysis, inactivate the enzyme in a water bath at 95-97℃ and cool to room temperature; S3, the enzymatic hydrolysate is filtered through a 9-11 kDa ultrafiltration membrane, and the permeate is collected; then it is concentrated through a 2-4 kDa nanofiltration membrane to obtain deer spleen amino peptide concentrate. S4. The deer spleen amino peptide concentrate is mixed and shaken with QCS-MSN-STI-RGD modified material at 3-5℃; after sterilization by microporous filter membrane, the composite solution is pre-frozen at -50~-52℃ and then freeze-dried at -50℃.

2. The method for preparing deer spleen amino peptide powder according to claim 1, characterized in that, In step S1, the volume of phosphate buffer is 3-5 times the volume of homogenate; the speed of the high-speed homogenizer is 10,000-12,000 rpm.

3. The method for preparing deer spleen amino peptide powder according to claim 1, characterized in that, In step S2, the centrifugation time is 15-20 min; the enzymatic hydrolysis time is 4-6 h; and the enzyme inactivation time is 15-20 min.

4. The method for preparing deer spleen amino peptide powder according to claim 1, characterized in that, In step S3, the permeate contains small molecule peptides with a molecular weight <10kDa; the nanofiltration membrane concentration pressure is 1.4-1.6MPa, and the nanofiltration membrane concentration temperature is 24-26℃.

5. The method for preparing deer spleen amino peptide powder according to claim 1, characterized in that, In step S4, the concentration of the deer spleen amino peptide concentrate is 9-11 mg / mL; the mass ratio of the deer spleen amino peptide concentrate to the QCS-MSN-STI-RGD modified material is (9-11):1; the mixing and oscillation speed is 150-200 rpm, and the oscillation time is 2-4 h; the pore size of the microporous filter membrane is 0.2-0.24 μm; the pre-freezing time is 4-6 h; the vacuum degree of freeze drying is 10-12 Pa, and the freeze drying time is 24-30 h.

6. The method for preparing deer spleen amino peptide powder according to any one of claims 1-5, characterized in that, The preparation steps of the QCS-MSN-STI-RGD modified material include: A1. Chitosan was dissolved in acetic acid solution to prepare a chitosan solution; 3-chloro-2-hydroxypropyltrimethylammonium chloride was added and the mixture was magnetically stirred at 40-42℃; after the reaction was completed, the mixture was dialyzed with deionized water until neutral and then freeze-dried to obtain quaternized ammonium salt chitosan. A2, a quaternized chitosan solution was mixed with a hexadecyltrimethylammonium bromide aqueous solution, tetraethyl orthosilicate was added, the pH was adjusted to 8.9-9.1, and the reaction was carried out at 58-62℃; after centrifugation and washing, the hexadecyltrimethylammonium bromide template was etched with NaOH solution, and then dialyzed and dried again to obtain a quaternized chitosan-mesoporous silica nanoparticle composite. A3. Quaternized chitosan-mesoporous silica nanoparticle complex was dispersed in phosphate buffer at pH 7.2-7.6, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was carried out at room temperature; then bovine trypsin inhibitor was added, and the reaction was carried out at 3.8-4.2℃; unreacted bovine trypsin inhibitor was removed by dialysis to obtain the QCS-MSN-STI complex. A4. Dissolve the QCS-MSN-STI complex in 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.8-6.2, add RGD peptide, carbodiimide, and N-hydroxysuccinimide, and react at room temperature; centrifuge, wash, and lyophilize.

7. The method for preparing deer spleen amino peptide powder according to claim 6, characterized in that, In step A1, the mass ratio of chitosan to 3-chloro-2-hydroxypropyltrimethylammonium chloride is 1:(2-4); the reaction time with magnetic stirring is 24-30 h.

8. The method for preparing deer spleen amino peptide powder according to claim 6, characterized in that, In step A2, the volume ratio of the quaternized chitosan solution to the hexadecyltrimethylammonium bromide aqueous solution is 1:5; the reaction time is 2-4 h; after centrifugation and washing, the NaOH solution is used to etch the CTAB template at a temperature of 60-62℃ for 3-6 h.

9. The method for preparing deer spleen amino peptide powder according to claim 6, characterized in that, In step A3, the reaction time at room temperature is 30-60 min; the reaction time at 3.8-4.2℃ is 12-14 h.

10. The method for preparing deer spleen amino peptide powder according to claim 6, characterized in that, In step A4, the molar ratio of the QCS-MSN-STI complex to the RGD peptide is 1:5; the reaction time is 2-4 h at room temperature.

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