Process for the preparation of high activity elastase inhibiting peptides

Abstract

The application belongs to the technical field of bioactive peptide preparation, and particularly relates to a preparation method of high-activity elastase inhibitory peptide. The preparation method of the high-activity elastase inhibitory peptide comprises the following steps: obtaining remodeled protein suspension by physically pretreating prepared protein suspension, then performing enzymolysis, separation and purification to obtain primary peptide, and finally performing affinity adsorption and desorption of the primary peptide by using a fixed enzyme functional membrane, so that the high-activity elastase inhibitory peptide is finally obtained. The coupling process of the physical remodeling, specific enzyme degradation and affinity purification is synergistic, and forms an efficient path of "structure opening-targeted cutting-precise capture", so that the inhibitory peptide obtained finally can achieve an inhibition rate of 77.35% on elastase. The antioxidant activity detection result shows that the clearance rates of DPPH, ABTS and hydroxyl radicals are 82.31%, 88.26% and 80.99%, respectively.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically relating to a method for preparing a highly active elastase inhibitory peptide. Background Technology

[0002] Existing elastase inhibitors are mainly derived from natural proteins and peptides. Although these naturally derived bioactive peptides have certain antioxidant and enzyme-inhibiting effects, their extraction rates and elastin inhibition rates are still limited due to the limitations of extraction methods. For example, CN118932002A uses ball milling and multi-step enzymatic hydrolysis, but its extraction rate depends on the pretreatment of raw materials and the preparation of intermediate products, making the overall process complex. At the same time, the ball milling process may degrade some active peptides and reduce the elastase inhibitory activity. CN120248035A uses a single alkaline protease to hydrolyze hawthorn fish glue. Due to the limited number of enzymatic hydrolysis sites, the highest inhibition rate of the finally screened active peptide IDGF is only 66.47% (at a concentration of 20 mM), which is difficult to achieve the expected effect. CN120138097A uses ionic liquid treatment, which is beneficial for removing impurities, but it is costly, difficult to recover, and may have an environmental impact, which is not conducive to large-scale production. The enzymatic hydrolysate obtained by CN120574920A still needs to be subjected to cation exchange resin and gel chromatography to improve purity, but the process is complex, the yield is low, and the enrichment effect on small molecule peptides is limited.

[0003] In summary, the aforementioned methods currently suffer from several technical shortcomings, including low substrate utilization, insufficient exposure of the active sites of enzymatically digested fragments, lack of specificity in purification methods, and difficulties in the precise enrichment and targeted extraction of highly active inhibitory peptides. The main reasons for these problems are the complex composition and similar molecular weight and properties of the enzymatically digested products, making it difficult to achieve effective fractionation using conventional ultrafiltration, chromatography, and other separation methods; the internal masking phenomenon of the active sites of peptides leads to low adsorption and extraction efficiency; and the existing operating conditions exhibit uncontrollable pH, ionic strength, and temperature, making targeted extraction difficult. These reasons ultimately result in low purity and unstable activity of the final products, failing to meet the demands of high-end biopharmaceuticals and skincare products. Therefore, precise enrichment and targeted extraction remain significant bottlenecks in current technologies.

[0004] To address the aforementioned technical deficiencies, this invention provides a method for preparing highly active elastase inhibitory peptides. The method is characterized by a series of steps including synergistic pretreatment, enzymatic hydrolysis, precise enrichment and targeted extraction, and freeze-drying, which efficiently and controllably yields high-purity, highly active peptide powder. This significantly improves upon the problems of low enzymatic hydrolysis efficiency, insufficient exposure of active sites, and lack of specificity in purification found in existing technologies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing highly active elastase inhibitory peptides. This method induces protein conformational changes through multiple physical field pretreatments, combined with specific enzyme synergistic hydrolysis and functional membrane affinity capture of specific immobilized enzymes, thereby achieving efficient enrichment of elastase inhibitory peptides. The resulting inhibitory peptides exhibit an inhibitory activity of up to approximately 78%, and antioxidant tests show that the inhibitory peptides possess extremely strong free radical scavenging capabilities (ABTS scavenging rate reaches 88.26%).

[0006] The method for preparing highly active elastase inhibitory peptides provided by this invention comprises the following steps:

[0007] S1: Take animal tissue, crush, defatted, and filtered. Add alkaline solution to the obtained precipitate I, react, cool, wash until neutral, centrifuge to obtain precipitate II, dry, and obtain protein powder.

[0008] S2: The protein powder obtained in S1 is formulated into a protein suspension, and then pretreated to obtain a remodeled protein suspension; the pretreatment includes at least one of ultrasound, microwave, ultra-high pressure, pulsed electric field, and irradiation.

[0009] S3: Add enzyme preparation to the reconstituted protein suspension in S2, perform enzymatic hydrolysis, inactivate the enzyme, and obtain the enzymatic hydrolysate;

[0010] S4: Separate and purify the enzymatic hydrolysate from S3, dry it, and obtain the primary purified peptide powder;

[0011] S5: The primary purified peptide powder from S4 is prepared into a peptide solution. An immobilized enzyme functional membrane is used to affinity adsorb and desorb the peptide solution to obtain a highly active elastase inhibitory peptide.

[0012] Preferably, in S2, the preprocessing conditions are as follows:

[0013] Ultrasound treatment is performed at 200-600W for 10-40 minutes.

[0014] Microwaves are used at a power of 300-800W for a duration of 30-180 seconds.

[0015] The ultra-high pressure pretreatment pressure is 100 ~ 600 MPa, and the time is 5 ~ 30 min;

[0016] The field strength of the pulsed electric field is 10 ~ 40 kV / cm, and the total time is 1 ~ 20 ms;

[0017] The irradiation dose is 2 kGy ~ 10 kGy, and the time is 10 ~ 60 s;

[0018] Microwave-ultrasound synergistic processing: microwave 400-600W, 120-180s; ultrasound 300-500W, 10-30min, ultrasound 5-10s, interval 5-10sn;

[0019] Ultra-high voltage-microwave co-processing: Set ultra-high voltage to 200-500MPa for 10-25min; microwave to 400-600W for 120-180s.

[0020] Ultra-high voltage-pulse coordinated processing: Set ultra-high voltage to 200-500MPa, 10-25min; pulse to 15-30kV / cm, 5-15ms.

[0021] Ultrasound-irradiation synergistic treatment: ultrasound 300-500W, 10-30min, ultrasound 5-10s, interval 5-10sn; irradiation dose 3-8kGy, 20-50s.

[0022] Preferably, in S3, the enzyme preparation is selected from at least one of pancreatic enzyme, trypsin, chymotrypsin, elastase, and alkaline protease. The amount of enzyme preparation is 1.0 to 3.0 wt% of the reconstituted protein suspension. The enzymatic hydrolysis conditions are: pH 6 to 10, 35 to 60°C, 12 to 22 h; the enzyme inactivation conditions are: 95 to 100°C, 5 to 10 min.

[0023] Preferably, in step S4, the separation and purification steps are as follows:

[0024] The enzymatic hydrolysate in S3 is subjected to a combined treatment, wherein the combined treatment is selected from any of the following methods: centrifugation and microfiltration, centrifugation and ultrafiltration, centrifugation and nanofiltration, centrifugation and chromatography, microfiltration and ultrafiltration, microfiltration and nanofiltration, microfiltration and chromatography, ultrafiltration and nanofiltration, ultrafiltration and chromatography, or nanofiltration and chromatography; then, the active peptide components with a molecular weight less than 3000 Da are collected and dried to obtain primary purified peptide powder.

[0025] Preferably, in S5, the method for preparing the functional membrane for immobilizing the enzyme is as follows:

[0026] (1) Preparation of nanofiber membrane: Take a nanofiber membrane, wherein the nanofiber membrane is prepared by co-spinning the backbone material polycaprolactone and an amino-containing natural material, m 聚己内酯 :m 氨基材料 =1:0.2-0.8; the amino-containing natural material is selected from at least one of chitosan, gelatin, zein, silk fibroin, and aminoated lignin;

[0027] (2) Modification: The nanofiber membrane is immersed in a crosslinking agent accounting for 5-20% of its mass for 1-4 hours to carry out surface functionalization modification, thereby obtaining a modified nanofiber membrane;

[0028] The crosslinking agent is selected from at least one of glutaraldehyde, epichlorohydrin, polyethylene glycol diacrylate, polydopamine, and genipin;

[0029] (3) Immobilization: The modified nanofiber membrane is immersed in an immobilized enzyme solution with a concentration of 1~20 mg / mL and a volume of 5~30 mL per square centimeter, and immobilized at 2~8°C for 1~3 h; the immobilized enzyme solution is selected from at least one of trypsin, elastase, alkaline protease, complex protease, and bromelain.

[0030] After immobilization, the modified nanofiber membrane was removed and its surface was repeatedly rinsed with PBS buffer (pH 6.8–7.2) to completely remove physically adsorbed free enzyme, resulting in an immobilized enzyme loading of 5–40 mg / cm³. 2 Immobilized enzyme functional membranes.

[0031] Preferably, in step S5, the affinity adsorption step is as follows:

[0032] Take the primary purified peptide powder from S4 and add it to PBS buffer with a pH of 6.8 to 7.2 to prepare a peptide solution with a concentration of 5 to 20 mg / mL.

[0033] Cutting size is 1~9 cm 2 The immobilized enzyme functional membrane was completely immersed in 5-25 mL of the above peptide solution and placed in a constant temperature environment of 4-25°C for static adsorption for 1-4 hours.

[0034] In S5, the desorption steps are as follows:

[0035] After adsorption is complete, remove the adsorbed nanofiber membrane, wash it repeatedly with deionized water, and then perform any of the following steps:

[0036] (1) Treat with ultrasonic power of 50-200W for 20-60 minutes.

[0037] Or: (2) Immerse the washed nanofiber membrane in acidic buffer solution of pH 3 ~ 6 and alkaline buffer solution of pH 8 ~ 10 for 20 ~ 60 min, collect the adsorption solution, dry it, and obtain high-purity elastase inhibitory peptide powder.

[0038] Or: (3) Immerse the washed nanofiber membrane in ethanol, acetonitrile, isopropanol or acetone solution for 20 to 60 minutes, collect the adsorption solution, dry it, and obtain high-purity elastase inhibitory peptide powder.

[0039] Or: (4) Temperature gradient method: The washed nanofiber membrane is immersed in phosphate buffer and treated with different temperature gradients. The temperature gradient is slowly increased from 4°C to 50°C, the gradient change rate is 1~10°C per hour, the total treatment time is 20~60 min, the adsorbent is collected, dried, and high-purity elastase inhibitory peptide powder is obtained.

[0040] Or (5) Ionic strength variation method: Immerse the washed nanofiber membrane in brine eluent and gradually adjust the ionic strength of the eluent to desorb the peptide from the nanofiber membrane; the ionic strength is slowly increased from 0.01~0.1M to 0.5~1.0M, the total treatment time is 20~60min, the adsorbent is collected, dried, and high-purity elastase inhibitory peptide powder is obtained.

[0041] Preferably, in S1, the animal tissue is selected from at least one of bovine heart tube, carotid artery, pig heart, horse ligament, and skipjack tuna heart bulb;

[0042] The degreasing steps are as follows: Mix the raw material and the organic solvent at a mass-volume ratio of 1g:(5~20)mL and soak for 12~36h. The organic solvent is selected from at least one of acetone, anhydrous ethanol, petroleum ether, and xylene.

[0043] The reaction steps are as follows: The mixture is then heated and stirred at 70-100℃ for 0.5-3 hours at a mass-to-volume ratio of precipitate I to alkaline solution of 1 g:(5-20) mL. The alkaline solution is selected from at least one of the following: potassium hydroxide solution, sodium hydroxide solution, sodium carbonate solution, and ammonia water with a concentration of 0.05-2 M. The pH during the reaction is 9-13. After the reaction, the mixture is cooled to room temperature.

[0044] Wash with deionized water until neutral, then centrifuge at 5000-12000 rpm for 5-15 min to obtain precipitate II;

[0045] The drying process is selected from any one of the following: oven drying, freeze drying, spray drying, vacuum drying, and supercritical fluid drying.

[0046] Preferably, in S2, the protein powder from S1 is taken and added to deionized water at a solid-liquid ratio of 1 g: (8~15) mL to prepare a protein suspension.

[0047] In S2, the preprocessing is as follows: microwave-ultrasound co-processing: microwave is set to 400-600W, with a total duration of 120-180s; ultrasound is set to 300-500W, for 10-30min, with a 5-10s interval after each 5-10s ultrasound session.

[0048] The beneficial effects of this invention are as follows:

[0049] (1) Conformation-inducing effect significantly improves enzymatic hydrolysis efficiency and active site exposure.

[0050] Before enzymatic hydrolysis, physical pretreatment methods such as ultrasound and microwave are used to effectively break down the tight cross-linking network and highly entangled spatial conformation between protein molecules. Compared with direct enzymatic hydrolysis, physical pretreatment induces the effective exposure of hydrophobic groups and restriction enzyme sites inside the protein, significantly increasing the contact probability between the substrate and the protease. This results in a significant increase in the proportion of small molecule active peptides (<3000 Da) in the hydrolysis product, thereby enhancing the initiation activity of the repressor peptide from the source.

[0051] (2) Targeted enzymatic hydrolysis enables precise release and miniaturization of the active sequence.

[0052] This invention, based on conformational remodeling, utilizes the high specificity of protease catalysis to directionally cleave exposed restriction sites. Compared to chemical hydrolysis, this process, under mild conditions, maximizes the preservation of specific amino acid sequences with inhibitory functions, preventing the inactivation of active sites. Simultaneously, by precisely controlling enzymatic hydrolysis parameters, large protein molecules are efficiently converted into low-molecular-weight active peptides with high solubility and strong diffusivity. This not only improves the bioavailability of the peptides, but also ensures that their molecular weight distribution characteristics closely match the pore size and affinity sites of subsequent nanofiber membranes, laying the foundation for high-throughput purification.

[0053] (3) Affinity adsorption technology enables precise enrichment and high-purity acquisition of target peptides.

[0054] Unlike traditional ultrafiltration or chromatography techniques that rely solely on molecular weight, this invention utilizes the bioaffinity properties of enzymes immobilized on the surface of nanofiber membranes to "precisely capture" elastase inhibitory peptides with specific binding capabilities from complex enzymatic hydrolysates. This purification method based on the principle of bioaffinity effectively removes inactive impurities and large protein molecules, significantly improving the purity and bioactivity of the product.

[0055] (4) The synergistic effect of process coupling enables high-efficiency cascade preparation

[0056] The "physical remodeling-specific enzyme degradation-affinity purification" coupled process of this invention constructs an interlocking cascade system. Physical remodeling acts as a "key" to unlock the protein's spatial conformation, significantly improving enzymatic accessibility; specific enzyme degradation acts as "precision scissors" to achieve targeted release of active sequences and precise molecular weight control; and affinity purification acts as a "smart magnet" to specifically enrich highly active peptides. These three processes synergistically form a highly efficient pathway of "structure opening-targeted cleavage-precision capture," collectively solving a series of bottlenecks in traditional technologies, such as low enzymatic hydrolysis efficiency, insufficient yield of active peptides, and low product activity. This achieves efficient conversion and performance enhancement from raw materials to highly active end products. Attached Figure Description

[0057] Figure 1 This is a flowchart of the method of the present invention;

[0058] Figure 2 This is a comparison chart of solubility, elastase inhibitory activity, specific activity, DPPH radical scavenging rate, ABTS radical scavenging rate, and hydroxyl radical scavenging rate in Examples 1-8 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0059] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0060] Example 1

[0061] The specific steps for preparing a highly active elastase inhibitory peptide are as follows:

[0062] (1) Protein preparation

[0063] In this Example 1, bovine heart tube is used as an example to illustrate the preparation process of animal protein powder:

[0064] Fresh bovine heart tube tissue was collected, fat and fascia were removed, and the tissue was pulverized. Petroleum ether solution was added at a weight-to-volume ratio of 1 g:10 mL, and the mixture was soaked at room temperature for 24 hours to defatted. After defatting, the precipitate was obtained by filtration. 0.5 M ammonia solution was added at a weight-to-volume ratio of precipitate to alkali solution of 1 g:15 mL, and the mixture was heated at 90°C with continuous stirring for 1.5 hours. After the solution cooled naturally to room temperature, it was repeatedly washed with deionized water until neutral. The precipitate was collected by centrifugation at 8000 rpm for 10 minutes, pre-frozen at -80°C, transferred to a vacuum, and dried at -0.08 MPa for 8 hours. The freeze-dried product yielded high-purity insoluble protein powder.

[0065] (2) Physical pretreatment

[0066] Take the protein powder from (1) and add it to deionized water at a solid-liquid ratio of 1g:12mL to prepare a protein suspension. Microwave treatment was performed with a power of 500W and a total treatment time of 180s. After microwave treatment, the protein suspension was further treated with a probe-type ultrasonic cell disruptor with a power of 400W and a total treatment time of 20min. To prevent excessive denaturation of the protein due to temperature rise during treatment, the container was placed in an ice-water bath to maintain a low temperature, and the operation was performed in a pulse mode (e.g., 5s of ultrasound followed by 5s of intermittent pulses). After treatment, the conformationally remodeled protein suspension was collected and stored at 4°C for later use.

[0067] (3) Enzymatic hydrolysis and purification

[0068] In the conformationally remodeled protein suspension from step (1), trypsin and elastase (mass ratio 1:1) were added at 2.0% of the substrate mass. The pH of the system was adjusted to 8.5 using 1.0 mol / L NaOH solution. Subsequently, the reaction system was placed in a 45°C constant temperature water bath for 16 h for enzymatic hydrolysis. After the reaction was completed, the mixture was heated in a 95°C water bath for 10 min to inactivate the enzyme protein.

[0069] The enzyme-inactivated hydrolysate was centrifuged at 10,000 rpm for 15 min in a high-speed refrigerated centrifuge to remove unhydrolyzed large protein molecules and insoluble impurities. The supernatant was collected and pre-filtered using a 0.45 μm microporous membrane to remove small suspended solids. The filtrate was then transferred to an ultrafiltration system and subjected to pressure-driven fractional filtration using an ultrafiltration membrane with a molecular weight cutoff (MWCO) of 3000 Da. The fraction permeated through the membrane (molecular weight <3000 Da) was collected; this fraction is the active component rich in oligopeptides. The collected filtrate was dried to obtain a loose, dry, primary purified peptide powder.

[0070] (4) Affinity adsorption and desorption of nanofiber membranes

[0071] Polycaprolactone (PCL) nanofiber membranes were selected as the framework material and co-dissolved with chitosan in a mixed solvent of N,N-dimethylformamide (DMF) and acetic acid (mPCL:mchitosan = 1:0.3). The resulting PCL-CS nanofiber membranes were prepared by co-spinning. The PCL-CS nanofiber membranes were then immersed in a 5% glutaraldehyde solution for 3 hours for surface functionalization modification. The modified PAN membranes were then immersed in 25 mL of a 5 mg / mL trypsin solution. Immobilization was carried out at 4°C for 2 hours, utilizing the covalent binding of the active groups on the membrane surface to the enzyme molecules. After immobilization, the functional membrane was removed and repeatedly rinsed with phosphate-buffered saline (PBS, pH 7.4) to thoroughly remove physically adsorbed free enzyme. The final immobilized trypsin functional membrane was then stored at 4°C under humid conditions, protected from light, for later use.

[0072] Accurately weigh the freeze-dried primary purified peptide powder from (3) and prepare a peptide solution with a concentration of 10 mg / mL using phosphate-buffered saline (PBS, pH 7.4). Cut to a size of 1 cm. 2An immobilized enzyme functional membrane was completely immersed in 10 mL of the above peptide solution. The system was placed in a constant temperature environment at 20°C for 2 h for adsorption. After adsorption, the functional membrane was removed and washed three times with a large amount of deionized water to thoroughly remove impurity peptides that were physically adsorbed in the membrane pores and non-specifically bound to the surface. Subsequently, the washed functional membrane was immersed in a glycine-HCl buffer solution at pH 2.5 and treated at room temperature for 45 min. The low pH environment disrupts the electrostatic attraction or hydrogen bonds between peptide molecules and ligands on the membrane surface, achieving efficient desorption of the target peptide. The eluent was collected and freeze-dried to obtain high-purity elastase inhibitory peptide powder.

[0073] Example 2

[0074] The difference between this embodiment and Example 1 is that, in step (1), the animal tissue material selected is skipjack tuna heart arterial bulb tissue; the selected alkali is sodium hydroxide solution with a concentration of 0.1M, added according to a solid-liquid ratio of 1g:10mL, heated at 95°C and continuously stirred for 45min; in step (2), the physical pretreatment method is microwave treatment, the microwave power is set to 500W, the total treatment time is 180s, and a protein suspension after conformational remodeling is obtained; in step (3), trypsin and alkaline protease are added to the protein suspension after conformational remodeling at a ratio of 1:1 at 2.0% of the substrate mass, and the remaining steps are the same as in Example 1.

[0075] Example 3

[0076] The only difference between this embodiment and Example 1 is that in step (1), the animal tissue material selected is horse ligament tissue, which is added to ethanol solution at a ratio of 1g:10mL and soaked at room temperature for 24h for defatting; in step (2), the physical pretreatment method adopts ultra-high pressure-microwave synergistic treatment (set ultra-high pressure 400MPa, 20min; microwave 500W, 180s), and after protein conformation remodeling is achieved under strict temperature control, it is stored at 4℃ for later use; in step (3), chymotrypsin and elastase are added to the protein suspension after conformation remodeling at 1.5% of the substrate mass, with a mass ratio of 2:1, and the reaction system is placed in a constant temperature water bath at 45°C for 15h for enzymatic hydrolysis. The remaining steps are exactly the same as in Example 1.

[0077] Example 4

[0078] The difference between this embodiment and Example 1 is that in step (2), the protein powder is prepared into a suspension at a ratio of 1g:12mL, placed in an ice-water bath, and subjected to a combined ultrasonic-irradiation treatment. The ultrasonic treatment is performed at 400W for 20min, followed by 5s of ultrasonic treatment and a 5s interval; the irradiation dose is 5kGy for 30ns. After achieving conformational remodeling of the protein under strict temperature control, it is stored at 4℃ for later use. In step (3), trypsin and chymotrypsin are added to the protein suspension after conformational remodeling at a ratio of 1:1 at 2.0% of the substrate mass, and the pH of the system is adjusted to 9 using 1.0mol / L NaOH solution. Subsequently, the reaction system was placed in a 45°C constant temperature water bath for 16 hours for enzymatic hydrolysis; in step (4), the modified nanofiber membrane was immersed in 10 mL of 20 mg / mL chymotrypsin solution and immobilized at 4°C for 1 hour. The enzyme was immobilized by covalent bonding between the active groups on the membrane surface and the enzyme molecules; after adsorption, the nanofiber membrane was removed and washed 1-3 times to remove unadsorbed impurities. Physical ultrasound was used with an ultrasound power of 400 W for 10 minutes to achieve desorption. The desorbed solution was collected to obtain high-purity elastase inhibitory peptide powder. The remaining steps were exactly the same as in Example 1.

[0079] Example 5

[0080] The difference between this embodiment and Example 1 is that in step (2), the protein powder is prepared into a suspension at a ratio of 1g:12mL, placed in an ice-water bath, and subjected to ultra-high pressure-pulse synergistic treatment (ultra-high pressure 400MPa, 20min; pulse 20kV / cm, 10ms). After the protein conformation is remodeled under strict temperature control, it is stored at 4℃ for later use. In step (3), elastase and chymotrypsin are added to the protein suspension after conformation remodeling at a ratio of 1.2:1 at 1.0% of the substrate mass. The pH of the system is adjusted to 9.5 using 1.0mol / L NaOH solution. Subsequently, the reaction system was placed in a 45°C constant temperature water bath for 14 hours for enzymatic hydrolysis; in step (4), the modified nanofiber membrane was immersed in 10 mL of a solution of 20 mg / mL elastase and chymotrypsin, and immobilized at 4°C for 2 hours. Enzyme immobilization was achieved by covalently binding the active groups on the membrane surface with the enzyme molecules; after adsorption, the nanofiber membrane was removed and washed 1-3 times to remove unadsorbed impurities. The pH gradient method was used, and the adsorbed nanofiber membrane was alternately treated with a pH 4.0 acetate-sodium acetate buffer and a pH 9.5 sodium bicarbonate-sodium carbonate buffer at room temperature for a total time of 20-60 minutes to achieve mild and efficient dissociation of the target peptide. Desorption was achieved, the desorbed solution was collected, and high-purity elastase inhibitory peptide powder was obtained. The remaining steps were exactly the same as in Example 1.

[0081] Example 6

[0082] The difference between this embodiment and Example 1 is that in step (3), trypsin and elastase are added to the protein suspension after conformational remodeling at 2.0% of the substrate mass, in a 1:1 ratio, and the pH of the system is adjusted to 10 using 1.0 mol / L NaOH solution. The reaction system is then placed in a 37°C constant temperature water bath for 12 h for enzymatic hydrolysis. After centrifuging to remove macromolecular impurities, the hydrolysate is pre-filtered using a 0.45 μm microporous membrane. Next, the filtrate is separated using a reversed-phase C18 column, and components of different molecular weights are obtained through gradient elution. The absorbance is monitored (214 nm or 280 nm), and the fraction containing low molecular weight elastase inhibitory peptides is collected. Finally, the enriched peptide components were pre-frozen at -80°C and purified elastase inhibitor peptide powder was obtained by vacuum freeze-drying. In step (4), the modified nanofiber membrane was immersed in 20 mL of 10 mg / mL elastase solution and immobilized at 4°C for 1 h. The enzyme was immobilized by covalent bonding between the active groups on the membrane surface and the enzyme molecules. After adsorption, the nanofiber membrane was removed and washed 1-3 times to remove unadsorbed impurities. Then, the washed functional membrane was immersed in 0.5 M NaCl and continuously treated at room temperature for 1 h to achieve desorption. The desorption solution was collected to obtain high-purity elastase inhibitor peptide powder. The remaining steps were exactly the same as in Example 1.

[0083] Example 7

[0084] The difference between this embodiment and Example 1 is that in step (4), polycaprolactone (PCL) nanofiber membrane is selected as the substrate, and zein is dissolved together in a mixed solvent of DMF and acetic acid (mPCL:mzein = 1:0.7). The PCL-Zein nanofiber membrane is prepared by co-spinning. The nanofiber membrane is soaked in 5% crosslinking agent genipin for 3 hours to perform surface functionalization modification. The modified nanofiber membrane is immersed in 10 mL of trypsin and elastase solution of 20 mg / mL and immobilized at 4°C for 1 hour. The enzyme is immobilized by covalently binding the active groups on the membrane surface with the enzyme molecules. After adsorption, the nanofiber membrane is taken out and washed 1-3 times to remove unadsorbed impurities. Physical ultrasound is used with an ultrasonic power of 200W for 20 minutes to achieve desorption. The desorbed liquid is collected to obtain high-purity elastase inhibitory peptide powder. The remaining steps are exactly the same as in Example 1.

[0085] Example 8

[0086] The difference between this embodiment and Example 1 is that in step (4), the modified nanofiber membrane is immersed in 10 mL of a 20 mg / mL trypsin solution. Immobilization is performed at 4°C for 3 h, utilizing the covalent bonding between the active groups on the membrane surface and the enzyme molecules to achieve enzyme immobilization. Accurately weigh the freeze-dried primary purified peptide powder and prepare a 20 mg / mL peptide solution using a pH 8.0 Tirs-HCl buffer. Cut an immobilized enzyme functional membrane to a size of 9 cm², completely immerse it in 20 mL of the above peptide solution, and place the system in a constant temperature environment at 20°C for 3 h for adsorption. After adsorption, remove the functional membrane and wash it repeatedly three times with a large amount of deionized water to thoroughly remove impurity peptides physically adsorbed in the membrane pores and non-specifically bound to the surface. Subsequently, immerse the washed functional membrane in a 2M NaCl solution and continue treatment at room temperature for 45 min to achieve efficient desorption of the target peptide. Collect the eluent, freeze-dry it, and obtain high-purity elastase inhibitor peptide powder.

[0087] Comparative Example 1

[0088] This comparative example illustrates the effect of physical pretreatment on protein conformational remodeling and enzymatic hydrolysis efficiency. The procedure differs from Example 1 in that the protein was added directly to deionized water at a solid-liquid ratio of 1g:12mL without physical pretreatment, stirred thoroughly, and then directly subjected to subsequent enzymatic hydrolysis. The remaining steps are the same as in Example 1.

[0089] Comparative Example 2

[0090] This comparative example illustrates the superiority of bioactive peptides obtained through enzymatic hydrolysis. The procedure differs from Example 1 in that the elastase inhibitor peptide is extracted using acid hydrolysis. The raw material is placed in a 6 mol / L HCl solution and reacted at 110°C for 12 hours, followed by neutralization with a strong alkali and desalting extraction. The remaining steps are the same as in Example 1.

[0091] Comparative Example 3

[0092] This comparative example illustrates the specific affinity of the immobilized enzyme membrane. The procedure differs from Example 1 in that the primary purified peptide powder was not purified using a nanofiber membrane; the remaining steps are the same as in Example 1.

[0093] Comparative Example 4

[0094] This comparative example illustrates the effect of simultaneously introducing a physical energy field during enzymatic hydrolysis on the preparation of elastase inhibitory peptides. The steps differ from Example 1 in that the protein pretreatment step (2) is omitted, and instead, the protein suspension is enzymatically hydrolyzed while simultaneously subjected to ultrasonic and irradiation treatments; the remaining steps are the same as in Example 1.

[0095] Experimental Example

[0096] (a) Performance Testing

[0097] To verify the dual advantages of the method of the present invention in producing highly active elastase inhibitory peptides and simultaneously enhancing elastase inhibitory activity and antioxidant activity, a systematic evaluation was conducted on the high-purity elastase inhibitory peptide powders prepared in Examples 1-8 and Comparative Examples 1-3, including remodeled protein solubility, elastase inhibitory activity, specific activity, and antioxidant indicators. The specific detection methods are as follows:

[0098] (1) Solubility

[0099] The remodeled protein samples obtained through different physical treatments were prepared into initial suspensions of 10 mg / mL. After thorough equilibration by stirring at 25°C for 30 min, they were centrifuged at 10000 r / min for 15 min. Subsequently, the supernatant of each group was accurately aspirated and diluted to the linear range. 1 mL of the diluted solution (with deionized water as a blank) was vortexed with 5.0 mL of Coomassie Brilliant Blue G-250 reagent, and after standing in the dark for 15 min, the absorbance was measured at a wavelength of 595 nm. The protein content in the supernatant was calculated according to the bovine serum albumin (BSA) standard curve, and the solubility was calculated using the following formula:

[0100] Solubility (%) = (Supernatant protein concentration / Initial protein concentration) × 100%

[0101] (2) Determination of elastase inhibitory activity

[0102] The lyophilized hydrolysate was redissolved in Tris-HCl buffer (0.1 M, pH 8.0) to achieve a final peptide concentration of 2.0 mg / mL. 200 μL of sample and 50 μL of elastase were added sequentially to a 96-well plate, mixed thoroughly, and incubated at 25 °C for 10 min. Then, 50 μL of N-succinyl-Ala-Ala-Ala-pNA (1 mmol / L) was added, and the absorbance was measured at 405 nm, denoted as A. c0 A s0 The plate was placed in a full-wavelength scanner and incubated at 25°C for 10 min. Immediately afterwards, the absorbance after the reaction was measured at 405 nm and recorded as A. c A s Tris-HCl buffer was used as a control. Elastase inhibitory activity was calculated using the following formula:

[0103] elastase inhibitory activity (%) = 1 - (A S -A S0 ) / (A C -A C0 )×100%

[0104] In the formula: A s0A represents the absorbance of the sample group before the reaction. s A represents the absorbance of the sample group after the reaction. c0 A represents the absorbance of the control group before the reaction. c The absorbance is the value of the control group after the reaction.

[0105] (3) Total peptide content and specific activity

[0106] Accurately weigh bovine serum albumin (BSA) standard and prepare a series of standard solutions (0-200 μL), with gradually increasing concentrations, to construct a standard curve. Place 1 mL of the peptide solution (or standard solution) in a test tube. Add 5.0 mL of Lowry's reagent A (prepared from 4% Na₂CO₃, 0.2 mol / L NaOH, 1% CuSO₄, and 2% potassium sodium tartrate in a specific ratio) to the test tube, mix thoroughly, and let stand at room temperature for 10 minutes to complete the biuret reaction. Then, quickly add 0.5 mL of Lowry's reagent B (Folin-phenol working solution) to each test tube, vortex immediately, and let stand in the dark for 30 minutes. After the reaction, measure the absorbance of each tube at 750 nm using a UV spectrophotometer. Calculate the peptide concentration in the peptide solution based on the bovine serum albumin (BSA) standard curve.

[0107] Specific activity = Measured elastase inhibitory activity (%) / Total peptide mass concentration (mg / mL)

[0108] (4) Antioxidant activity

[0109] Antioxidant activity was determined using DPPH (2,2-diphenyl-1-propyl-4-sulfonylhydrazine), ABTS (2,2-azido-3-ethyl-6-benzothiazoline-1-diammonium salt), and hydroxyl radical and reducing power assays.

[0110] 2 mL of the emulsion sample to be tested was thoroughly mixed with 2 mL of 0.1 mM DPPH ethanol solution and reacted for 30 min under light-protected conditions. After the reaction, the absorbance of the sample was measured at 517 nm using a UV-Vis spectrophotometer (labeled A1). Simultaneously, an equal volume of ethanol was used as a control group (absorbance A2) to replace the sample, and an equal volume of ethanol was used as a blank group (absorbance A3). The DPPH free radical scavenging rate was calculated using the following formula:

[0111] DPPH free radical scavenging activity (%) = 1 - (A1 - A2) / A3 × 100%

[0112] An ABTS solution was prepared by mixing 7 mM ABTS and 4.9 mM K₂S₂O₈ at a 1:1 volume ratio and reacting in the dark for 12–16 hours. This solution was then diluted with ethanol to achieve an absorbance of 0.7 ± 0.02 at 734 nm. 20 μL of the sample was mixed with 2 mL of the diluted ABTS solution. The mixture was incubated in the dark at room temperature for 30 min, and the absorbance was measured at 734 nm (A1). The sample was replaced with distilled water as a control (A2), and zero-point calibration was performed using 1 mL of anhydrous ethanol and 1 mL of distilled water. The ABTS radical scavenging activity was calculated using the following formula:

[0113] ABTS free radical scavenging activity (%) = 1 - A1 / A2 × 100%

[0114] Take 1 mL of sample and mix it with 5 mL of FeSO4 (9 mM) and 10 μL of salicylic acid (9 mM, dissolved in ethanol), then add 0.5 mL of H2O2 (8.8 mM) to initiate the reaction. Incubate the mixture at 37 °C for 30 min. Measure the absorbance at 510 nm and record it as A1. Replace the sample with distilled water as the control group (A2). Replace H2O2 with distilled water as the blank group (A3). Calculate the scavenging activity of hydroxyl radicals using the following formula:

[0115] Free radical scavenging activity (%) = 1 - (A1 - A2) / A3 × 100%.

[0116] (II) Test Results

[0117] Table 1 Results of protein solubility test project Solubility (%) Examples 1, 6, 7, 8; Comparative Examples 2, 3, 4 50.26±1.04 Example 2 34.54±0.68 Example 3 46.51±1.68 Example 4 35.65±1.07 Example 5 42.85±0.86 Comparative Example 1 20.90±0.69

[0118] Experimental data show that physical pretreatment has a significant effect on inducing and improving the solubility of proteins. The untreated raw material (Comparative Example 1) has the lowest solubility due to its compact structure. However, physical pretreatment such as ultrasound, microwave, ultra-high pressure, and pulse can effectively break the intermolecular forces and extend the structure. The alternating internal and external energy fields can thoroughly dismantle the stubborn protein crystal structure, resulting in a significant increase in solubility compared to the comparative example, up to 50.26%.

[0119] Table 2 Results of elastase inhibitory activity test project Elastase inhibitory activity (%) at a concentration of 2.0 mg / mL Example 1 77.35 ± 0.82 Example 2 64.96 ± 0.58 Example 3 74.43 ± 0.93 Example 4 68.88 ± 0.52 Example 5 62.59 ± 0.90 Example 6 76.37 ± 0.79 Example 7 61.68 ± 1.07 Example 8 57.85 ± 0.68 Comparative Example 1 46.31 ± 1.10 Comparative Example 2 22.90 ± 2.18 Comparative Example 3 41.76 ± 0.57 Comparative Example 4 31.18 ± 1.12

[0120] Analysis of the experimental data revealed a significant gradient in the elastase inhibitory activity of each group of samples. The results indicated that Examples 1-8 exhibited high elastase inhibitory activity, demonstrating that the synergistic treatment of physical pretreatment, protease hydrolysis, and nanofiber membrane affinity purification can efficiently identify and enrich elastase inhibitory peptides. In contrast, Comparative Examples 1 and 4 (without physical treatment) and Comparative Example 3 (without membrane purification) showed significantly reduced activity, while Comparative Example 2, treated with strong acid hydrolysis, exhibited the lowest activity due to the destruction of functional groups. This result strongly demonstrates the superior advantages of the coupled process of "physical remodeling + dual-enzyme degradation + specific adsorption" in the preparation of highly active inhibitory peptides.

[0121] Table 3 Specific activity test results project Specific activity (%) Example 1 15.12 ± 0.17 Example 2 8.72 ± 0.07 Example 3 13.04 ± 0.61 Example 4 8.97 ± 0.53 Example 5 7.74 ± 0.10 Example 6 14.04 ± 0.17 Example 7 7.61 ± 0.13 Example 8 7.05 ± 0.03 Comparative Example 1 5.58 ± 0.20 Comparative Example 2 2.41 ± 0.23 Comparative Example 3 5.03 ± 0.07 Comparative Example 4 3.26 ± 0.15

[0122] The specific activity data clearly outlines the contribution of process optimization to the improvement of product purity and activity. The results strongly demonstrate that the examples, through specific affinity purification using nanofiber membranes, successfully achieved the leap from a "mixture of heteropeptides" to a "high-purity active peptide," with a maximum specific activity more than three times that of the group without membrane purification (Comparative Example 3). In contrast, the protein without physical pretreatment (Comparative Example 1) had incomplete enzymatic digestion due to insufficient unfolding of its internal structure and insufficient exposure of enzymatic sites, resulting in a significant decrease in its specific activity. In Comparative Example 4, which simultaneously applied ultrasonic-microwave treatment during enzymatic digestion, although the physical energy field accelerated the reaction rate to some extent, the ultrasonic cavitation effect and the local high temperature and electromagnetic disturbances caused by microwaves could adversely affect the spatial conformation and stability of the enzyme, leading to fluctuations in enzyme activity, difficulty in precisely controlling the enzymatic digestion process, and consequently, excessive degradation or structural damage of the active peptides, resulting in a specific activity significantly lower than that of the examples. The acid hydrolysis method (Comparative Example 2), due to its harsh reaction conditions, complex product composition, and damage to the structure of some active peptides, had the lowest specific activity. The above results further establish "physical conformational remodeling synergistic bioaffinity purification" as the core technical route for preparing high-abundance, high-purity elastase inhibitory peptides, which has significant advantages in improving specific activity and stability.

[0123] Table 4 Results of Antioxidant Performance Test project DPPH clearance rate (%) ABTS clearance rate (%) Hydroxyl radical scavenging rate (%) Example 1 82.31 ± 0.68 88.26 ± 0.43 80.99 ± 0.51 Example 2 74.29 ± 0.88 84.22 ± 0.63 66.79 ± 0.66 Example 3 79.80 ± 0.38 83.42 ± 0.90 71.22 ± 0.28 Example 4 75.26 ± 0.29 82.19 ± 0.38 69.08 ± 0.92 Example 5 74.19 ± 0.68 79.06 ± 0.66 66.03 ± 0.83 Example 6 81.60 ± 0.65 86.80 ± 0.50 73.11 ± 0.21 Example 7 72.55 ± 0.45 77.89 ± 0.63 66.26 ± 0.92 Example 8 72.08 ± 0.56 79.80 ± 0.42 68.69 ± 0.66 Comparative Example 1 48.71 ± 0.74 52.19 ± 0.82 41.98 ± 0.76 Comparative Example 2 22.93 ± 0.51 30.96 ± 0.55 25.36 ± 0.50 Comparative Example 3 70.07 ± 0.56 76.87 ± 0.68 64.06 ± 0.10 Comparative Example 4 36.25 ± 0.48 48.32 ± 1.02 31.29 ± 0.78

[0124] The antioxidant test results showed that the free radical scavenging ability of each group of samples was highly consistent. Compared with Comparative Example 3 and Comparative Example 1, the example, through physical remodeling and affinity adsorption technology, not only achieved highly efficient and specific inhibition of elastase, but also simultaneously enriched the core peptide with extremely strong antioxidant properties, so that the scavenging rates of DPPH, ABTS and hydroxyl radicals reached 82.31%, 88.26% and 80.99% respectively. The data further demonstrated the advanced nature of the physical conformation remodeling coupled with bioaffinity purification process, proving that the inhibitory peptide prepared by this process can not only accurately protect the elastic fibers of the skin, but also provide comprehensive antioxidant protection by scavenging a variety of free radicals.

[0125] This study systematically demonstrated the core advantages of the coupled "physical remodeling-dual-enzyme degradation-affinity purification" process in preparing highly active elastase inhibitory peptides through comprehensive evaluation of various indicators. Experimental results showed that, compared with the comparative example, solubility, inhibitory activity, and specific activity all exhibited a significant positive correlation increasing trend. Specifically, in Examples 1-8, through physical pretreatment and synergistic treatment using both methods, the protein solubility reached a maximum of 50.26%, a 1.4-fold increase compared to Comparative Example 1, laying a structural foundation for efficient enzymatic hydrolysis. Furthermore, thanks to the specific affinity capture of the immobilized enzyme nanofiber membrane, the inhibitory activity reached a maximum of 77.35%, and its specific activity (15.12) was 3.2 times that of the unpurified group (Comparative Example 3), achieving precise enrichment of the target peptide. Simultaneously, antioxidant tests verified that the peptide possessed extremely strong free radical scavenging ability (ABTS scavenging rate reached 88.26%), proving that this process effectively preserved and enhanced the bioactive center of the peptide while improving inhibitory specificity. In summary, the process of this invention, through the deep coupling of conformational remodeling and bioaffinity, successfully prepared a multifunctional elastase inhibitory peptide with high purity, high inhibitory activity, and strong antioxidant properties, which is significantly superior to traditional physical fractionation or chemical hydrolysis methods.

Claims

1. A method for preparing highly active elastase inhibitory peptides, characterized in that, Includes the following steps: S1: Take animal tissue, crush, defatted, and filtered. Add alkaline solution to the obtained precipitate I, react, cool, wash until neutral, centrifuge to obtain precipitate II, dry, and obtain protein powder. S2: The protein powder obtained in S1 is formulated into a protein suspension, and then pretreated to obtain a remodeled protein suspension; the pretreatment includes at least one of ultrasound, microwave, ultra-high pressure, pulsed electric field, and irradiation. S3: Add enzyme preparation to the reconstituted protein suspension in S2, perform enzymatic hydrolysis, inactivate the enzyme, and obtain the enzymatic hydrolysate; S4: Separate and purify the enzymatic hydrolysate from S3, dry it, and obtain the primary purified peptide powder; S5: The primary purified peptide powder from S4 is prepared into a peptide solution. An immobilized enzyme functional membrane is used to affinity adsorb and desorb the peptide solution to obtain a highly active elastase inhibitory peptide.

2. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S2, the preprocessing conditions are as follows: Ultrasound treatment is performed at 200-600W for 10-40 minutes. Microwaves are used at a power of 300-800W for a duration of 30-180 seconds. The ultra-high pressure pretreatment pressure is 100 ~ 600 MPa, and the time is 5 ~ 30 min; The field strength of the pulsed electric field is 10 ~ 40 kV / cm, and the total time is 1 ~ 20 ms; The irradiation dose is 2 kGy ~ 10 kGy, and the time is 10 ~ 60 s; Microwave-ultrasound synergistic processing: microwave 400-600W, 120-180s; ultrasound 300-500W, 10-30min, ultrasound 5-10s, interval 5-10sn; Ultra-high pressure-microwave co-processing: Set ultra-high pressure to 200-500MPa for 10-25 minutes; Microwave 400-600W, 120-180s; Ultra-high voltage-pulse coordinated processing: Set ultra-high voltage to 200-500MPa, 10-25min; pulse to 15-30kV / cm, 5-15ms. Ultrasound-irradiation synergistic treatment: ultrasound 300-500W, 10-30min, ultrasound 5-10s, interval 5-10sn; irradiation dose 3-8kGy, 20-50s.

3. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S3, the enzyme preparation is selected from at least one of pancreatic enzyme, trypsin, chymotrypsin, elastase, and alkaline protease. The amount of enzyme preparation is 1.0~3.0 wt% of the reconstituted protein suspension. The enzymatic hydrolysis conditions are: pH 6~10, 35~60℃, 12~22h; the enzyme inactivation conditions are: 95~100℃, 5~10min.

4. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S4, the separation and purification steps are as follows: The enzymatic hydrolysate in S3 is subjected to a combined treatment, wherein the combined treatment is selected from any of the following methods: centrifugation and microfiltration, centrifugation and ultrafiltration, centrifugation and nanofiltration, centrifugation and chromatography, microfiltration and ultrafiltration, microfiltration and nanofiltration, microfiltration and chromatography, ultrafiltration and nanofiltration, ultrafiltration and chromatography, or nanofiltration and chromatography; then, the active peptide components with a molecular weight less than 3000 Da are collected and dried to obtain primary purified peptide powder.

5. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S5, the preparation method of the functional membrane for immobilized enzyme is as follows: (1) Preparation of nanofiber membrane: Take a nanofiber membrane, wherein the nanofiber membrane is prepared by co-spinning the backbone material polycaprolactone and an amino-containing natural material, m 聚己内酯 :m 氨基材料 =1:0.2-0.8; the amino-containing natural material is selected from at least one of chitosan, gelatin, zein, silk fibroin, and aminoated lignin; (2) Modification: The nanofiber membrane is immersed in a crosslinking agent accounting for 5-20% of its mass for 1-4 hours to carry out surface functionalization modification, thereby obtaining a modified nanofiber membrane; The crosslinking agent is selected from at least one of glutaraldehyde, epichlorohydrin, polyethylene glycol diacrylate, polydopamine, and genipin; (3) Immobilization: The modified nanofiber membrane is immersed in an immobilized enzyme solution with a concentration of 1~20 mg / mL and a volume of 5~30 mL per square centimeter, and immobilized at 2~8°C for 1~3 h; the immobilized enzyme solution is selected from at least one of trypsin, elastase, alkaline protease, complex protease, and bromelain. After immobilization, the modified nanofiber membrane was removed and its surface was repeatedly rinsed with PBS buffer (pH 6.8–7.2) to completely remove physically adsorbed free enzyme, resulting in an immobilized enzyme loading of 5–40 mg / cm³. 2 Immobilized enzyme functional membranes.

6. The method for preparing the highly active elastase inhibitory peptide as described in claim 5, characterized in that, In S5, the affinity adsorption step is as follows: Take the primary purified peptide powder from S4 and add it to PBS buffer with a pH of 6.8 to 7.2 to prepare a peptide solution with a concentration of 5 to 20 mg / mL. Cutting size is 1~9 cm 2 The immobilized enzyme functional membrane was completely immersed in 5-25 mL of the above peptide solution and placed in a constant temperature environment of 4-25°C for static adsorption for 1-4 hours.

7. The method for preparing the highly active elastase inhibitory peptide as described in claim 6, characterized in that, In S5, the desorption steps are as follows: After adsorption is complete, remove the adsorbed nanofiber membrane, wash it repeatedly with deionized water, and then perform any of the following steps: (1) Treat with ultrasonic power of 50-200W for 20-60 minutes. Or: (2) Immerse the washed nanofiber membrane in acidic buffer solution of pH 3 ~ 6 and alkaline buffer solution of pH 8 ~ 10 for 20 ~ 60 min, collect the adsorption solution, dry it, and obtain high-purity elastase inhibitory peptide powder. Or: (3) Immerse the washed nanofiber membrane in ethanol, acetonitrile, isopropanol or acetone solution for 20 to 60 minutes, collect the adsorption solution, dry it, and obtain high-purity elastase inhibitory peptide powder. Or: (4) Temperature gradient method: The washed nanofiber membrane is immersed in phosphate buffer and treated with different temperature gradients. The temperature gradient is slowly increased from 4°C to 50°C, the gradient change rate is 1~10°C per hour, the total treatment time is 20~60 min, the adsorbent is collected, dried, and high-purity elastase inhibitory peptide powder is obtained. Or (5) Ionic strength variation method: Immerse the washed nanofiber membrane in brine eluent and gradually adjust the ionic strength of the eluent to desorb the peptide from the nanofiber membrane; the ionic strength is slowly increased from 0.01~0.1M to 0.5~1.0M, the total treatment time is 20~60min, the adsorbent is collected, dried, and high-purity elastase inhibitory peptide powder is obtained.

8. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S1, the animal tissue is selected from at least one of the following: bovine heart tube, carotid artery, pig heart, horse ligament, and skipjack tuna heart bulb; The degreasing steps are as follows: Mix the raw material and the organic solvent at a mass-volume ratio of 1g:(5~20)mL and soak for 12~36h. The organic solvent is selected from at least one of acetone, anhydrous ethanol, petroleum ether, and xylene. The reaction steps are as follows: The mixture is then heated and stirred at 70-100℃ for 0.5-3 hours at a mass-to-volume ratio of precipitate I to alkaline solution of 1 g:(5-20) mL. The alkaline solution is selected from at least one of the following: potassium hydroxide solution, sodium hydroxide solution, sodium carbonate solution, and ammonia water with a concentration of 0.05-2 M. The pH during the reaction is 9-13. After the reaction, the mixture is cooled to room temperature. Wash with deionized water until neutral, then centrifuge at 5000-12000 rpm for 5-15 min to obtain precipitate II; The drying process is selected from any one of the following: oven drying, freeze drying, spray drying, vacuum drying, and supercritical fluid drying.

9. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S2, take the protein powder from S1 and add it to deionized water at a solid-liquid ratio of 1 g: (8~15) mL to prepare a protein suspension.

10. The method for preparing the highly active elastase inhibitory peptide as described in claim 1, characterized in that, In S2, the preprocessing is as follows: microwave-ultrasound co-processing: microwave is set to 400-600W, with a total duration of 120-180s; ultrasound is set to 300-500W, for 10-30min, with a 5-10s interval after each 5-10s ultrasound session.

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