A targeted extraction and purification process of white snail glycosaminoglycan-growth factor complex active

Abstract

The application discloses a kind of white snail glycosaminoglycan-growth factor composite active substance directional extraction purification process, belong to biological active substance extraction technical field.Core breakthrough is in at: through-10 ℃ acetone circulation degreasing and 20kHz ultrasonic coordination papain-neutral protease compound system (mass ratio 1:0.8), realize glycosaminoglycan and growth factor synchronous efficient release, extraction rate reaches 97.2%;Using DEAE-Sepharose Fast Flow and Q Sepharose Fast Flow double column chromatography gradient elution, combined with 1000Da ultrafiltration membrane refining, composite active substance purity is ≥99.6%;Through-Vc mild depolymerization technology regulates glycosaminoglycan molecular weight to 3.8~23.8kDa, retains its heparin-like structure activity;Using porous medium enhanced vacuum freeze-drying process, so that composite active substance activity retention rate is ≥98.5%.Product glycosaminoglycan content is ≥35mg / g, growth factor (EGF class) activity is ≥1200IU / mg, DPPH free radical clearance rate is ≥65%, to skin fibroblast proliferation promotion rate is ≥72%, no organic solvent residue and heavy metal pollution.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction technology, and in particular to a targeted extraction and purification process for a complex active substance of white jade snail glycosaminoglycan-growth factor. Background Technology

[0002] The white jade snail (scientific name: Achatina Fulica) is rich in glycosaminoglycans and growth factors, among other complex active substances. Glycosaminoglycans (AFG) have heparin-like structural features, formed by the alternating linkage of L-iduronic acid sulfate and D-acetylglucosamine, and possess anti-inflammatory, antioxidant, and tissue repair-promoting biological activities. Growth factors (such as EGF) can significantly promote the proliferation of skin fibroblasts and accelerate wound healing, making them core raw materials in the fields of medical aesthetics and biomedicine.

[0003] However, existing extraction processes suffer from four major technological bottlenecks: Simultaneous extraction of active ingredients is difficult: Traditional single enzymatic or alkaline hydrolysis methods cannot release glycosaminoglycans and growth factors at the same time, resulting in an extraction rate of less than 70% for one of the components, and easy separation and loss.

[0004] Low purification precision: Conventional single-column chromatography or simple filtration is insufficient to completely remove impurities such as extraneous proteins and sensitizing impurities, resulting in product purity generally below 95%, which limits its application in the medical aesthetics field.

[0005] Severe structural damage: Degradation by strong acids and alkalis or high-temperature treatment can destroy the spatial structure of sulfate groups and growth factors in glycosaminoglycans, resulting in a loss of more than 30% of biological activity.

[0006] Activity decay during drying process: Traditional vacuum freeze drying has a long time and low heat and mass transfer efficiency, resulting in an activity retention rate of only about 80% for composite active materials, and it is also prone to clumping, which affects solubility.

[0007] While there have been attempts at improvement within the industry, such as using enzymatic hydrolysis-column chromatography to extract glycosaminoglycans, the yield is only 0.88%, and simultaneous enrichment with growth factors has not been achieved. Single ion exchange chromatography purification has increased product purity to 97%, but the process is cumbersome and costly, making industrial-scale production difficult. Conventional freeze-drying processes, while retaining some activity, have drying cycles exceeding 30 hours, resulting in low production efficiency. Therefore, developing a targeted extraction and purification process for the glycosaminoglycan-growth factor complex active ingredient from *Solanum lyratum* that simultaneously extracts efficiently, achieves high purification precision, preserves the structure well, and maintains stable activity after drying has become a crucial technology urgently needing to be addressed in the industry. Summary of the Invention

[0008] In view of this, the present invention provides a targeted extraction and purification process for the active compound of white snail amine polysaccharide-growth factor, so as to solve or alleviate one of the technical problems existing in the prior art, and at least provide a beneficial option.

[0009] The technical solution of this invention is implemented as follows: a targeted extraction and purification process for the active compound of white jade snail glycosaminoglycan-growth factor complex includes the following steps: Step S1, Raw Material Pretreatment and Low-Temperature Degreasing: Select healthy, live white jade snails weighing 70-90g and subject them to a 3-day fasting and quiet rearing period. The quiet rearing environment should be 18-20℃, relative humidity 60%-65%, and ventilation rate 0.8m³ / h. Rinse twice daily with sterile saline. Aseptically separate the shell, soft tissue, digestive glands, and mucus. Mix them at a mass ratio of soft tissue:digestive glands:mucus = 5:3:2, add 8 times the volume of -10℃ acetone, stir and defatted for 30 minutes, then centrifuge at 4℃ and 10000rpm for 20 minutes and collect the precipitate. Repeat the above defatting operation twice. Redissolve the final precipitate with sterile water at 4℃ and adjust the pH of the system to 6.5-7.0 to obtain the raw material suspension. Step S2, Ultrasonic-Coordinated Targeted Hydrolysis with Compound Enzyme: The raw material suspension is transferred to a low-temperature ultrasonic reactor, and ultrasound with a frequency of 20kHz and a power of 250W is applied. The ultrasonic mode is set to 5s working / 5s intermittent, with a power density of 0.5W / cm², and the system temperature is controlled at 40~45℃. A compound enzyme preparation is added, which consists of papain with an enzyme activity ≥1500U / mg and neutral protease with an enzyme activity ≥1800U / mg in a mass ratio of 1:0.8. The amount of enzyme added is 1.2%~1.8% of the total weight of the raw materials, and the solid-liquid ratio of the hydrolysis system is controlled at 1:6~1:8 (g / mL). Hydrolysis is carried out at a constant temperature for 5~6 hours, with stirring at a rate of 80r / min for 8 minutes every 40 minutes. After hydrolysis, the enzyme is inactivated in an 85℃ water bath for 10 minutes to obtain the hydrolysate. Step S3, Gradient ion exchange chromatography purification: The hydrolysate was centrifuged at 4℃ and 12000rpm for 30min. The supernatant was filtered through a 0.22μm filter membrane and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated with 0.05M Tris-HCl buffer at pH 7.2. The column diameter to column height ratio was 1:15, and the sample volume was 1 / 10 of the column volume. Elution was performed sequentially with distilled water, 0.2M, and 0.5M sodium chloride aqueous solution at a flow rate of 1.5mL / min, each with a column volume of 3. The elution peaks were monitored using the phenol-sulfuric acid colorimetric method, and the 0.5M eluent was collected. The collected solution was then loaded onto a Q Sepharose Fast Flow column and eluted with 0.8M sodium chloride aqueous solution at a volume of 3. The eluent was collected to obtain the crude pure solution. Step S4, Membrane separation purification and gentle depolymerization: The crude pure product solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da at a pressure of 0.3~0.4 MPa and a temperature of 4~6℃. The temperature fluctuation of the feed solution is controlled to not exceed ±1℃. The permeate is collected. 11 mM ascorbic acid is added to the permeate. After it is fully dissolved, a 2% hydrogen peroxide solution is added, wherein the volume ratio of hydrogen peroxide to ascorbic acid is 1:3. The reaction is carried out at a constant temperature of 37℃ for 3 hours to obtain the depolymerized composite active solution. Step S5, Vacuum freeze-drying with porous media: The composite active liquid and mannitol-maltodextrin composite carrier are mixed evenly at a mass ratio of 10:1, wherein the mass ratio of mannitol to maltodextrin in the composite carrier is 1:1. The mixture is spread on a freeze-drying tray with a thickness ≤4mm and placed in a vacuum freeze dryer. First, it is pre-frozen at -55℃ for 7h, and the vacuum degree is adjusted to ≤5Pa. Gradient porous media is added, wherein the porosity of the gradient porous media is 60%~80% and the pore size is 50~500μm. The temperature is raised to -15℃ for sublimation drying for 14h, and then raised to 20℃ for desorption drying for 9h to obtain the finished product of white snail glycosaminoglycan-growth factor composite active product.

[0010] Preferably, during the fasting and rest period described in step S1, the environment is kept ventilated at a rate of 0.8 m³ / h; during the defatting process, an ice-water bath is used to control the temperature to ensure that the local temperature does not rise and to prevent the active ingredients from being deactivated by heat.

[0011] Preferably, the total enzyme activity of the compound enzyme preparation in step S2 is ≥1650U / mg. The ultrasonic action targets and hydrolyzes the protein matrix encapsulating the active ingredients through cavitation effect, avoiding damage to the glycosaminoglycan sulfate groups and the active centers of growth factors.

[0012] Preferably, the DEAE-Sepharose Fast Flow chromatography column and the Q Sepharose Fast Flow chromatography column in step S3 form a dual-column tandem system, which achieves precise separation of impurities and target active substances by elution with gradient salt solution, and the removal rate of sensitizing impurities reaches 100%.

[0013] Preferably, the ultrafiltration process in step S4 is carried out at a low temperature of 4-6°C, using hydrogen peroxide and ascorbic acid. The -Vc system is used to gently control the molecular weight of glycosaminoglycans to 3.8~23.8kDa, and no additional catalyst removal is required after the reaction is complete.

[0014] Preferably, the gradient porous medium in step S5 has a porosity of 70% and a pore size of 200 μm; the condenser temperature during freeze-drying is ≤-60℃; the resulting product has a water content of ≤2.5% and a solubility of ≥99.8%.

[0015] Preferably, the composite active ingredient has a purity of ≥99.6%, an activity retention rate of ≥98.5%, a glycosaminoglycan content of ≥35mg / g, a growth factor activity of ≥1200IU / mg, a DPPH free radical scavenging rate of ≥65%, and is free from sensitizing impurities, organic solvent residues, and heavy metal contamination.

[0016] As a preferred embodiment, it exhibits a loose and porous structure under a scanning electron microscope, without clumping, and its activity decreases by ≤5% after 12 months of storage at room temperature.

[0017] Preferably, the composite active ingredient is added to medical aesthetic repair dressings, anti-aging creams or wound healing gels at a mass percentage of 5% to 15% for skin barrier repair, anti-inflammatory and antioxidant effects and tissue repair.

[0018] As a preferred option, when added to medical aesthetic repair dressings, users can achieve a skin barrier repair rate of 62.5% and an inflammation reduction rate of 58.7% after 14 days of use, with no allergic reactions.

[0019] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: 1. Simultaneous and efficient extraction: Ultrasonic synergistic compound enzyme directional hydrolysis technology enables the simultaneous enrichment of glycosaminoglycans and growth factors, with a comprehensive extraction rate of ≥97.2%, which is much higher than the 75%~85% of traditional processes.

[0020] High purification precision: The combination of gradient dual-column chromatography and membrane separation results in a protein content of ≤0.1%, with no sensitizing reaction, meeting the stringent requirements of high-end medical aesthetic products.

[0021] The structure remains intact: -Vc's gentle depolymerization avoids the loss of sulfate groups and the denaturation of growth factors, resulting in a glycosaminoglycan heparin structure retention rate of ≥99% and a growth factor activity of ≥1200 IU / mg, demonstrating significantly superior bioactivity compared to existing technologies.

[0022] Balancing drying efficiency and activity stability: Porous media-enhanced freeze drying shortens the drying cycle to less than 23 hours, with an activity retention rate of ≥98.5%, a finished product moisture content of ≤2.5%, and an activity decay of ≤5% after 12 months of storage at room temperature.

[0023] The process is green and easy to industrialize: no toxic or harmful reagents are used throughout the process, and all equipment used is standard industry equipment, reducing production costs by more than 35%, making it suitable for large-scale mass production. The product has wide applicability and can be applied to multiple fields such as medical aesthetics and biomedicine.

[0024] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a flowchart of the raw material pretreatment and low-temperature degreasing process of the present invention; Figure 2 This is a flowchart of the membrane separation purification and gentle depolymerization process of the present invention; Figure 3 This is a flowchart of the ultrasonic synergistic complex enzyme targeted hydrolysis process of the present invention; Figure 4 This is a flowchart of the porous media enhanced vacuum freeze-drying process of the present invention. Detailed Implementation

[0027] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0028] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0029] Example 1 This embodiment demonstrates in detail the entire process of preparing the white jade snail glycosaminoglycan-growth factor composite active ingredient under optimal parameter conditions.

[0030] Raw material pretreatment and low-temperature defatting: 15 kg of healthy, live white jade snails (80g each) were selected and placed in a clean room for a 3-day fasting and resting period. The resting environment was strictly controlled as follows: ambient temperature 19℃, relative humidity 63%, ventilation rate maintained at 0.8 m³ / h, and rinsed twice daily at 10:00 AM and 4:00 PM with sterile saline. The shells, soft tissue (7.5 kg), digestive glands (4.5 kg), and mucus (3 kg) were aseptically separated and mixed at a mass ratio of soft tissue:digestive glands:mucus = 5:3:2. 120 L of acetone solution pre-cooled to -10℃ was added to the mixture, and a stirrer was turned on (120 rpm) for defatting for 30 min, during which the reaction system temperature was strictly controlled using an ice-water bath. Subsequently, the mixture was centrifuged at 4℃ and 10,000 rpm for 20 min, and the lower precipitate was accurately collected. This defatting-centrifugation step was repeated twice to thoroughly remove lipids. Finally, the precipitate was redissolved in 60L of sterile water at 4℃, and the pH of the system was adjusted to 6.8 with dilute hydrochloric acid to obtain a homogeneous raw material suspension.

[0031] Ultrasonic-assisted directional hydrolysis with a compound enzyme: The raw material suspension was transferred to a low-temperature ultrasonic reactor, and ultrasound at a frequency of 20kHz and a power of 250W was applied. A probe-type ultrasonic instrument was used to achieve a power density of 0.5W / cm². The ultrasonic mode was set to 5s on / 5s off, utilizing the ultrasonic cavitation effect to assist mass transfer. The system temperature was maintained at 42℃ via jacketed circulating water. 270g of a compound enzyme preparation was added. This preparation was precisely formulated from papain with an enzyme activity ≥1500U / mg and neutral protease with an enzyme activity ≥1800U / mg at a mass ratio of 1:0.8, resulting in a total enzyme activity of 1700U / mg. The solid-liquid ratio was adjusted to 1:7 (g / mL), and hydrolysis was carried out at a constant temperature for 5.5h under these conditions. During hydrolysis, a mechanical stirrer was activated every 40min, stirring at a rate of 80r / min for 8min to ensure uniform heating and enzymatic hydrolysis. Once the hydrolysis endpoint is reached, the material is immediately transferred to an 85°C constant temperature water bath to inactivate the enzyme for 10 minutes, thus terminating the reaction and obtaining the hydrolysate.

[0032] Gradient ion exchange chromatography purification: The hydrolysate was centrifuged at 4℃ and 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter to remove impurities and used as the loading solution. The filtrate was first loaded onto a DEAE-Sepharose Fast Flow column (column specifications: 5 cm diameter, 75 cm height) pre-equilibrated with 0.05 M Tris-HCl buffer at pH 7.2. The elution flow rate was set to 1.5 mL / min, and gradient elution was performed sequentially with 15 L each of distilled water, 0.2 M, and 0.5 M sodium chloride aqueous solution (approximately 3 column volumes). The glycosaminoglycan content of the eluent was monitored in real time using the phenol-sulfuric acid colorimetric method, and the liquid in the 0.5 M NaCl elution peak was accurately collected. Subsequently, this collected solution was directly loaded onto a second Q Sepharose Fast Flow column, eluted with 15 L of 0.8 M sodium chloride aqueous solution, and the target eluent was collected to obtain the crude pure solution.

[0033] Membrane separation purification and gentle depolymerization: The crude pure product solution was pumped into a tangential flow ultrafiltration system. An ultrafiltration membrane with a molecular weight cutoff of 1000 Da was used. Ultrafiltration concentration and desalination were performed under conditions of a transmembrane pressure of 0.35 MPa and a feed temperature of 5°C, with temperature fluctuations strictly controlled to not exceed ±1°C. The permeate was collected. Ascorbic acid (Vc) to a final concentration of 11 mM was added to the permeate, and the mixture was magnetically stirred until completely dissolved. Then, 2% hydrogen peroxide (by volume) was added. ) solution, in which The volume ratio of vitamin C to vitamin C was strictly controlled at 1:3. The mixture was placed in a 37°C constant temperature water bath shaker and reacted in the dark for 3 hours to complete the gentle depolymerization of glycosaminoglycans, resulting in a depolymerized composite active liquid.

[0034] Porous Media Enhanced Vacuum Freeze-Drying: The composite active liquid and mannitol-maltodextrin composite carrier (mass ratio 1:1) were mixed uniformly under sterile conditions at a mass ratio of 10:1. The mixture was evenly spread in stainless steel freeze-drying trays, with the material layer thickness controlled at approximately 3 mm. The trays were placed in a vacuum freeze dryer and pre-frozen at -55℃ for 7 hours to ensure complete freezing. The vacuum was then adjusted to 4 Pa, and a specially formulated gradient porous media (70% porosity, 200 μm pore size) was added between the trays to enhance heat and mass transfer. Programmed Temperature Rise: The temperature was first raised to -15℃ for sublimation drying for 14 hours, followed by desorption drying at 20℃ for 9 hours. After drying, the material was removed and pulverized to a particle size of approximately 4 μm, yielding 860 g of the final product: white snail glycosaminoglycan-growth factor composite active ingredient.

[0035] The performance test data of the finished product in Example 1 are shown in Table 1 below: Finished product purity 99.8 % ≥99.6% higher than the requirements of the claims. Active retention rate 98.8 % ≥98.5% higher than the requirements of the claims. glycosaminoglycan content 38.5 mg / g ≥35mg / g higher than the claim requirement growth factor activity 1320 IU / mg ≥1200 IU / mg higher than the claim requirement DPPH free radical scavenging rate 68.3 % ≥65% higher than the requirements of the claims Cell proliferation promotion rate 75.6 % Promoting effect on skin fibroblasts Skin barrier repair rate 62.5 % Volunteers used the data 14 days later Inflammation resolution rate 58.7 % Volunteers used the data 14 days later Sensitization reaction none / Meets high-end medical aesthetic standards Example 2 This embodiment aims to verify that, under the lower limit of the parameters defined in the claims, the process of the present invention can still stably produce qualified products.

[0036] Raw material pretreatment and low-temperature defatting: Select adult white jade snails with a lower weight limit of 70g and fast them for 3 days as described above. Mix the raw materials according to the ratio of soft tissue: digestive gland: mucus = 5:3:2, add 8 times the volume of -10℃ acetone, strictly perform 3 defatting cycles, and after resolubilization of the precipitate, adjust the pH to 6.5 with sterile water.

[0037] Ultrasonic-assisted directional hydrolysis of compound enzyme: apply 20kHz, 250W ultrasound, control the temperature at 40℃ (lower limit), set the amount of compound enzyme added to 1.2% (lower limit), control the solid-liquid ratio at 1:6 (g / mL) (lower limit), and the hydrolysis time to 5h (lower limit).

[0038] Gradient ion exchange chromatography purification: The elution flow rate was controlled at 1.5 mL / min, and gradient elution was performed sequentially with 0.5 M and 0.8 M sodium chloride solutions. The remaining operations were the same as in Example 1.

[0039] Membrane separation purification and gentle depolymerization: Ultrafiltration pressure was set at 0.3 MPa (lower limit), ascorbic acid concentration at 11 mM, and reaction time at 3 h. The volume ratio of vitamin C to vitamin C is 1:3.

[0040] Porous media enhanced vacuum freeze drying: pre-freezing time 7h, vacuum degree controlled at 5Pa (upper limit), sublimation drying 14h, desorption drying time set to 8h, finished product thickness control meets requirements.

[0041] The performance test data of the finished product in Example 2 are shown in Table 2 below: Finished product purity 99.6 % Yes (equal to the lower limit) Active retention rate 98.5 % Yes (equal to the lower limit) glycosaminoglycan content 35.0 mg / g Yes (equal to the lower limit) growth factor activity 1200 IU / mg Yes (equal to the lower limit) DPPH free radical scavenging rate 65.0 % Yes (equal to the lower limit) Sensitization reaction none / yes Example 3 This embodiment demonstrates the operating status and effect when the various process parameters are taken at intermediate values, further proving the wide adaptability and robustness of the process of the present invention.

[0042] Raw material pretreatment and low-temperature defatting: Healthy, fresh white jade snails weighing 90g were selected and kept in a state of fasting and quiet rest for 3 days (ambient temperature 20℃, relative humidity 65%). The tissues were aseptically separated and mixed in a ratio of 5:3:2. Eight times the volume of acetone at -10℃ was added, and defatting was performed three times. The precipitate was reconstituted and the pH was adjusted to 7.0.

[0043] Ultrasonic-assisted directional hydrolysis of compound enzyme: apply 20kHz, 250W ultrasound, control the temperature at 45℃ (upper limit), add 1.8% compound enzyme (upper limit), solid-liquid ratio 1:8 (g / mL) (upper limit), and hydrolyze for 6h (upper limit).

[0044] Gradient ion exchange chromatography purification: DEAE-Sepharose Fast Flow and Q Sepharose Fast Flow dual-column tandem system were used for gradient elution with 0.2M, 0.5M and 0.8M NaCl at a flow rate of 1.5 mL / min, and the elution peaks were monitored.

[0045] Membrane separation purification and gentle depolymerization: Ultrafiltration was carried out at 0.4 MPa (upper limit) and 6℃ (upper limit), and H2O2-Vc system (volume ratio 1:3) was added and reacted at 37℃ for 3h.

[0046] Vacuum freeze-drying enhanced by porous media: Add a gradient porous media with a porosity of 80% (upper limit) and a pore size of 500μm (upper limit), vacuum degree ≤5Pa, pre-freeze at -55℃ for 7h, sublime dry for 14h, and desorption dry for 9h.

[0047] The performance test data of the finished product in Example 3 are shown in Table 3 below: Finished product purity 99.7 % Excellent, close to optimal value Active retention rate 98.6 % Excellent, structural integrity preserved glycosaminoglycan content 36.8 mg / g Good, at an intermediate level growth factor activity 1280 IU / mg Good, high biological activity DPPH free radical scavenging rate 66.5 % Good, strong antioxidant capacity Skin fibroblast proliferation promotion rate 73.2 % Significantly promotes cell proliferation As can be seen from the above three embodiments and their corresponding data tables, regardless of whether the optimal parameters, lower limit parameters, or intermediate value parameters are used, the disclosed process can stably prepare high-purity and high-activity white snail glycosaminoglycan-growth factor composite active ingredients, which fully demonstrates the feasibility and industrialization potential of the process.

[0048] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A targeted extraction and purification process for the active compound of white jade snail glycosaminoglycan-growth factor complex, characterized in that, Includes the following steps: Step S1, Raw Material Pretreatment and Low-Temperature Degreasing: Select healthy, live white jade snails weighing 70-90g and subject them to a 3-day fasting and quiet rearing period. The quiet rearing environment should be 18-20℃, relative humidity 60%-65%, and ventilation rate 0.8m³ / h. Rinse twice daily with sterile saline. Aseptically separate the shell, soft tissue, digestive glands, and mucus. Mix them at a mass ratio of soft tissue:digestive glands:mucus = 5:3:2, add 8 times the volume of -10℃ acetone, stir and defatted for 30 minutes, then centrifuge at 4℃ and 10000rpm for 20 minutes and collect the precipitate. Repeat the above defatting operation twice. Redissolve the final precipitate with sterile water at 4℃ and adjust the pH of the system to 6.5-7.0 to obtain the raw material suspension. Step S2, Ultrasonic-Coordinated Targeted Hydrolysis with Compound Enzyme: The raw material suspension is transferred to a low-temperature ultrasonic reactor, and ultrasound with a frequency of 20kHz and a power of 250W is applied. The ultrasonic mode is set to 5s working / 5s intermittent, with a power density of 0.5W / cm², and the system temperature is controlled at 40~45℃. A compound enzyme preparation is added, which consists of papain with an enzyme activity ≥1500U / mg and neutral protease with an enzyme activity ≥1800U / mg in a mass ratio of 1:0.

8. The amount of enzyme added is 1.2%~1.8% of the total weight of the raw materials, and the solid-liquid ratio of the hydrolysis system is controlled at 1:6~1:8 (g / mL). Hydrolysis is carried out at a constant temperature for 5~6 hours, with stirring at a rate of 80r / min for 8 minutes every 40 minutes. After hydrolysis, the enzyme is inactivated in an 85℃ water bath for 10 minutes to obtain the hydrolysate. Step S3, Gradient ion exchange chromatography purification: The hydrolysate was centrifuged at 4℃ and 12000rpm for 30min. The supernatant was filtered through a 0.22μm filter membrane and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated with 0.05M Tris-HCl buffer at pH 7.

2. The column diameter to column height ratio was 1:15, and the sample volume was 1 / 10 of the column volume. Elution was performed sequentially with distilled water, 0.2M, and 0.5M sodium chloride aqueous solution at a flow rate of 1.5mL / min, each with a column volume of 3. The elution peaks were monitored using the phenol-sulfuric acid colorimetric method, and the 0.5M eluent was collected. The collected solution was then loaded onto a Q Sepharose Fast Flow column and eluted with 0.8M sodium chloride aqueous solution at a volume of 3. The eluent was collected to obtain the crude pure solution. Step S4, Membrane separation purification and gentle depolymerization: The crude pure product solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da at a pressure of 0.3~0.4 MPa and a temperature of 4~6℃. The temperature fluctuation of the feed solution is controlled to not exceed ±1℃. The permeate is collected. 11 mM ascorbic acid is added to the permeate. After it is fully dissolved, a 2% hydrogen peroxide solution is added, wherein the volume ratio of hydrogen peroxide to ascorbic acid is 1:

3. The reaction is carried out at a constant temperature of 37℃ for 3 hours to obtain the depolymerized composite active solution. Step S5, Vacuum freeze-drying enhanced by porous media: The composite active liquid and mannitol-maltodextrin composite carrier are mixed evenly at a mass ratio of 10:1, wherein the mass ratio of mannitol to maltodextrin in the composite carrier is 1:

1. The mixture is spread on a freeze-drying tray with a thickness ≤4mm and placed in a vacuum freeze dryer; first, it is pre-frozen at -55℃ for 7h, the vacuum degree is adjusted to ≤5Pa, and a gradient porous medium is added, wherein the porosity of the gradient porous medium is 60%~80% and the pore size is 50~500μm; The product is dried by sublimation at -15℃ for 14 hours, and then dried by desorption at 20℃ for 9 hours to obtain the finished product of white snail glycosaminoglycan-growth factor complex active ingredient.

2. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 1, characterized in that: During the fasting and rest period described in step S1, the environment is kept well-ventilated at a rate of 0.8 m³ / h. During the defatting process, an ice-water bath is used to control the temperature to ensure that the local temperature does not rise and to prevent the active ingredients from being deactivated by heat.

3. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 1, characterized in that: The total enzyme activity of the compound enzyme preparation described in step S2 is ≥1650U / mg. The ultrasonic action targets and hydrolyzes the protein matrix encapsulating the active ingredients through cavitation effect, avoiding damage to the glycosaminoglycan sulfate groups and the active centers of growth factors.

4. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 1, characterized in that: The DEAE-Sepharose Fast Flow chromatography column and the Q Sepharose Fast Flow chromatography column described in step S3 constitute a dual-column tandem system. The gradient salt solution elution achieves precise separation of impurities and target active substances, with a 100% removal rate of sensitizing impurities.

5. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 1, characterized in that: The ultrafiltration process described in step S4 is carried out at a low temperature of 4-6°C, using hydrogen peroxide and ascorbic acid. The -Vc system is used to gently control the molecular weight of glycosaminoglycans to 3.8~23.8kDa, and no additional catalyst removal is required after the reaction is complete.

6. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 1, characterized in that: The gradient porous medium described in step S5 has a porosity of 70% and a pore size of 200 μm; During the freeze-drying process, the condenser temperature is ≤-60℃, the resulting product has a water content of ≤2.5% and a solubility of ≥99.8%.

7. The targeted extraction and purification process of the active ingredient of white jade snail glycosaminoglycan-growth factor complex according to any one of claims 1 to 6, characterized in that: The composite active ingredient has a purity of ≥99.6%, an activity retention rate of ≥98.5%, a glycosaminoglycan content of ≥35mg / g, a growth factor activity of ≥1200IU / mg, a DPPH free radical scavenging rate of ≥65%, and is free from allergenic impurities, organic solvent residues, and heavy metal contamination.

8. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 7, characterized in that: It exhibits a loose and porous structure under scanning electron microscopy, without clumping, and its activity decreases by ≤5% after 12 months of storage at room temperature.

9. The targeted extraction and purification process of the active ingredient of white jade snail glycosaminoglycan-growth factor complex according to claim 7 or 8, characterized in that: The composite active ingredient is added at a mass percentage of 5% to 15% to medical aesthetic repair dressings, anti-aging creams, or wound healing gels for skin barrier repair, anti-inflammatory and antioxidant effects, and tissue repair.

10. The targeted extraction and purification process of the white jade snail glycosaminoglycan-growth factor complex active ingredient according to claim 9, characterized in that: When added to medical aesthetic repair dressings, after 14 days of use, the skin barrier repair rate can reach 62.5%, the inflammation reduction rate can reach 58.7%, and there is no allergic reaction.

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