Method for extracting crocodile methyl peptide with anti-radiation effect

Abstract

The invention relates to the technical field of polypeptide extraction, and discloses a method for extracting crocodile nail peptide with an anti-radiation effect, which comprises the following steps: degreasing and crushing crocodile nails to obtain fine powder, and performing solid-phase kneading on the fine powder and an enzymatic extraction auxiliary composition under an anhydrous condition; adding water, homogenizing, and treating by a high-voltage pulse electric field to obtain a flexible precursor; sequentially adding keratinase and alkaline protease to carry out constant pH relay hydrolysis in a nitrogen-filled micro-positive pressure environment; cooling, adjusting the pH to an isoelectric point, performing coagulation, separating supernate, performing gradient elution through macroporous adsorption resin, and performing vacuum concentration to remove alcohol; finally, the concentrated solution is screened through 3000 Da and 500 Da ultrafiltration membrane bags in sequence, trapped fluid is collected, freeze drying is conducted, and the product is obtained. According to the preparation method disclosed by the invention, the dissolution rate and the purity of the crocodile methyl peptide with the anti-radiation effect are effectively improved by combining solid-phase kneading, a high-voltage electric field with double-enzyme hydrolysis and physical screening, and inactivation and loss of active ingredients are avoided.

Description

A method for extracting crocodile methyl peptide with anti-radiation properties Technical Field

[0001] This invention relates to the field of polypeptide extraction technology, specifically to a method for extracting crocodile methyl peptide with anti-radiation properties. Background Technology

[0002] Crocodile scales are the scaly tissue on the surface of a crocodile's body, rich in keratin. Their internal polypeptide chains form a dense cross-linked structure through disulfide and hydrogen bonds. Degrading this large protein molecule yields crocodile scale peptides. Crocodile scale peptides contain characteristic amino acids that can scavenge free radicals generated by ultraviolet or electron radiation in the environment, exhibiting anti-radiation effects and corresponding biological value in repairing damaged skin barriers.

[0003] Against the backdrop of continuously increasing environmental radiation, natural ingredients with both anti-radiation and tissue repair functions have a basis for application in health products, skin care products, and medical dressings. Compared with chemical protective agents that are prone to causing skin irritation and plant extracts with limited effects, developing crocodile peptides as a natural anti-radiation ingredient has become a research and development direction for the industry, which can be used to meet the actual needs of daily radiation protection for the human body.

[0004] Existing crocodile shell extraction processes have technical limitations in practical applications. Due to the high stability of crocodile shell keratin structure, conventional physical fragmentation or single enzymatic hydrolysis techniques are insufficient to break its intermolecular cross-linking structure, resulting in incomplete keratinase hydrolysis and low dissolution rate of target peptides. Furthermore, single degradation methods result in the presence of unbroken protein fragments and impurities in the extract, leading to poor peptide purity. In addition, existing processes lack control over the reaction environment and temperature, causing oxidative inactivation and loss of heat-sensitive active ingredients during extraction.

[0005] Therefore, this invention proposes a method for extracting crocodile methyl peptides with anti-radiation properties to overcome the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for extracting alligator cartilage peptides with anti-radiation properties, which solves the problems of insufficient keratinase hydrolysis, low peptide dissolution rate, poor purity, and easy loss of heat-sensitive active ingredients in existing alligator cartilage active polypeptide extraction processes.

[0007] To achieve the above objectives, the present invention provides a method for extracting alligator ketokinase with anti-radiation properties, comprising the following steps: S1: Degreasing, washing, drying, and pulverizing degreased alligator ketokinase to obtain degreased alligator ketokinase fine powder; S2: Kneading the degreased alligator ketokinase fine powder with an enzyme-catalyzed synergistic extraction auxiliary composition in anhydrous solid phase to obtain a solid-phase kneading mixture; S3: Adding water to the solid-phase kneading mixture for homogenization to obtain a homogeneous ultrafine suspension slurry; S4: [The text abruptly ends here, so the translation stops as well.] S5: Apply a high-voltage electric pulse to the slurry to obtain a flexible precursor mixture; S6: Add recombinant keratinase to the flexible precursor mixture and react at a constant pH to obtain a first-stage enzymatic hydrolysate; S7: Add alkaline protease to the first-stage enzymatic hydrolysate for hydrolysis and enzyme inactivation to obtain an inactivated mixture; S8: Adjust the pH of the inactivated mixture to the isoelectric point for coagulation to obtain an isoelectric point coagulation solution; S9: Centrifuge the isoelectric point coagulation solution, and take the supernatant obtained by centrifugation for adsorption elution and concentration to remove alcohol to obtain a polypeptide concentrate; S10: Collect the retentate by ultrafiltration sieving of the polypeptide concentrate, and freeze-dry to obtain crocodile peptide.

[0008] By adopting the above technical solution, the present invention uses a combination of mechanical shearing, electric field polarization and relay enzymatic hydrolysis to promote the gradual depolymerization and precise cleavage of highly cross-linked keratin inside the crocodile shell.

[0009] To address the dense physical structure of crocodile carapace, an auxiliary composition was introduced after defatting and pulverizing, followed by solid-phase kneading under water-free conditions. By eliminating the competitive interference of free water on the hydrogen bond network, the composition can directly penetrate the interior of the keratin fibers, disrupting hydrogen bonds between peptide chains and reducing disulfide bonds, leading to initial depolymerization in the dense crystalline regions. Simultaneously, the unwound local spatial conformation is stabilized by components such as tetrahydropyrimidine, preventing irreversible aggregation of the polypeptide backbone due to hydrophobic interactions.

[0010] After the dense physical structure was broken down, the homogenized slurry was placed in a high-voltage pulsed electric field to further expose the internal enzyme cleavage sites. The alternating electric field caused changes in the transmembrane potential and the dipole moment of the protein molecules, overcoming the residual non-covalent interactions within the polypeptide chain and forcing the protein structure to further unfold and transform into a flexible precursor conformation. This significantly reduced the steric hindrance for subsequent enzymatic degradation at the physical level.

[0011] Once the substrate is converted into a flexible structure, it enters the dual-enzyme relay hydrolysis stage. The recombinant keratinase preferentially recognizes and cleaves peptide bonds in specific keratinized regions of the macromolecule, completing the initial degradation. Immediately afterwards, the alkaline protease performs extensive endohydrolysis, cleaving the long-chain polypeptide into small peptide fragments with anti-radiation activity. Maintaining a constant pH throughout this process eliminates the inhibition of enzyme catalytic activity by local pH fluctuations.

[0012] After hydrolysis, the mixture undergoes isoelectric point coagulation to precipitate large molecular impurities. The target peptide is then enriched by adsorption elution, followed by ultrafiltration to remove specific fragments and freeze-drying. This gentle extraction method effectively avoids thermal denaturation caused by strong acids, alkalis, or high temperatures, preserving the peptide's radiation-resistant spatial conformation.

[0013] Preferably, in step S2, the enzymatic synergistic extraction auxiliary composition is made from the following components in parts by weight: 55-65 parts of anhydrous L-arginine and L-lactic acid eutectic solvent, 20-25 parts of L-ascorbic acid, and 15-20 parts of tetrahydropyrimidine.

[0014] By employing the above technical solution, the composition prepared in a specific ratio exhibits matched flow and permeation properties in a solid-phase system. The eutectic solvent provides a dense array of hydrogen bond donors and acceptors, a fixed amount of L-ascorbic acid is responsible for cleaving disulfide bonds, while tetrahydropyrimidine plays a role in maintaining the conformation of the nascent polypeptide chain. This combination not only improves the efficiency of mechanical shear force transfer but also avoids the additional burden on subsequent separation processes caused by excessive addition.

[0015] Preferably, the method for preparing the anhydrous L-arginine and L-lactic acid eutectic solvent is as follows: 87.1 to 261.3 parts by weight of L-arginine and 90.1 to 270.3 parts by weight of L-lactic acid are added to 20 to 80 parts by weight of water, and stirred at a constant temperature of 65 to 75°C for 2 to 3 hours, followed by dehydration at 45 to 55°C and a vacuum of 0.07 to 0.09 MPa for 12 to 15 hours.

[0016] By adopting the above technical solution, the residual water in the reaction is removed by vacuum dehydration after aqueous synthesis. This not only avoids carbonization and browning of the components in the high-temperature molten state, but also eliminates abnormal swelling of the keratin surface caused by water during the solid-phase kneading stage, thus ensuring that the solvent can smoothly penetrate into the internal pores.

[0017] Preferably, in step S1, the degreasing involves immersing the cleaned crocodile shell in a sodium carbonate aqueous solution with a mass concentration of 4%–6% for 2–4 hours; the washing is stopped when the conductivity of the washing effluent drops to 0.7–0.9 mS / cm; the drying is carried out at 50–60°C until the moisture content is below 5%; and the pulverization is performed by ultra-fine pulverization and passing through a 300–400 mesh sieve.

[0018] By employing the above technical solution, sodium carbonate can saponify the lipid components adhering to the surface of crocodile carapace, increasing the hydrophilicity of the substrate surface. Conductivity monitoring clarifies the elution endpoint of inorganic ions, preventing residual salt ions from interfering with the electric field distribution of the high-voltage pulse. Ultrafine grinding further expands the contact area for the solid-phase reaction.

[0019] Preferably, in step S2, 7.2 to 10.8 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition are added to 90 parts by weight of the defatted alligator shell powder; the conditions for the anhydrous solid-phase kneading are: kneading at 20 to 25°C and a rotation speed of 60 to 80 r / min for 30 to 40 min.

[0020] By employing the above technical solution and setting reasonable substrate-reagent ratios and processing parameters, the shear stress generated by the equipment can precisely overcome the high frictional resistance in an anhydrous system, driving the composition to penetrate the keratin fibers. This control also prevents mechanical heating caused by excessive rotation speed, avoiding localized carbonization or denaturation of proteins.

[0021] Preferably, in step S3, 1800-2700 parts by weight of deionized water are added for every 90 parts by weight of the defatted alligator shell powder, and the homogenization treatment is performed 1-2 times using a high-pressure homogenizer at a processing pressure of 40-60 MPa; in step S4, the application of high-voltage electric pulses is performed by pumping the homogenized ultrafine suspension slurry into a high-voltage pulse electric field treatment chamber, and applying high-voltage electric pulses with an electric field strength of 10-18 kV / cm, a pulse width of 20-40 μs, and a pulse number of 8-12 at a fluid linear velocity of 10-15 mL / s and a temperature of 20-25℃.

[0022] By employing the above technical solution, the cavitation effect generated by high-pressure homogenization reduces particle size and forms a stable suspension. When the material passes through the processing chamber at a specific linear velocity, the electrical pulse energy within a defined range is sufficient to break the residual non-covalent interactions within the molecules, maintaining the flexible extension state of the peptide chain, without causing the covalent peptide bonds in the primary structure to break due to energy overload.

[0023] Preferably, in step S5, the reaction at a constant pH is carried out in a reaction vessel under a nitrogen atmosphere maintaining a slight positive pressure of 0.02–0.05 MPa. For every 90 parts by weight of the defatted alligator shell powder, 15.18–33.61 parts by weight of recombinant keratinase are added. The constant pH is 7.5–8.5, the reaction temperature is 45–50°C, and the reaction time is 2.0–3.0 h. In step S6, 9.49–56.02 parts by weight of alkaline protease are added to the first-stage enzymatic hydrolysate, and hydrolysis is carried out for 0.5–3.0 h. The enzyme inactivation condition is to raise the temperature to 70–80°C and maintain it for 5–10 min.

[0024] By employing the above technical solution, nitrogen-filled micro-positive pressure operation isolates oxygen, preventing the oxidative inactivation of sensitive residues exposed during enzymatic hydrolysis, thereby maintaining the structural integrity of the peptide as a radiation-resistant electron donor. The two enzymes relay catalysis at their respective suitable constant pH and temperature, while the defined time and dosage define the enzymatic digestion process, preventing over-hydrolysis and the generation of inactive free amino acids.

[0025] Preferably, in step S7, the inactivated mixture is cooled to 15-20°C, hydrochloric acid solution is added dropwise to adjust the pH of the inactivated mixture to 4.5-5.0, and the mixture is allowed to stand at 15°C for 20-30 minutes to carry out the flocculation.

[0026] By employing the above technical solution, the system is adjusted to near the isoelectric point of the impurity proteins, reducing their electrostatic repulsion and the thickness of the hydration layer. Combined with low-temperature conditions, this promotes the aggregation and precipitation of large molecular impurity proteins dominated by hydrophobic interactions, achieving preliminary separation of the target product from the impurity proteins and reducing the impurity load for subsequent resin adsorption.

[0027] Preferably, in step S8, the centrifugation conditions are: centrifugation at 4000–5000 r / min for 10–15 min; the adsorption elution is performed as follows: the supernatant is pumped into a macroporous adsorption resin column, and eluted sequentially with deionized water at a flow rate of 3.0–4.0 BV / h for 2.0–3.0 BV, with 5%–10% ethanol at a flow rate of 3.0–4.0 BV / h for 1.0–2.0 BV, and with 20%–30% ethanol aqueous solution at a flow rate of 1.5–2.0 BV / h for 3.0–4.0 BV; the concentration and de-alcoholization is performed by collecting the eluent of the 20%–30% ethanol aqueous solution and concentrating it under reduced pressure at 40–50°C.

[0028] By employing the above technical solution, macroporous resins utilize polarity differences for separation, while deionized water and low-concentration ethanol wash away inorganic salts and impurities with low polarity. The 20% to 30% ethanol aqueous solution eluent is enriched with target peptides that have moderate polarity and a high content of hydrophobic amino acids. These peptides often possess strong cell membrane penetration and free radical scavenging capabilities.

[0029] Preferably, in step S9, the ultrafiltration screening process involves: collecting the permeate through a 3000 Da ultrafiltration membrane, and then collecting the retentate through a 500 Da ultrafiltration membrane; the freeze-drying conditions are: cold trap temperature -40°C to -35°C, vacuum degree 10 to 30 Pa, and heating rate 1.0 to 2.0°C / min.

[0030] By employing the above technical solution, two-stage ultrafiltration sieving can accurately retain oligopeptide components with molecular weights in the range of 500 Da to 3000 Da. The peptide structure in this range conforms to the structure-activity relationship for anti-radiation activity, while eliminating large molecular protein residues and free amino acids. Vacuum freeze-drying parameters avoid the peptide bond breakage problem caused by conventional high temperatures, thus ensuring the biological activity of the final alligator carapace peptides in solid form.

[0031] This invention provides a method for extracting crocodile keratin peptides with anti-radiation properties. It offers the following advantages: 1. The crocodile keratin peptide extraction method of this invention utilizes an auxiliary composition containing a eutectic solvent for solid-phase kneading, combined with high-voltage pulsed electric field treatment, to disrupt the hydrogen bonds and disulfide bonds between dense keratin molecules. This process loosens the spatial conformation of the macromolecules, allowing the internal enzymatic cleavage sites to be fully exposed, directly overcoming the deficiency of insufficient keratinase hydrolysis in existing processes. This solves the problem of low peptide dissolution rate, achieving efficient separation and acquisition of the target component.

[0032] 2. This invention achieves product degradation and separation through a combination of dual-enzyme relay hydrolysis, isoelectric point coagulation, and gradient elution with macroporous resin. Targeted enzymatic hydrolysis, coupled with physical sieving using a specific molecular weight ultrafiltration membrane, removes impurities such as proteins and non-target macromolecules, solving the problem of poor purity in previous extracts. The resulting alligator acetylcholine peptides within a specific molecular weight range maintain their structural integrity and exhibit clear anti-radiation effects, meeting the needs for repairing skin barrier damage caused by daily radiation.

[0033] 3. This invention utilizes enzymatic hydrolysis under a slightly positive nitrogen pressure environment and employs freeze-drying technology at the end to treat the peptide concentrate, thus isolating oxygen and avoiding high-temperature heating. This process prevents the oxidative inactivation of sensitive amino acid residues and solves the problem of easy loss of heat-sensitive active ingredients in traditional processes. This design ensures the stability of the peptide's spatial conformation, guaranteeing that the obtained peptides can exert their corresponding biological activities in subsequent applications. Attached Figure Description

[0034] Figure 1 is a bar chart showing the results of measuring the free thiol content and surface hydrophobicity index of alligator keratin in each group in Test Example 1 of the present invention; wherein, (a) shows the differences in free thiol content in Examples 1 to 3 and Comparative Examples 1 and 7; (b) shows the distribution of surface hydrophobicity index in Examples 1 to 3 and Comparative Examples 1 and 7; Figure 2 is a bar chart showing the results of measuring the redox potential and peptide thiol retention rate in each group during isothermal enzymatic hydrolysis in Test Example 2 of the present invention; wherein, (a) shows the differences in free thiol content in Examples 1 to 3 and Comparative Examples 1 and 7. (a) Comparison of redox potentials of Example 3 and Comparative Example 3 at 1.0h, 2.0h and the end of hydrolysis; (b) Comparison of peptide thiol retention rates of Examples 1 to 3 and Comparative Example 3 at 1.0h, 2.0h and the end of hydrolysis; Figure 3 is a bar chart of peptide dissolution rate, total yield and product purity test results of each group in Test Example 3 of the present invention; wherein, (a) shows the differences in peptide dissolution rates of Examples 1 to 3 and Comparative Examples 1, 2, 4, 6 and 7; (b) shows the differences in peptide dissolution rates of Examples 1 to 3 and Comparative Examples 1, 2, 4, 6 and 7. Example 3, and the overall yield distribution of the freeze-dried powders finally obtained from Comparative Examples 1, 2, 4, 6, and 7; (c) shows the product purity comparison of the freeze-dried powders of Examples 1 to 3, and Comparative Examples 1, 2, 4, 6, and 7 in the range of 500-3000 Da; Figure 4 is a bar chart of the results of the purified alligator shell anti-radiation active peptides obtained in each group of Test Example 4 of the present invention in the in vitro antioxidant and anti-radiation activity tests; wherein, (a) shows the results of Example 1 (a) The differences in DPPH radical scavenging rates of the test solutions in Examples 1 to 3, and Comparative Examples 1, 3, and 5; (b) The comparison of hydroxyl radical scavenging rates of the test solutions in Examples 1 to 3, and Comparative Examples 1, 3, and 5; (c) The retention rates of plasmid supercoiled DNA in the test solutions in Examples 1 to 3, and Comparative Examples 1, 3, and 5; Figure 5 is a graph showing the repair effect of each test solution in Test Example 5 of the present invention on the survival rate of UVB-damaged cells. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0037] Fresh crocodile shells are manually cleaned of any attached meat and impurities from the mud and sand, then air-dried at room temperature for later use; L-arginine (CAS No.: 74-79-3), purity ≥98%; L-lactic acid (CAS No.: 79-33-4), purity ≥98%; L-ascorbic acid (CAS No.: 50-81-7), food grade, purity ≥99%. Tetrahydropyrimidine (CAS No.: 96702-03-3), purity ≥99%; recombinant keratinase, prepared by fermentation and purification of Bacillus subtilis engineered strain WB600-pMA0911-ker carrying the ker gene, with enzyme activity of 500 U / mL to 550 U / mL; alkaline protease (CAS No.: 9014-01-1), with enzyme activity of 200,000 U / g; macroporous adsorption resin, selected from AB-8 type weakly polar styrene copolymer, with a crosslinking degree of 8% to 10%, a particle size range of 0.3 mm to 1.25 mm, a water content of 60% to 70%, and a specific surface area of ​​480 m². 2 / g to 520m 2 / g, with an average pore size of 13nm to 14nm.

[0038] Preparation Example 1: This preparation example provides a method for preparing an enzyme-catalyzed synergistic extraction auxiliary composition, including the following steps: Step 1: 174.2 parts by weight of L-arginine and 180.2 parts by weight of L-lactic acid are added to a three-necked flask equipped with a reflux condenser, and 48 parts by weight of deionized water are added. The mixture is placed in a constant temperature magnetic stirring bath at 70°C and stirred at 450 r / min for 2.5 h to convert it into a uniform and transparent liquid. Then, it is transferred to a vacuum drying oven and dehydrated at 50°C and a vacuum degree of 0.08 MPa for 13.5 h to obtain an anhydrous L-arginine and L-lactic acid eutectic solvent.

[0039] Step 2: Weigh 60 parts by weight of the anhydrous L-arginine and L-lactic acid eutectic solvent obtained in Step 1, 22 parts by weight of L-ascorbic acid and 18 parts by weight of tetrahydropyrimidine, and place them in a high-shear disperser at 22°C and an anhydrous operating environment. Shear and mix at a speed of 3250 r / min for 12 min to form a composite gel paste, which is the enzyme-catalyzed synergistic extraction auxiliary composition.

[0040] Preparation Example 2: This preparation example provides a method for preparing an enzyme-catalyzed synergistic extraction auxiliary composition, including the following steps: Step 1: 87.1 parts by weight of L-arginine and 90.1 parts by weight of L-lactic acid are added to a three-necked flask equipped with a reflux condenser, and 20 parts by weight of deionized water are added. The mixture is placed in a constant temperature magnetic stirring bath at 65°C and stirred at 400 r / min for 2 h to convert it into a uniform and transparent liquid. Then, it is transferred to a vacuum drying oven and dehydrated at 45°C and a vacuum degree of 0.07 MPa for 12 h to obtain an anhydrous L-arginine and L-lactic acid eutectic solvent.

[0041] Step 2: Weigh 55 parts by weight of the anhydrous L-arginine and L-lactic acid eutectic solvent obtained in Step 1, 25 parts by weight of L-ascorbic acid and 20 parts by weight of tetrahydropyrimidine, and place them in a high-shear disperser at 20°C and an anhydrous operating environment. Shear and mix at 3000 r / min for 10 min to form a composite gel paste, which is the enzyme-catalyzed synergistic extraction auxiliary composition.

[0042] Preparation Example 3: This preparation example provides a method for preparing an enzyme-catalyzed synergistic extraction auxiliary composition, including the following steps: Step 1: 261.3 parts by weight of L-arginine and 270.3 parts by weight of L-lactic acid are added to a three-necked flask equipped with a reflux condenser, and 80 parts by weight of deionized water are added. The mixture is placed in a constant temperature magnetic stirring bath at 75°C and stirred at 500 r / min for 3 h to convert it into a uniform and transparent liquid. Then, it is transferred to a vacuum drying oven and dehydrated at 55°C and a vacuum degree of 0.09 MPa for 15 h to obtain an anhydrous L-arginine and L-lactic acid eutectic solvent.

[0043] Step 2: Weigh 65 parts by weight of the anhydrous L-arginine and L-lactic acid eutectic solvent obtained in Step 1, 20 parts by weight of L-ascorbic acid and 15 parts by weight of tetrahydropyrimidine, and place them in a high-shear disperser at 25°C and an anhydrous operating environment. Shear and mix at 3500 r / min for 15 min to form a composite gel paste, which is the enzyme-catalyzed synergistic extraction auxiliary composition.

[0044] Example 1: This example provides a method for extracting crocodile cartilage peptide with anti-radiation effect, including the following steps: Step 1: Immerse 100 parts by weight of crocodile cartilage (after removing impurities) in 1000 parts by weight of a 5% sodium carbonate aqueous solution at room temperature for 3 hours to remove surface grease. After rinsing, rinse repeatedly with deionized water. Stop washing when the conductivity of the washing effluent drops to 0.8 mS / cm. Place the crocodile cartilage in a vacuum drying oven at 55°C and dry until the moisture content is less than 5%. Then, pulverize it into ultrafine powder and pass it through a 350-mesh sieve to obtain degreased crocodile cartilage fine powder.

[0045] Step 2: Take 90 parts by weight of the defatted crocodile shell powder obtained in Step 1 and place it in a high-power kneader with a wall scraping function. Under closed conditions and without free water, 9 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition prepared in Preparation Example 1 are evenly squeezed in through a screw extrusion pump. The mixture is kneaded under high shear at 22°C and 70 r / min for 35 min to obtain a solid-phase kneading mixture.

[0046] Step 3: Add 2250 parts by weight of deionized water to the solid kneaded mixture obtained in Step 2 and stir to mix. Then, continuously pump the solid kneaded mixture after adding water into a high-pressure homogenizer and perform two cycles under a pressure of 50 MPa to obtain a homogenized ultrafine suspension slurry with no sharp corners and an electrical conductivity of less than 1.0 mS / cm.

[0047] Step 4: The homogeneous ultrafine suspension slurry obtained in Step 3 is continuously pumped into the high-voltage pulse electric field treatment chamber at a fluid linear velocity of 12 mL / s. The temperature of the homogeneous ultrafine suspension slurry is controlled at 22℃. A high-voltage electric pulse with an electric field strength of 15 kV / cm, a pulse width of 30 μs, and a pulse number of 10 is applied to obtain a flexible precursor mixture.

[0048] Step 5: Transfer the flexible precursor mixture obtained in Step 4 to a jacketed reactor equipped with automatic titration control (a conventional existing technology that monitors in real time via an online pH probe and automatically replenishes the liquid via a pump). Evacuate the reactor and fill it with high-purity nitrogen to maintain a slight positive pressure of 0.03 MPa. Add 23.49 parts by weight of recombinant keratinase. Set the temperature to 48°C. Add 0.1 mol / L sodium hydroxide solution dropwise using the automatic titration control described above to forcefully maintain the pH of the flexible precursor mixture at a constant 8.0. Stir the reaction continuously for 2.5 hours under sealed protection to obtain the first-stage enzymatic hydrolysate.

[0049] Step Six: Under the conditions of maintaining a slight positive pressure of nitrogen, a temperature of 48°C, and maintaining the pH of the first-stage enzymatic hydrolysate at 8.0 by adding 0.1 mol / L sodium hydroxide solution through the above-mentioned automatic titration control, 23.49 parts by weight of alkaline protease were directly added to the first-stage enzymatic hydrolysate prepared in Step Five, and hydrolyzed for 2.0 h. After the hydrolysis was completed, the jacket was rapidly heated to 75°C and maintained for 8 min to completely inactivate the enzyme, thus obtaining the inactivated mixture.

[0050] Step 7: Cool the inactivated mixture obtained in Step 6 to 18°C ​​using a jacket, release the seal, and slowly add 1.0 mol / L hydrochloric acid solution dropwise under low-speed stirring at 50 r / min until the pH of the inactivated mixture drops to 4.8. Stop stirring and let it stand at 15°C for 25 min to promote hydrophobic aggregation of macromolecular proteins, thus obtaining the isoelectric point aggregated solution.

[0051] Step 8: Centrifuge the isoelectric point coagulation solution obtained in Step 7 at 4500 r / min for 12 min, collect the acidic supernatant with pH maintained at 4.8, without adjusting back to neutral, and directly pump the acidic supernatant into a macroporous adsorption resin column pre-eluted with 95% ethanol and washed with deionized water to neutrality at a flow rate of 2.5 BV / h for adsorption. After adsorption saturation, perform gradient elution sequentially: elute with deionized water at a flow rate of 3.5 BV / h for 2.5 BV, elute with 8% ethanol at a flow rate of 3.5 BV / h for 1.5 BV, and elute with 25% ethanol aqueous solution at a flow rate of 1.8 BV / h for 3.5 BV. Collect the 25% ethanol eluent and concentrate it under reduced pressure at 45℃ to remove the alcohol, thus obtaining the polypeptide concentrate.

[0052] Step 9: The concentrated peptide solution obtained in Step 8 is passed through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 3000 Da to collect the permeate, and then through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 500 Da to collect the retentate. The retentate of the 500 Da ultrafiltration membrane is placed in a freeze dryer and freeze-dried under the conditions of cold trap temperature -38℃, vacuum degree 20Pa, and heating rate 1.5℃ / min to obtain the purified alligator carapace anti-radiation active peptide.

[0053] Example 2: This example provides a method for extracting crocodile cartilage peptide with anti-radiation effect, including the following steps: Step 1: Immerse 100 parts by weight of crocodile cartilage (after removing impurities) in 1000 parts by weight of sodium carbonate aqueous solution with a mass concentration of 4% for 2 hours at room temperature to remove surface grease. After rinsing, rinse repeatedly with deionized water. Stop washing when the conductivity of the washing effluent drops to 0.9 mS / cm. Place the above crocodile cartilage in a vacuum drying oven and dry at 50°C until the moisture content is less than 5%. Grind into ultrafine powder and pass through a 300-mesh sieve to obtain degreased crocodile cartilage fine powder.

[0054] Step 2: Take 90 parts by weight of the defatted crocodile shell powder obtained in Step 1 and place it in a high-power kneader with a wall scraping function. Under closed conditions and without free water, 7.2 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition prepared in Preparation Example 2 is uniformly squeezed in through a screw extrusion pump. The mixture is kneaded under high shear at 20°C and 60 r / min for 30 min to obtain a solid-phase kneaded mixture.

[0055] Step 3: Add 1800 parts by weight of deionized water to the solid kneaded mixture obtained in Step 2 and stir to mix. Then, continuously pump the solid kneaded mixture after adding water into a high-pressure homogenizer and process it once under a pressure of 40 MPa to obtain a homogenized ultrafine suspension slurry with no sharp corners and an electrical conductivity of less than 1.0 mS / cm.

[0056] Step 4: The homogeneous ultrafine suspension slurry obtained in Step 3 is continuously pumped into the high-voltage pulse electric field treatment chamber at a fluid linear velocity of 10 mL / s. The temperature of the homogeneous ultrafine suspension slurry is controlled at 20℃. A high-voltage electric pulse with an electric field strength of 10 kV / cm, a pulse width of 20 μs, and a pulse number of 8 is applied to obtain a flexible precursor mixture.

[0057] Step 5: Transfer the flexible precursor mixture obtained in Step 4 to a jacketed reactor with automatic titration control. Evacuate the reactor and fill it with high-purity nitrogen to maintain a slight positive pressure of 0.02 MPa. Add 15.18 parts by weight of recombinant keratinase. Set the temperature to 45°C. Add 0.1 mol / L sodium hydroxide solution dropwise using the automatic titration control to force the pH of the flexible precursor mixture to remain constant at 7.5. Stir the reaction continuously for 2.0 h under sealed protection to obtain the first-stage enzymatic hydrolysate.

[0058] Step Six: Under the conditions of maintaining a slight positive pressure of nitrogen, a temperature of 45°C, and maintaining the pH of the first-stage enzymatic hydrolysate at 7.5 by adding 0.1 mol / L sodium hydroxide solution through the above-mentioned automatic titration control, add 9.49 parts by weight of alkaline protease directly to the first-stage enzymatic hydrolysate prepared in Step Five, and continue hydrolysis for 0.5 h. After hydrolysis is completed, heat the jacket to 70°C and maintain it for 5 min to completely inactivate the enzyme, and obtain the inactivated mixture.

[0059] Step 7: Cool the inactivated mixture obtained in Step 6 to 15°C using a jacket, release the seal, and slowly add 1.0 mol / L hydrochloric acid solution dropwise under low-speed stirring at 50 r / min until the pH of the inactivated mixture drops to 5.0. Stop stirring and let it stand at 15°C for 20 min to promote hydrophobic aggregation of macromolecular proteins, thus obtaining the isoelectric point aggregated solution.

[0060] Step 8: Centrifuge the isoelectric point coagulation solution obtained in Step 7 at 4000 r / min for 10 min, collect the acidic supernatant with pH maintained at 5.0, without returning to neutral, and directly pump the acidic supernatant into a macroporous adsorption resin column pre-eluted with 95% ethanol and washed with deionized water to neutrality at a flow rate of 2.0 BV / h for adsorption. After adsorption saturation, perform gradient elution sequentially: elute with deionized water at a flow rate of 3.0 BV / h for 2.0 BV, elute with 5% ethanol at a flow rate of 3.0 BV / h for 1.0 BV, and elute with 20% ethanol aqueous solution at a flow rate of 1.5 BV / h for 3.0 BV. Collect the 20% ethanol eluent and concentrate it under reduced pressure at 40℃ to remove the alcohol, thus obtaining the polypeptide concentrate.

[0061] Step 9: The concentrated peptide solution obtained in Step 8 is passed through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 3000 Da to collect the permeate, and then through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 500 Da to collect the retentate. The retentate of the 500 Da ultrafiltration membrane is placed in a freeze dryer and freeze-dried under the conditions of cold trap temperature -35℃, vacuum degree 10Pa, and heating rate 1.0℃ / min to obtain the purified alligator carapace anti-radiation active peptide.

[0062] Example 3: This example provides a method for extracting crocodile cartilage peptides with anti-radiation properties, including the following steps: Step 1: Immerse 100 parts by weight of crocodile cartilage (after removing impurities) in 1000 parts by weight of a 6% sodium carbonate aqueous solution at room temperature for 4 hours to remove surface grease. After rinsing, rinse repeatedly with deionized water. Stop washing when the conductivity of the washing effluent drops to 0.7 mS / cm. Place the crocodile cartilage in a vacuum drying oven and dry at 60°C until the moisture content is less than 5%. Then, pulverize it into ultrafine powder and pass it through a 400-mesh sieve to obtain degreased crocodile cartilage fine powder.

[0063] Step 2: Take 90 parts by weight of the defatted crocodile shell powder obtained in Step 1 and place it in a high-power kneader with a wall scraping function. Under closed conditions and without free water, 10.8 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition prepared in Preparation Example 3 is uniformly squeezed in through a screw extrusion pump. The mixture is kneaded under high shear at 25°C and 80 r / min for 40 min to obtain a solid-phase kneading mixture.

[0064] Step 3: Add 2700 parts by weight of deionized water to the solid kneaded mixture obtained in Step 2 and stir to mix. Then, continuously pump the solid kneaded mixture after adding water into a high-pressure homogenizer and perform two cycles under a pressure of 60 MPa to obtain a homogenized ultrafine suspension slurry with no sharp corners and an electrical conductivity of less than 1.0 mS / cm.

[0065] Step 4: The homogeneous ultrafine suspension slurry obtained in Step 3 is continuously pumped into the high-voltage pulse electric field treatment chamber at a fluid linear velocity of 15 mL / s. The temperature of the homogeneous ultrafine suspension slurry is controlled at 25℃. A high-voltage electric pulse with an electric field strength of 18 kV / cm, a pulse width of 40 μs, and a pulse number of 12 is applied to obtain a flexible precursor mixture.

[0066] Step 5: Transfer the flexible precursor mixture obtained in Step 4 to a jacketed reactor with automatic titration control. Evacuate the reactor and fill it with high-purity nitrogen to maintain a slight positive pressure of 0.05 MPa. Add 33.61 parts by weight of recombinant keratinase. Set the temperature to 50°C. Add 0.1 mol / L sodium hydroxide solution dropwise using the automatic titration control to force the pH of the flexible precursor mixture to remain constant at 8.5. Stir the reaction continuously for 3.0 h under sealed protection to obtain the first-stage enzymatic hydrolysate.

[0067] Step Six: Under the conditions of maintaining a slight positive pressure of nitrogen, a temperature of 50℃, and maintaining the pH of the first-stage enzymatic hydrolysate at 8.5 by adding 0.1 mol / L sodium hydroxide solution through the above-mentioned automatic titration control, add 56.02 parts by weight of alkaline protease directly to the first-stage enzymatic hydrolysate prepared in Step Five, and continue hydrolysis for 3.0 h. After hydrolysis is completed, heat the jacket to 80℃ and maintain it for 10 min to completely inactivate the enzyme, and obtain the inactivated mixture.

[0068] Step 7: Cool the inactivated mixture obtained in Step 6 to 20°C using a jacket, release the seal, and slowly add 1.0 mol / L hydrochloric acid solution dropwise under low-speed stirring at 50 r / min until the pH of the inactivated mixture drops to 4.5. Stop stirring and let it stand at 15°C for 30 min to promote hydrophobic aggregation of macromolecular proteins, thus obtaining the isoelectric point aggregated solution.

[0069] Step 8: Centrifuge the isoelectric point coagulation solution obtained in Step 7 at 5000 r / min for 15 min, collect the acidic supernatant with pH maintained at 4.5, without returning to neutral, and directly pump the acidic supernatant into a macroporous adsorption resin column pre-eluted with 95% ethanol and washed with deionized water to neutrality at a flow rate of 3.0 BV / h for adsorption. After adsorption saturation, perform gradient elution sequentially: elute with deionized water at a flow rate of 4.0 BV / h for 3.0 BV, elute with 10% ethanol at a flow rate of 4.0 BV / h for 2.0 BV, and elute with 30% ethanol aqueous solution at a flow rate of 2.0 BV / h for 4.0 BV. Collect the 30% ethanol eluent and concentrate it under reduced pressure at 50℃ to remove the alcohol, thus obtaining the polypeptide concentrate.

[0070] Step 9: The concentrated peptide solution obtained in Step 8 is passed through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 3000 Da to collect the permeate, and then through a tangential flow ultrafiltration membrane with a molecular weight cutoff of 500 Da to collect the retentate. The retentate of the 500 Da ultrafiltration membrane is placed in a freeze dryer and freeze-dried under the conditions of cold trap temperature -40℃, vacuum degree 30Pa, and heating rate 2.0℃ / min to obtain the purified alligator carapace anti-radiation active peptide.

[0071] Comparative Example 1: Compared with Example 1, the difference is that the process of kneading the anhydrous solid phase first and then adding water for impact in steps two and three was not performed. Instead, 90 parts by weight of defatted alligator shell powder, 9 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition prepared in Example 1, and 2250 parts by weight of deionized water were directly stirred and mixed. The mixture was then continuously pumped into a high-pressure homogenizer and circulated twice under a pressure of 50 MPa to obtain a suspension slurry. All other aspects were the same.

[0072] Comparative Example 2: Compared with Example 1, the difference is the absence of step four, that is, the homogeneous ultrafine suspension slurry was not pumped into the high-voltage pulse electric field treatment chamber for high-voltage electric pulse treatment, but was directly transferred to the jacketed reactor for subsequent operations, and the rest were the same.

[0073] Comparative Example 3: Compared with Example 1, the difference is that in steps five and six, the jacketed reactor was not evacuated and was not filled with high-purity nitrogen to maintain a slight positive pressure. The reaction system was carried out under constant temperature stirring reaction and relay hydrolysis under normal pressure air open conditions. All other aspects are the same.

[0074] Comparative Example 4: Compared with Example 1, the difference is that in step eight, after collecting the acidic supernatant with pH maintained at 4.8, the pH of the acidic supernatant was first adjusted back to neutral using sodium hydroxide solution, and then pumped into a macroporous adsorption resin column for adsorption and elution. The rest are the same.

[0075] Comparative Example 5: Compared with Example 1, the difference is that the physical exclusion and purification step of the two-stage tangential flow ultrafiltration membrane in step nine is missing. Instead, the polypeptide concentrate obtained in step eight is directly placed in a freeze dryer for freeze drying. All other aspects are the same.

[0076] Comparative Example 6: Compared with Example 1, the difference is that in steps five and six, recombinant keratinase was not added. Instead, in step five, 23.49 parts by weight of alkaline protease was directly added to replace the recombinant keratinase for isothermal stirring reaction. In step six, no enzyme was added, so that the total isothermal stirring reaction time of the alkaline protease reached 4.5 hours. All other steps were the same.

[0077] Comparative Example 7: Compared with Example 1, the difference is that the low temperature high pressure pulse electric field treatment process in step four was not performed. Instead, the homogeneous ultrafine suspension slurry obtained in step three was heated and boiled at 80°C for 30 minutes. After the treatment, it was used as a precursor mixture and transferred to a jacketed reactor for subsequent operations. All other aspects were the same.

[0078] Test Example 1: Experimental Objective: To verify the actual disintegration effect of the combined treatment of anhydrous solid-phase targeted intercalation and low-temperature high-voltage pulsed electric field on the steric hindrance of dense keratin in crocodile carapace, as well as the promoting effect on the exposure of deep disulfide bonds and internal hydrophobic groups in the process of this invention.

[0079] Experimental Procedure: Flexible precursor mixtures prepared in step four of Examples 1, 2, and 3, as well as the homogenized suspension slurry from Comparative Example 1 and the precursor mixture from Comparative Example 7 after boiling at 80°C, were extracted as test samples. The precipitates from each group of test samples were centrifuged, washed with phosphate buffer (pH 8.0), and resuspended. The crude protein concentration in each suspension was adjusted to a uniform 2.0 mg / mL, which was then used as the standardized assay solution.

[0080] The free thiol content in each group of samples was determined using the DTNB colorimetric method. 4.0 mL of the standardized assay solution was accurately transferred to a stoppered test tube, and 0.05 mL of 4 mg / mL DTNB colorimetric reagent (pre-dissolved in pH 8.0 phosphate buffer) was added. The mixture was shaken thoroughly for 25 min at room temperature in the dark. After the reaction, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was placed in a UV-Vis spectrophotometer to measure the absorbance at 412 nm. Simultaneously, a buffer solution without the test sample was used as a blank control. The free thiol content was calculated using the cysteine ​​standard curve, with the number of micromoles of free thiol per gram of protein in each sample as the free thiol content.

[0081] The surface hydrophobicity index of each group of samples was characterized using the ANS fluorescent probe method. Standardized assay solutions were serially diluted with phosphate buffer to create a series of concentration gradients from 0.2 mg / mL to 1.0 mg / mL. 20 μL of 8.0 mmol / L ANS probe solution was added to each 4.0 mL aliquot of the diluted sample, and the samples were incubated in the dark for 15 min. The samples were then transferred to a fluorescence spectrophotometer, and the relative fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. A scatter plot was plotted with protein concentration on the x-axis and the corresponding relative fluorescence intensity on the y-axis. The initial slope of the fitted line was used as the surface hydrophobicity index (H0).

[0082] For the tests on free thiol content and surface hydrophobicity index, the data for all groups are the average and standard deviation of three parallel determinations.

[0083] Experimental results (Table 1): Table 1: Determination of free thiol content and surface hydrophobicity index of crocodile keratin in each group

[0084] Test Conclusions: Based on the data in Table 1 and Figure 1, the Example Group and the Comparative Group showed significant numerical differences in the two core indicators characterizing the degree of protein conformational unwinding: free thiol groups and surface hydrophobicity. When processing the samples from Examples 1 to 3, we observed that the solution after complete processing exhibited excellent suspension stability, which is directly related to the large exposure of its internal polar and hydrophobic groups. The free thiol content in the Example Group generally remained above 38 μmol / g, and the surface hydrophobicity index exceeded 2400, proving that the high-concentration, low-eutectic solvent introduced by the present invention through "anhydrous solid-phase kneading" successfully squeezed into the keratin micropores, and under the subsequent dielectric polarization effect of the high-voltage pulsed electric field, the disulfide bonds and hydrophobic residues deeply embedded in the folded structure were fully stretched into the surface aqueous phase.

[0085] Different process parameters directly affect the degree of molecular structure unfolding. Example 3, under higher kneading speed, homogenization pressure, and stronger electric field parameters, achieved the highest unfolding efficiency, with a free thiol content of 49.51 μmol / g. In contrast, Comparative Example 1, without anhydrous solid-phase kneading targeting intercalation, directly mixed and homogenized the fine powder with water and reagents, resulting in a precipitous drop in free thiol content to 18.29 μmol / g. This indicates that conventional macroscopic fluid homogenization shearing cannot penetrate the repulsive hydration layer formed by polar water molecules on the keratin surface, and cannot reach and dismantle its dense internal hydrogen and disulfide bond network. Comparative Example 7's data is even more extreme. Using traditional 80°C high-temperature boiling instead of low-temperature high-pressure pulse treatment not only failed to promote structural unwinding but also caused subunit aggregation and secondary hydrophobic deposition due to the thermosensitive effect, leading to a significant shrinkage of the surface hydrophobicity index to 987.5. Previously exposed active sites were re-encapsulated. These test indicators intuitively and conclusively demonstrate the absolute necessity and scientific basis of the physicochemical cascade process of this invention in removing the spatial steric hindrance of crocodile shells.

[0086] Test Example 2: Experimental Objective: To verify the actual effect of maintaining a slight positive pressure by filling the isothermal enzymatic hydrolysis process with high-purity nitrogen gas and adding auxiliary compositions on maintaining a low redox potential and protecting the heat-sensitive thiol groups from oxidative cross-linking.

[0087] Experimental Procedure: Examples 1, 2, 3, and Comparative Example 3 were selected as test subjects. The moment when each group of examples and comparative examples entered the jacketed reactor to begin the isothermal stirring reaction was recorded as the initial time (0h). Since the hydrolysis completion time of each group was different, online recording and sampling were performed at 1.0h, 2.0h, and the end of complete hydrolysis for each group (4.5h for Example 1 and Comparative Example 3, 2.5h for Example 2, and 6.0h for Example 3).

[0088] The redox potential (ORP) of each reaction solution was monitored using a portable redox potentiometer. At set time points, the electrode probe of the redox potentiometer was inserted to a fixed depth below the liquid surface in the jacketed reactor of each group. After the reading stabilized, the potential value of each group was recorded. The values ​​recorded at 1.0 h, 2.0 h, and the end of complete hydrolysis for each group were taken as the 1.0 h ORP, 2.0 h ORP, and the ORP at the end of hydrolysis for each group, respectively.

[0089] The retention rate of peptide thiol groups in the isothermal enzymatic hydrolysis solution in the jacketed reactor of each group was determined at each time point. At each time point specified above, 5.0 mL of the isothermal enzymatic hydrolysis solution of each group was drawn from the jacketed reactor, and pre-cooled 10% trichloroacetic acid solution was quickly added to terminate the reaction and precipitate large undissociated proteins. The solution was centrifuged at 10000 r / min for 15 min at 4℃, and the supernatant of each group was collected. The absorbance of the supernatant of each group was measured at a wavelength of 412 nm using the DTNB colorimetric method, and the free thiol content in the supernatant of each group was calculated in conjunction with the cysteine ​​standard curve. The total amount of free thiol groups in the flexible precursor mixture of each group before adding the enzyme preparation at the initial time (0 h) was used as the baseline (100%), and the percentage of the total amount of free thiol groups measured in the supernatant of each group at each time point was calculated relative to the baseline value. The percentage values ​​calculated after 1.0h, 2.0h and the end of complete hydrolysis for each group were used as the peptide thiol retention rate at 1.0h, 2.0h and the end of hydrolysis for each group, respectively.

[0090] For the above tests on redox potential and peptide thiol retention rate, the data recorded for each group at all time points were the average and standard deviation of three parallel determinations.

[0091] Experimental results (see Table 2): Table 2: Results of redox potential and peptide thiol retention rate determination in each group during isothermal enzymatic hydrolysis

[0092] Test Conclusion: According to the data in Table 2 and Figure 2, there are significant differences in the redox state and thiol stability of the isothermal enzymatic hydrolysis reaction solution under different treatment conditions. In actual studies of peptide enzymatic extraction, a phenomenon is often observed: as the reaction time increases, free thiol groups exposed to the aqueous phase are easily interfered with by trace amounts of oxygen in the environment, resulting in irreversible disulfide crosslinking. This crosslinking not only leads to the loss of antioxidant and antiradiation activity of the target product, but also causes abnormal aggregation of peptide fragments. Observation of the data of Examples 1 to 3 shows that, under the conditions of maintaining a slight positive pressure by filling the jacketed reactor with high-purity nitrogen and adding auxiliary compositions, the redox potential of the reaction solution in each example can be stably maintained in the negative potential range of -130mV to -170mV at 1.0h, 2.0h, and at the end of hydrolysis. This continuous reducing environment effectively limits the spontaneous oxidation process of thiol groups. Specifically, in Example 1, the peptide thiol retention rate still reached 87.5% at the end of the 4.5-hour hydrolysis. In Example 3, despite the large amount of enzyme added and the extended hydrolysis time to 6.0 hours, the peptide thiol retention rate also remained at 85.2% at the end of the reaction, indicating that the above-mentioned antioxidant intervention measures played a substantial protective role in the long-term reaction.

[0093] In contrast, Comparative Example 3 underwent a isothermal stirring reaction under open air at atmospheric pressure, eliminating physical barriers. The redox potential of its reaction solution showed a significant upward trend, rising from 45.8 mV at 1.0 h to 115.6 mV at the end of hydrolysis. This oxidative environment directly accelerated the dissipation of thiol groups, resulting in a sharp decrease in the thiol retention rate of the peptides in Comparative Example 3 to 32.7% at the end of hydrolysis. This numerical decrease indicates that a large number of physiologically active sulfur-containing peptides underwent structural denaturation during the extraction stage. The comparative results of the above data confirm the necessity of using a combined strategy of nitrogen-filled micro-positive pressure and auxiliary composition in long-duration peptide enzymatic hydrolysis processes. This combination can reduce the oxidative loss of heat-sensitive active groups by intervening in the potential environment of the reaction system.

[0094] Test Example 3: Experimental Objective: To verify the actual effect of the process of the present invention in improving the dissolution rate of the final polypeptide product, the total yield of dry powder, and the purity of the target molecular weight range in terms of physicochemical synergistic pretreatment to break the substrate structure, dual-enzyme relay hydrolysis, and in-situ acid chromatography purification.

[0095] Experimental Procedure: Examples 1, 2, and 3, as well as Comparative Examples 1, 2, 4, 6, and 7, were selected as test subjects. After centrifugation in step eight for each group, samples were taken from the acidic supernatant collected at the top of the centrifuge tubes to determine the polypeptide dissolution rate. Each group then proceeded to step nine, followed by lyophilization, to obtain purified alligator shell anti-radiation active polypeptides. The purified alligator shell anti-radiation active polypeptides (lyophilized powder) obtained from each group were collected to determine the total yield and product purity.

[0096] To determine the polypeptide dissolution rate, accurately transfer 2.0 mL of the acidic supernatant collected from each group, and determine the total nitrogen content of the acidic supernatant of each group using the Kjeldahl method. Multiply by the protein conversion factor (6.25) to calculate the mass of soluble polypeptides in the acidic supernatant of each group. Calculate the ratio of the mass of soluble polypeptides in the acidic supernatant of each group to the total crude protein content of the defatted alligator shell powder initially added in step two of each group, and use the percentage of this ratio as the polypeptide dissolution rate.

[0097] The total yield was determined by accurately weighing the final mass of the purified alligator carapace anti-radiation active peptides collected from each group after freeze-drying, i.e., obtaining the purified alligator carapace anti-radiation active peptides, using an analytical balance. The ratio of the mass of the purified alligator carapace anti-radiation active peptides in each group to the mass of the defatted alligator carapace powder initially added in step two of each group was calculated, and the percentage of this ratio was taken as the total yield.

[0098] To determine product purity, 0.1 g of purified alligator shell anti-radiation active peptides from each group were accurately weighed and dissolved in 10 mL of mobile phase (a mixture of 0.1% trifluoroacetic acid aqueous solution and acetonitrile). The solution was filtered through a 0.22 μm microporous membrane and analyzed by high-performance liquid chromatography (HPLC). A size exclusion column was used, and the detection wavelength was set to 214 nm. Chromatograms were recorded, and the percentage of the total area of ​​chromatographic peaks with molecular weights in the 500-3000 Da range was calculated using the area normalization method. The percentage of the chromatographic peak area in the 500-3000 Da range was used as the product purity.

[0099] For the tests of peptide dissolution rate, total yield and product purity, the data for each group are the average and standard deviation of three parallel determinations.

[0100] Experimental Results (Table 3): Table 3: Results of peptide dissolution rate, total yield and product purity determination for each experimental group

[0101] Test Conclusions: According to the data in Table 3 and Figure 3, the selection of pretreatment process and the control of pH in the chromatography stage have a significant impact on the extraction efficiency and purity of the target peptides. In conventional experiments of keratin hydrolysis, it can be observed that if the substrate structure is not fully expanded, a large amount of undegraded keratin precipitate usually remains at the bottom of the reactor after enzymatic hydrolysis. In Examples 1 to 3, due to the use of anhydrous solid-phase kneading combined with a high-voltage pulsed electric field, the peptide dissolution rate all exceeded 82%, with Example 3 achieving a peptide dissolution rate of 91.05% and a total yield of 70.48%. This is because the dense disulfide and hydrogen bond network was pre-disrupted, allowing the subsequent dual-enzyme relay hydrolysis to more easily cleave the exposed peptide bonds, converting insoluble keratin into soluble peptides.

[0102] Examining the data characteristics of the comparative examples further confirms the role of each process step. Comparative Example 1 lacked anhydrous solid-phase kneading and intercalation, and Comparative Example 2 did not introduce a high-voltage pulsed electric field. The peptide dissolution rates of the two groups decreased to 51.26% and 56.41%, respectively, indicating that a single treatment method is insufficient to completely overcome the wetting barrier of the aqueous environment on highly folded proteins. Comparative Example 7 used 80℃ high-temperature boiling instead of low-temperature electric field treatment, resulting in secondary aggregation and hardening of the protein upon heating, masking the enzyme cleavage sites, and further reducing its peptide dissolution rate to 45.18%. Regarding enzyme selection, Comparative Example 6 relied solely on a single alkaline protease for hydrolysis. The specific cleavage site limitation of a single enzyme prevented some long-chain peptides from being deeply cleaved, resulting in a final total yield of only 51.76%.

[0103] Regarding product purity distribution, the product purity of Examples 1 to 3 remained consistently above 91%, demonstrating the stability of in-situ acidic chromatography in impurity separation. The sample of Comparative Example 4 was processed according to conventional procedures, with the pH of the acidic supernatant pre-adjusted to neutral using sodium hydroxide before resin adsorption. In practice, this pH adjustment operation resulted in localized supersaturation of trace metal ions and phosphate ions in the feed solution, leading to the precipitation of tiny inorganic salt crystals. These crystals partially adhered to the resin pore surface, reducing the resin's adsorption capacity for the target peptide and co-eluting with the peptide during subsequent elution, ultimately causing the product purity of Comparative Example 4 to decrease to 78.53%. The examples retained the low pH of the acidic supernatant for direct column adsorption, avoiding interference from inorganic salt precipitation and maintaining high product purity while improving the overall yield.

[0104] Test Example 4: Experimental Objective: To verify the ability of the alligator cartilage polypeptide prepared by the process of this invention to scavenge free radicals in the in vitro environment, as well as the structural protection effect on plasmid deoxyribonucleic acid (DNA) attacked by ultraviolet radiation, and to confirm the degree to which each operation in the production process retains the activity of the final product.

[0105] Experimental Procedure: Examples 1, 2, and 3, as well as Comparative Examples 1, 3, and 5, were selected as test subjects. The purified alligator carapace anti-radiation active peptides (lyophilized powder) obtained from each group were accurately weighed, and deionized water was used to prepare test solutions with a uniform peptide concentration of 5.0 mg / mL for each group.

[0106] The scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals was determined. 2.0 mL of each test solution was transferred to separate stoppered test tubes, and 2.0 mL of 0.1 mmol / L anhydrous ethanol solution of DPPH was added to each tube. The mixtures were shaken thoroughly and incubated at room temperature in the dark for 30 min. After the reaction, the absorbance was measured at 517 nm using a UV-Vis spectrophotometer. An equal volume of deionized water was used as a blank control, and an equal volume of anhydrous ethanol was used as a background subtraction agent. The scavenging ratio of each test solution for DPPH free radicals was calculated based on the absorbance difference, and this calculated scavenging ratio was taken as the DPPH free radical scavenging rate.

[0107] To determine the hydroxyl radical scavenging rate, 1.0 mL of each test solution, 1.0 mL of 9.0 mmol / L ferrous sulfate solution, and 1.0 mL of 9.0 mmol / L salicylic acid-ethanol solution were added sequentially to different test tubes. Finally, 1.0 mL of 8.8 mmol / L hydrogen peroxide solution was added to each test tube to initiate the reaction. Each mixture was incubated at 37°C for 30 min, and the absorbance was measured at 510 nm. Similarly, a deionized water blank control group and a background group without chromogenic reagent were set up. The scavenging ratio of each group was calculated based on the change in absorbance, and this calculated scavenging ratio was used as the hydroxyl radical scavenging rate.

[0108] The retention rate of supercoiled deoxyribonucleic acid (DNA) was determined using pBR322 plasmid DNA (an E. coli cloning vector used as a common model of DNA damage). 5 μL of pBR322 plasmid DNA solution (50 μg / mL) was added to each of the microcentrifuge tubes, along with 10 μL of the respective assay solution. After mixing, the tubes were vertically irradiated under a high-power short-wave ultraviolet (UVC) lamp for 45 min (radiation dose set at 100 J / m²). 2After irradiation, samples from each group were subjected to 1% agarose gel electrophoresis. Following electrophoresis, a readily available gel imaging analysis system (a mature instrument for fluorescence signal acquisition and quantitative analysis after gel electrophoresis) was used to scan and quantify the fluorescence intensity of each band. Using the fluorescence intensity of the supercoiled structure of unirradiated normal plasmid DNA as a 100% baseline, the ratio of the fluorescence intensity of the supercoiled structure after adding the assay solution and undergoing irradiation treatment to the baseline value was calculated. The percentage of this ratio was used as the supercoiled DNA retention rate.

[0109] For the tests on DPPH radical scavenging rate, hydroxyl radical scavenging rate and supercoiled DNA retention rate, the data for each group are the average and standard deviation of three parallel determinations.

[0110] Experimental results (see Table 4): Table 4: Results of in vitro anti-radiation and antioxidant activity assays of each group of peptides

[0111] Test Conclusions: According to the data in Table 4 and Figure 4, there are significant differences in the in vitro anti-radiation and antioxidant activities of alligator cartilage peptides prepared by different processes. In in vitro studies related to radiation damage, ultraviolet irradiation of aqueous solutions induces a large amount of free radicals. These free radicals easily attack the phosphodiester bonds of plasmid deoxyribonucleic acid (DNA), causing the originally tight supercoiled structure to break and transform into an open-ring or linear structure. The test results show that the test solutions of Examples 1 to 3 exhibit better protective intervention effects. Among them, the supercoiled DNA retention rate of Example 3 reached 81.12%, and the corresponding scavenging rates of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and hydroxyl radicals were also maintained at a high level. This is related to the use of anhydrous solid-phase intercalation and high-voltage pulsed electric field in the extraction process. This pretreatment method opens the dense disulfide bond structure of keratin, allowing the internal sulfur-containing groups and other active sites to enter the liquid phase with the peptide, providing electron donors for neutralizing ultraviolet radiation-induced free radicals in the in vitro test environment.

[0112] Observing the data from the control groups further verifies the actual effectiveness of specific protective measures in the process. Comparative Example 1 lacked prior physicochemical pretreatment; the incompletely dissociated substrate limited the release of core active groups, resulting in low radiation resistance and a DNA retention rate of only 48.91%. Comparative Example 3 did not employ inert gas protection or a micro-positive pressure environment during enzymatic hydrolysis; open heating caused oxidative cross-linking of the reducing thiol groups in the peptides. Due to the loss of key antioxidant groups, the free radical scavenging ability of the assay solution in Comparative Example 3 significantly decreased, with a DPPH free radical scavenging rate of only 36.58% and a supercoiled DNA retention rate dropping to 24.65%, essentially losing its protective effect against radiation damage. Furthermore, Comparative Example 5 lacked a two-stage tangential flow ultrafiltration physical exclusion cleavage purification step, resulting in the presence of large molecular impurities and inactive amino acid monomers in the product. This diluted the effective peptide concentration per unit mass of powder, leading to a supercoiled DNA retention rate of only 55.33%. The above data indicate that the anti-radiation activity of peptide products depends not only on the amino acid composition of the raw material itself, but also on the degree of protein structure expansion during the extraction process, the protective effect against oxidation of thermosensitive groups, and the precision of end-cleavage purification.

[0113] Test Example 5: Experimental Objective: To verify the ability of each group of test solutions to penetrate the stratum corneum and enter the living epidermis or dermis, as well as the actual repair effect on keratinocyte damage induced by UVB irradiation, and to confirm the influence of different preparation process steps on transdermal absorption and bioactivity of peptides.

[0114] Experimental Procedure: Examples 1, 2, and 3, as well as Comparative Examples 2, 4, and 6, were selected as test subjects. The purified alligator carapace anti-radiation active peptides (lyophilized powder) obtained from each group were accurately weighed and prepared into test solutions with a uniform peptide concentration of 10.0 mg / mL using phosphate-buffered saline (PBS, a commonly used salt solution for maintaining pH stability, pH 7.4).

[0115] Transdermal absorption rates were determined using a standard Franz diffusion cell apparatus for in vitro transdermal experiments. Isolated porcine ear skin (previously treated with dehairing and subcutaneous fat removal to serve as a standard permeation model substituted for human skin) was fixed between the supply and receiving chambers of the diffusion cell, with the stratum corneum facing the supply chamber. 1.0 mL of the test solution was added to the supply chamber of several different diffusion cells, while the receiving chamber was filled with phosphate-buffered saline (PBS) maintained at a constant temperature of 32°C, and the mixture was continuously stirred magnetically.

[0116] At 12 and 24 hours after the start of the experiment, 0.5 mL of the absorption solution was drawn from the receiving chamber (an equal volume of fresh phosphate buffer at the same temperature was immediately added after each draw). The drawn absorption solution was filtered through a 0.22 μm microporous membrane, and the mass concentration of the permeated peptides was determined using high-performance liquid chromatography (HPLC, a conventional instrument used for the separation and quantitative analysis of components in a mixture). The percentage was calculated based on the initial total amount of peptides in the supply chamber and the cumulative amount of peptides permeated in the receiving chamber at 12 hours; this calculated percentage was used as the transdermal absorption rate at 12 hours. Similarly, the percentage was calculated based on the cumulative amount of peptides permeated at 24 hours; this calculated percentage was used as the transdermal absorption rate at 24 hours.

[0117] To assess the skin barrier repair efficacy, immortalized human epidermal keratinocytes (HaCaT cells, an epidermal cell line commonly used in in vitro dermatological studies) were seeded into multiple wells of a 96-well culture plate and incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, the old culture medium was discarded, and a new culture medium at a concentration of 30 mJ / cm³ was used. 2 Medium-wave ultraviolet (UVB, ultraviolet light with wavelengths between 280-315nm, often used to construct skin photodamage models) was used to vertically irradiate cells to construct cell radiation damage models.

[0118] Immediately after irradiation, fresh culture medium containing each group of test solutions (final concentration diluted to 100 μg / mL) was added to different wells, and the cells were cultured for another 24 hours. Normal cells that were not irradiated with UVB and did not receive any test solutions were set up as a control group, and model cells that were irradiated with UVB but did not receive any test solutions were set up.

[0119] After culture, the working solution of the Cell Proliferation and Toxicity Assay Kit (CCK-8, a colorimetric assay reagent based on water-soluble tetrazolium salt) was added to each well, and incubated for 2 hours. The optical density (OD, a physical quantity representing the degree of light absorption by a substance) of each well was measured at 450 nm using a microplate reader. Using the OD value of the blank group cells as a 100% baseline, the relative percentage of the OD value of damaged cells after adding the test solution was calculated to the baseline. This relative percentage was used as the UVB-damaged cell survival rate. All test data were obtained as the mean and standard deviation from three independent parallel experiments.

[0120] Experimental results (see Table 5): Table 5: Results of transdermal peptide absorption rate and UVB-damaged cell survival rate measurement in each group

[0121] Test Conclusions: Based on the data in Table 5 and Figure 5, the repair function of the peptide mixture at the in vitro cellular level is limited by its permeation efficiency across the stratum corneum and the composition of the mixture itself. In conventional transdermal studies, large peptides with uneven molecular weight distribution or steric hindrance usually have difficulty penetrating the lipid bilayer of the stratum corneum. The experimental results show that the test solutions of Examples 1 to 3 exhibited high permeation kinetics, with Example 3 showing a cumulative transdermal absorption rate of 76.28% at 24 hours, and a corresponding recovery rate of 88.64% for UVB-damaged cells. This data indicates that the pretreatment using anhydrous mechanochemical solid-phase intercalation combined with a high-voltage pulsed electric field effectively dissociated the disulfide cross-linked backbone of keratin, providing sufficient substrate exposure area for subsequent enzymatic hydrolysis. This treatment method helps generate oligopeptide fragments with concentrated and uniform molecular weight. Because these small molecular weight oligopeptides have a suitable water-lipid partition coefficient, they can enter the live epidermal model and exert cell repair effects in a shorter time.

[0122] The data from the comparative group further confirmed the limitations of different variable parameters in the initial extraction and purification process on peptide activity. In Comparative Example 2, due to the lack of a high-voltage pulsed electric field, the dense keratin network was not fully dissociated, limiting the action sites of endonucleases and leading to incomplete degradation. Its product retained a large number of medium- and long-chain peptides, which, in the transdermal test, were blocked by the physical barrier of the stratum corneum, resulting in a transdermal absorption rate of only 42.15% at 24 hours; simultaneously, due to insufficient exposure of key repair sites, the corresponding cell survival rate was only 54.21%. In Comparative Example 4, following standard procedures, the acidic supernatant was neutralized before chromatography, leading to the precipitation of trace metal ions and other inorganic salt crystals that interfered with resin adsorption. Because the finished product contained a large amount of impurities and inorganic salts, the absolute content of effective peptides was diluted at the same test solution concentration, resulting in lower overall absorption and cell survival rates. Furthermore, the actual repair effect of peptides after entering a damaged environment is directly related to the purity of their active ingredients. Comparative Example 6 used only alkaline protease in the hydrolysis stage without adding specific recombinant keratinase. Although its transdermal absorption rate reached 68.42% at 24 hours, the survival rate of UVB-damaged cells was only 63.45%. Under the same test solution concentration, Comparative Example 6 failed to significantly improve cell survival rate as the examples. This is because the cleavage site of a single enzyme is limited, and it cannot completely cleave dense keratin into characteristic oligopeptide sequences with high antioxidant and repair activities, resulting in an increased proportion of impurities or low-activity fragments in the final extract. The above analysis shows that combining physical field dissociation technology, specific dual-enzyme relay hydrolysis, and subsequent precise chromatographic separation technology is a necessary condition to ensure that alligator cartilage peptides simultaneously possess high transdermal absorption rate and high cell repair activity.

[0123] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting crocodile methyl peptide with anti-radiation properties, characterized in that, Includes the following steps: S1: Degrease, wash, dry, and pulverize the purified crocodile shell to obtain degreased crocodile shell fine powder; S2: Knead the degreased crocodile shell fine powder with an enzyme-catalyzed synergistic extraction auxiliary composition in an anhydrous solid phase to obtain a solid-phase kneading mixture; the enzyme-catalyzed synergistic extraction auxiliary composition is made of the following components in parts by weight: 55-65 parts of anhydrous L-arginine and L-lactic acid eutectic solvent, 20-25 parts of L-ascorbic acid, and 15-20 parts of tetrahydropyrimidine; S3: Add water to the solid-phase kneading mixture for homogenization to obtain a homogeneous ultrafine suspension slurry; S4: Apply a high-voltage electric pulse to the homogeneous ultrafine suspension slurry to obtain a flexible precursor mixture; S5: Add recombinant keratinase to the flexible precursor mixture and react at a constant pH to obtain the first-stage enzymatic hydrolysate; S6: Add alkaline protease to the first-stage enzymatic hydrolysate to hydrolyze and inactivate the enzyme, and obtain the inactivated mixture; S7: Adjust the pH of the inactivated mixture to the isoelectric point and perform coagulation to obtain an isoelectric point coagulation solution; S8: Centrifuge the isoelectric point coagulation solution, take the supernatant obtained by centrifugation, perform adsorption elution and concentration to remove alcohol, and obtain a polypeptide concentrate; S9: Collect the retentate by ultrafiltration sieving of the polypeptide concentrate, and freeze-dry it to obtain crocodile peptide.

2. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, The method for preparing the anhydrous L-arginine and L-lactic acid eutectic solvent is as follows: 87.1–261.3 parts by weight of L-arginine and 90.1–270.3 parts by weight of L-lactic acid are added to 20–80 parts by weight of water, and stirred at a constant temperature of 65–75°C for 2–3 hours. Subsequently, the mixture is dehydrated at 45–55°C and a vacuum of 0.07–0.09 MPa for 12–15 hours.

3. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S1, the degreasing involves immersing the cleaned crocodile shell in a sodium carbonate aqueous solution with a mass concentration of 4%–6% for 2–4 hours; the washing is stopped when the conductivity of the washing effluent drops to 0.7–0.9 mS / cm; the drying is carried out at 50–60°C until the moisture content is below 5%; and the pulverization is ultra-fine pulverization followed by sieving through a 300–400 mesh sieve.

4. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S2, 7.2 to 10.8 parts by weight of the enzyme-catalyzed synergistic extraction auxiliary composition are added to 90 parts by weight of the defatted crocodile shell powder; the conditions for the anhydrous solid phase kneading are: kneading at 20 to 25°C and a rotation speed of 60 to 80 r / min for 30 to 40 min.

5. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S3, 1800-2700 parts by weight of deionized water are added to every 90 parts by weight of the defatted alligator shell powder. The homogenization process is performed by using a high-pressure homogenizer at a processing pressure of 40-60 MPa for 1-2 times. In step S4, the application of high-voltage electric pulses is performed by pumping the homogenized ultrafine suspension slurry into a high-voltage pulse electric field treatment chamber and applying high-voltage electric pulses with an electric field strength of 10-18 kV / cm, a pulse width of 20-40 μs, and a pulse number of 8-12 at a fluid linear velocity of 10-15 mL / s and a temperature of 20-25℃.

6. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S5, the reaction at a constant pH is carried out in a reaction vessel under a nitrogen atmosphere maintaining a slight positive pressure of 0.02–0.05 MPa. For every 90 parts by weight of the defatted alligator shell powder, 15.18–33.61 parts by weight of recombinant keratinase are added. The constant pH is 7.5–8.5, the reaction temperature is 45–50°C, and the reaction time is 2.0–3.0 h. In step S6, 9.49–56.02 parts by weight of alkaline protease are added to the first-stage enzymatic hydrolysate, and hydrolysis is carried out for 0.5–3.0 h. The enzyme inactivation conditions are to raise the temperature to 70–80°C and maintain it for 5–10 min.

7. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S7, the inactivated mixture is cooled to 15-20°C, hydrochloric acid solution is added dropwise to adjust the pH of the inactivated mixture to 4.5-5.0, and the mixture is allowed to stand at 15°C for 20-30 minutes to carry out the flocculation.

8. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S8, the centrifugation conditions are: centrifugation at 4000–5000 r / min for 10–15 min; the adsorption elution is performed as follows: the supernatant is pumped into a macroporous adsorption resin column, and eluted sequentially with deionized water at a flow rate of 3.0–4.0 BV / h for 2.0–3.0 BV, with 5%–10% ethanol at a flow rate of 3.0–4.0 BV / h for 1.0–2.0 BV, and with 20%–30% ethanol aqueous solution at a flow rate of 1.5–2.0 BV / h for 3.0–4.0 BV; the concentration and de-alcoholization involves collecting the eluent of the 20%–30% ethanol aqueous solution and concentrating it under reduced pressure at 40–50°C.

9. The method for extracting crocodile methyl peptide with anti-radiation effect according to claim 1, characterized in that, In step S9, the ultrafiltration screening process involves: collecting the permeate through a 3000 Da ultrafiltration membrane, and then collecting the retentate through a 500 Da ultrafiltration membrane; the freeze-drying conditions are: cold trap temperature -40℃ to -35℃, vacuum degree 10 to 30 Pa, and heating rate 1.0 to 2.0℃ / min.

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