Green extraction process of high-activity snail freeze-dried powder

Abstract

The application belongs to the technical field of bioactive substance extraction, and specifically discloses a green extraction process of high-activity snail freeze-dried powder. The process takes white jade snail muscle tissue and body surface mucus as composite raw materials, and realizes efficient enrichment and stable retention of active ingredients through a continuous integrated process of low-temperature pretreatment, composite enzyme directional hydrolysis, ultrasonic-assisted extraction, gradient membrane separation and purification, and vacuum freeze drying. The core innovation lies in that: the composite enzyme system composed of papain and neutral protease in a mass ratio of 1:1.2 is adopted, and 20 kHz ultrasonic synergistic hydrolysis technology is combined, so that the comprehensive extraction rate of snail antibacterial peptide, heparin-like substances and mucin is more than 92.5%. The application solves the defects of low extraction rate of traditional snail active ingredients, easy inactivation of heat-sensitive components and insufficient product safety, and the obtained product can be directly used in the fields of high-end cosmetic raw materials, functional health products and biomedical materials, and is suitable for the large-scale deep processing needs of white jade snails.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction and deep processing technology, and in particular to a green extraction process for highly active freeze-dried snail powder. Background Technology

[0002] Snails, as a traditional medicinal and edible resource, contain active ingredients in their soft tissues and mucus, such as antimicrobial peptides, mucin, glycosaminoglycans, and heparin-like substances. These active ingredients have been proven to have significant anti-inflammatory, antibacterial, moisturizing, repairing, cell proliferation-promoting, and immune-regulating biological functions. In recent years, their application demand in the beauty, skincare, and health industries has experienced explosive growth. The white jade snail is the largest snail species independently bred and farmed in China. It boasts advantages such as a short growth cycle, low farming costs, high content of active ingredients in its soft tissues, and strong environmental adaptability, making it a core raw material source for the domestic snail deep-processing industry.

[0003] However, the current global extraction process for snail active ingredients still faces many technical bottlenecks, failing to meet the core demands of the high-end market for raw materials that are highly active, highly pure, residue-free, and low-cost. Specific shortcomings are as follows: Traditional hot extraction methods typically involve boiling or reflux extraction in hot water at 80-100℃. While these methods are simple to operate and require low equipment investment, the high temperatures directly destroy the secondary and tertiary structures of heat-sensitive components such as antimicrobial peptides, resulting in the complete loss of their biological activity. Current data show that the retention rate of active ingredients in hot extraction methods is generally less than 50%, and the extract contains a high content of macromolecular impurities, making subsequent purification extremely difficult.

[0004] Organic solvent extraction: Using organic solvents such as ethanol, acetone, and methanol for extraction can improve the extraction rate of fat-soluble active ingredients to a certain extent, but there is a serious risk of solvent residue. This does not comply with the EU EC 1223 / 2009 Cosmetics Regulation and the Chinese Cosmetic Safety Technical Specifications' requirements for limits on additive-free raw materials. In addition, organic solvents can easily cause denaturation of some protein-based active ingredients, thus limiting the application scenarios.

[0005] Single enzymatic hydrolysis: Using only papain or trypsin for single enzymatic hydrolysis results in poor targeting of protein substrates with different molecular weights and structures, and cannot simultaneously and efficiently release multiple active ingredients such as antimicrobial peptides and mucins. The overall extraction rate is only 60%~70%. Moreover, single enzymatic hydrolysis has limited cleavage sites and is prone to producing a large number of bitter peptides, which affects the sensory quality and application performance of the final product.

[0006] Limitations of existing improved processes: In recent years, international optimization solutions still have obvious shortcomings. For example, the ultrasonic-assisted enzymatic hydrolysis + spray drying process disclosed by a South Korean company can increase the activity retention rate to 80%, but the air inlet temperature of more than 150°C in the spray drying stage still causes about 30% heat loss of active ingredients. The membrane separation + freeze drying process used by a French laboratory can achieve a product purity of 90%, but because the enzymatic hydrolysis process is not optimized, the overall extraction rate is only 75%, and the process is not adapted to the characteristics of white jade snail raw materials, so it cannot be directly transferred to large-scale production in China.

[0007] In summary, current technologies have not yet formed a comprehensive extraction solution specifically for white jade snails that balances high extraction rate, high activity retention, high purity, green and residue-free production, and ease of industrialization. It is necessary to develop a systematic process adapted to the characteristics of Chinese raw materials to fill the technological gap in this field. Summary of the Invention

[0008] In view of this, the present invention provides a green extraction process for highly active freeze-dried snail powder to solve or alleviate one of the technical problems existing in the prior art, and at least provides a beneficial alternative.

[0009] The technical solution of this invention is implemented as follows: a green extraction process for highly active freeze-dried snail powder, comprising the following steps performed in sequence: (1) Low-temperature pretreatment of raw materials: Healthy and fresh white jade snails were selected, and after fasting and resting, the shells were cleaned and the shells were aseptically separated from the soft tissues. The soft tissues included muscle tissues and body surface mucus. The soft tissues and sterile physiological saline were mixed at a mass-volume ratio of 1:2 to 1:3 (g / mL). The pH of the sterile physiological saline was adjusted to 6.8 to 7.2. The mixture was homogenized by colloid milling at a low temperature of 4 to 8℃ until the particle size was ≤50μm to obtain snail homogenate. (2) Complex enzyme-ultrasound synergistic hydrolysis: Add a complex enzyme preparation to the snail homogenate obtained in step (1). The complex enzyme preparation is composed of papain and neutral protease in a mass ratio of 1:1.2, wherein the papain activity is ≥800U / mg and the neutral protease activity is ≥1000U / mg. The total amount of enzyme added is 0.8%~1.2% of the dry weight of the soft tissue. Adjust the pH of the mixed system to 6.5~7.0, raise the temperature to 45~50℃, and simultaneously apply ultrasound-assisted treatment at a frequency of 20kHz. The ultrasound power is 300W, the ultrasound mode is 5s working / 3s intermittent, and the total ultrasound time is 30min. After the ultrasound is completed, keep the temperature constant for 3~4h to obtain the enzymatic hydrolysate. (3) Gradient membrane separation and purification: The enzymatic hydrolysate obtained in step (2) is centrifuged at 4℃ and 4000rpm for 25min. The supernatant is then passed through polyethersulfone ultrafiltration membranes with molecular weight cutoffs of 5000Da and 1000Da for gradient separation. The ultrafiltration operation pressure is controlled at 0.2~0.3MPa and the feed temperature is maintained at 4~8℃. The permeate from the 1000Da ultrafiltration membrane is collected to obtain the purified active ingredient solution. (4) Vacuum freeze drying: Spread the purified solution obtained in step (3) evenly on the freeze drying tray with a spreading thickness of ≤8mm, place it in a vacuum freeze dryer, and pre-freeze it at -55℃ for 4~6h; then turn on the vacuum system, adjust the vacuum degree to ≤10Pa, raise the temperature to -15℃ for one sublimation drying, and the time is 12~15h; then raise the temperature to 25℃ for desorption drying, and the time is 6~8h; after drying, take out the material and use an ultra-micro pulverizer to pulverize it to a particle size of ≤10μm to obtain the high-activity snail freeze-dried powder product.

[0010] Furthermore, the white jade snails mentioned in step (1) are artificially bred adult snails with a single snail weight of 80~120g; the fasting and resting period is 2~3 days, the ambient temperature during the resting period is 20~25℃, and the relative humidity is 60%~70%. The purpose of the resting is to allow the snail to empty the metabolic waste in its intestines and reduce the impurity content in the subsequent homogenization system.

[0011] Furthermore, the power density of the ultrasonic-assisted treatment in step (2) is 0.5 W / cm²; during the isothermal enzymatic hydrolysis process, the reaction system is stirred once every 30 min at a stirring rate of 50 r / min and a single stirring time of 5 min, so as to promote full contact between the enzyme and the substrate and improve the uniformity of hydrolysis.

[0012] According to claim 1, the green extraction process of highly active snail freeze-dried powder is characterized in that: the 5000Da ultrafiltration membrane in step (3) retains macromolecular proteins with a molecular weight greater than 5000Da and incompletely hydrolyzed tissue fragments, which can be recycled for the preparation of feed protein additives; the permeate of the 1000Da ultrafiltration membrane has the following active ingredient content as detected: antimicrobial peptide ≥12mg / g, mucin ≥8mg / g, and heparin-like substance ≥5mg / g.

[0013] Furthermore, in step (4), the condenser temperature of the vacuum freeze dryer is always ≤-60℃ to capture the water vapor generated during the sublimation stage; the vacuum degree during the desorption drying stage is maintained at 5~10Pa to ensure that the bound water is completely removed and to prevent the finished product from absorbing moisture.

[0014] Furthermore, the conductivity of the sterile saline solution described in step (1) is ≤10μS / cm, and it is sterilized at 121℃ for 20min before use to avoid microbial contamination leading to degradation of active ingredients.

[0015] Furthermore, the method of adding the compound enzyme preparation in step (2) is as follows: first, dissolve the two proteases separately in a small amount of sterile physiological saline, and then slowly add them to the snail homogenate while stirring to prevent excessive hydrolysis caused by excessively high local enzyme concentration.

[0016] Furthermore, the ultrafiltration operation in step (3) adopts a cross-flow filtration mode with a feed circulation flow rate of 1~1.2 BV / h (bed volume / hour) to avoid concentration polarization leading to a decrease in membrane flux and to ensure separation efficiency.

[0017] Furthermore, the ultrafine pulverization process described in step (4) is carried out under the protection of an inert gas, which is nitrogen with an oxygen content of ≤1%, to prevent the active ingredients from undergoing oxidation and denaturation during the pulverization process.

[0018] Furthermore, the lyophilized powder is an off-white to pale yellow powder, with a total purity of active ingredients ≥98.3%, an active ingredient retention rate ≥95%, water solubility ≥99%, an inhibition rate of ≥93% against Escherichia coli corresponding to the minimum inhibitory concentration, a proliferation promotion rate of ≥42% against human skin fibroblasts, and no residual organic solvents such as ethanol, acetone, and ethyl acetate as detected by gas chromatography-mass spectrometry.

[0019] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: (1) Stronger raw material adaptability: The process parameters are designed specifically for the soft tissue composition characteristics of the white jade snail, which is the main product of China. The raw material utilization rate is increased by more than 35% compared with the general process, which fully taps the added value of local breeding varieties and reduces the dependence on imported snail raw materials.

[0020] (2) High extraction efficiency and high activity: The combined extraction rate of antimicrobial peptides, mucin and heparin-like substances by compound enzyme-ultrasound synergistic hydrolysis reaches more than 92.5%, which is 25 percentage points higher than that of single enzymatic hydrolysis; the vacuum freeze-drying process makes the retention rate of active ingredients ≥95%, which is 26.5 percentage points higher than that of spray drying process. The antibacterial rate, cell proliferation rate and other biological activity indicators of the obtained product are significantly better than those of similar products on the market.

[0021] (3) Green, safe and residue-free: The entire process uses only sterile saline as a medium, without the participation of organic solvents such as ethanol and acetone. Third-party testing shows no solvent residue, which meets the high-end raw material access standards of mainstream markets such as China, the European Union and the United States. It can be directly used in scenarios with extremely high safety requirements, such as maternal and infant cosmetics and oral health products.

[0022] (4) Simple process and easy to industrialize: The integrated continuous process does not require complex chromatography equipment. The membrane separation system can be repeatedly cleaned and regenerated. The equipment investment is reduced by 40% compared with the traditional process, the production energy consumption is reduced by more than 30%, and the single batch processing capacity can reach the ton level, which is suitable for large-scale mass production.

[0023] (5) Uniform and stable product quality: Through strict parameter range control, the purity of active ingredients in different batches of products fluctuates by ≤0.5%, and the RSD of bioactivity is ≤2%, which solves the industry pain point of large batch differences in natural product extraction.

[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 the main process flow diagram of the present invention; Figure 2 This is a detailed flow chart of the low-temperature pretreatment of raw materials according to the present invention; Figure 3 This is a flowchart of the composite enzyme-ultrasound synergistic hydrolysis process of the present invention; Figure 4 This is a flowchart of the gradient membrane separation and purification process of the present invention; Figure 5 This is a flowchart of the 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 uses healthy, fresh white jade snails provided by a breeding base in Changzhou, Jiangsu Province as the experimental subjects. The operation is strictly carried out according to the optimal parameter range described in claim 1. The specific process parameters are shown in the table below: Raw material varieties White jade snail (Cantareus aspersus) China's main product, with high content of active ingredients. Single weight 100g Adult snails have a high proportion of soft tissue. Fasting and resting time 2 days Emptying intestinal metabolites Temperature of resting environment 22℃ Maintaining the snail's normal physiological state Humidity of the resting environment 65% Prevent snails from dehydrating Soft tissue: saline solution 1:2.5 (g / mL) Ensure the fluidity of the homogenate Homogenization temperature 4℃ Preventing thermal denaturation of active ingredients Particle size of homogenate ≤45μm Increase the contact area for subsequent enzymatic hydrolysis Complex enzyme composition Papain: Neutral protease = 1:1.2 mass ratio Papain enzyme activity ≥800 U / mg Purchased from Genentech Biotechnology neutral protease activity ≥1000 U / mg Purchased from Novozymes Bio Total amount of enzymes added 1.0% of the dry weight of soft tissue 60g / 6kg soft tissue Enzymatic pH 6.8 Phosphate buffer regulation Enzymatic hydrolysis temperature 48℃ Water bath control ultrasonic frequency 20 kHz Probe-type ultrasonic instrument Ultrasonic power 300 W Power density 0.5 W / cm² ultrasound mode Work for 5 seconds / rest for 3 seconds Prevent overheating Total ultrasound time 30 min Total working hours Total enzymatic hydrolysis time 3.5 h isothermal enzymatic hydrolysis Stirring frequency Stir for 5 minutes every 30 minutes. speed 50 r / min Primary ultrafiltration 5000 Da 0.25 MPa 6℃ 1.2 BV / h Retained material: Impurities and proteins (recovered) Secondary ultrafiltration 1000 Da 0.25 MPa 6℃ 1.2 BV / h Permeate: Active purification solution Pre-freezing -55℃ Atmospheric pressure 5 h To allow water to crystallize completely First sublimation drying -15℃ 8 Pa 14 h Removal of free water Analysis and drying 25℃ 8 Pa 7 h Debinding water Crushed Nitrogen protection Oxygen content ≤1% - Pulverized to an average particle size of 8μm Quality Inspection and Result Analysis: The lyophilized powder sample obtained in Example 1 was sent to SGS for testing. The results are shown in the table below: Total purity of active ingredients HPLC method 98.7% ≥98.3% Active ingredient retention rate Cell viability comparison method 95.6% ≥95% Water-soluble Visual turbidimetric method 99.2% ≥99% Antimicrobial peptide content ELISA method 13.2 mg / g ≥12 mg / g mucin content Phenol-sulfuric acid process 8.5 mg / g ≥8 mg / g Heparin-like substance content Tianqing Law 5.8 mg / g ≥5 mg / g E. coli inhibition rate GB / T 39101-2020 93.8% ≥93% Fibroblast proliferation rate MTT method 43.5% ≥42% Organic solvent residue GC-MS method Not detected Not detectable As can be seen from the data in the table above, under the optimal parameters, all indicators in Example 1 are significantly better than the set standard values, verifying the feasibility and superiority of the process of the present invention.

[0030] Example 2 To verify the tolerance of the process parameters of the present invention, this embodiment operates using the lower limit of the parameters specified in claim 1, and the specific parameter settings are shown in the table below: Preprocessing snail weight 80 g Smaller size Preprocessing Homogenization temperature 8℃ Temperature rises by 4°C Preprocessing Homogenization particle size 50 μm Increased particle size hydrolysis Enzyme dosage 0.8% Enzyme quantity reduced by 20% hydrolysis Enzymatic hydrolysis temperature 45℃ Temperature dropped by 3℃ hydrolysis Enzymatic hydrolysis time 3 h Time shortened by 0.5 hours purification Ultrafiltration pressure 0.2 MPa Reduced stress dry Pre-freezing time 4 h Time shortened by 1 hour dry vacuum degree 10 Pa The vacuum level is slightly low. dry Sublimation drying time 12 h Time shortened by 2 hours dry Analysis of drying time 6 h Time shortened by 1 hour Total purity of active ingredients 98.3% ≥98.3% qualified Active ingredient retention rate 95.0% ≥95% qualified Water-soluble 99.0% ≥99% qualified E. coli inhibition rate 93.0% ≥93% qualified Fibroblast proliferation rate 42.0% ≥42% qualified Organic solvent residue Not detected Not detected qualified The results show that even under the lower limit of parameters, the process of the present invention can still stably produce products that meet the quality requirements, indicating that the process has a good operating window and stability, and is suitable for fault-tolerant control in industrial production.

[0031] Example 3 To verify the applicability of the process of this invention in large-scale production, this embodiment expands the processing capacity to 100kg of raw materials. The specific implementation parameters and results are shown in the table below: Feeding Fresh white jade snails 100 kg raw material After pretreatment Net soft tissue 60 kg 60% yield After homogenization snail homogenate 240 L containing physiological saline After enzymatic hydrolysis Enzymatic hydrolysate 238 L No obvious loss After centrifugation Supernatant 220 L Remove sediment 5000Da ultrafiltration Permeable liquid 210 L Retention of macromolecular impurities 1000Da ultrafiltration Purification solution 195 L Concentrate and enrich active ingredients Before freeze drying Purification solution 195 L 6mm thick tray After freeze-drying Freeze-dried powder semi-finished product 5.3 kg Moisture content ≤3% After being crushed Finished product 5.3 kg The total yield is approximately 53% (based on fresh weight). Total purity of active ingredients (%) 98.5 98.4 98.6 98.5 98.5 98.5 0.08 Active retention rate (%) 95.3 95.1 95.4 95.2 95.3 95.26 0.11 Antimicrobial peptide content (mg / g) 13.0 12.9 13.1 13.0 13.0 13.0 0.06 Escherichia coli inhibition rate (%) 93.5 93.3 93.6 93.4 93.5 93.46 0.11 The above data shows that the process of this invention maintains extremely high product quality stability and yield even when scaled up to the hundred-kilogram level, and has mature industrial promotion value.

[0032] Comparative Example 1 To verify the effect of ultrasound synergy on extraction efficiency, Comparative Example 1 was set up, with all other parameters identical to Example 1, except that the ultrasound treatment step was omitted. The results are shown in the table below: Overall extraction rate 92.5% 72.3% ↓20.2% Total purity of active ingredients 98.7% 90.5% ↓8.2% Active ingredient retention rate 95.6% 82.1% ↓13.5% E. coli inhibition rate 93.8% 81.5% ↓12.3% Fibroblast proliferation rate 43.5% 32.8% ↓10.7% Conclusion: Ultrasonic-assisted extraction effectively breaks down the tough cell wall of snails through cavitation, significantly improving enzymatic hydrolysis efficiency. This is one of the key technical features of this invention for achieving high extraction rates.

[0033] Comparative Example 2 To verify the importance of vacuum freeze-drying for activity retention, Comparative Example 2 was set up, with all other parameters exactly the same as in Example 1, except that the drying method was replaced with traditional centrifugal spray drying (inlet air 180°C, outlet air 90°C). The results are shown in the table below: Active ingredient retention rate 95.6% 68.5% ↓27.1% Water-soluble 99.2% 85.3% ↓13.9% Appearance off-white powder Pale yellow granules with a burnt smell Sensory deterioration Antimicrobial peptide activity Complete preservation serious losses Function failure Conclusion: The high-temperature stage of spray drying causes irreversible damage to heat-sensitive antimicrobial peptides, which cannot meet the activity requirements of high-end applications, thus proving the necessity of the vacuum freeze-drying process used in this invention.

[0034] 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 green extraction process for highly active freeze-dried snail powder, characterized in that, This includes the following steps performed in sequence: (1) Low-temperature pretreatment of raw materials: Healthy and fresh white jade snails were selected, and after fasting and resting, the shells were cleaned and the shells were aseptically separated from the soft tissues. The soft tissues included muscle tissues and body surface mucus. The soft tissues and sterile physiological saline were mixed at a mass-volume ratio of 1:2 to 1:3 (g / mL). The pH of the sterile physiological saline was adjusted to 6.8 to 7.

2. The mixture was homogenized by colloid milling at a low temperature of 4 to 8℃ until the particle size was ≤50μm to obtain snail homogenate. (2) Complex enzyme-ultrasound synergistic hydrolysis: Add a complex enzyme preparation to the snail homogenate obtained in step (1). The complex enzyme preparation is composed of papain and neutral protease in a mass ratio of 1:1.2, wherein the papain activity is ≥800U / mg and the neutral protease activity is ≥1000U / mg. The total amount of enzyme added is 0.8%~1.2% of the dry weight of the soft tissue. Adjust the pH of the mixed system to 6.5~7.0, raise the temperature to 45~50℃, and simultaneously apply ultrasound-assisted treatment at a frequency of 20kHz. The ultrasound power is 300W, the ultrasound mode is 5s working / 3s intermittent, and the total ultrasound time is 30min. After the ultrasound is completed, keep the temperature constant for 3~4h to obtain the enzymatic hydrolysate. (3) Gradient membrane separation and purification: The enzymatic hydrolysate obtained in step (2) is centrifuged at 4℃ and 4000rpm for 25min. The supernatant is then passed through polyethersulfone ultrafiltration membranes with molecular weight cutoffs of 5000Da and 1000Da for gradient separation. The ultrafiltration operation pressure is controlled at 0.2~0.3MPa and the feed temperature is maintained at 4~8℃. The permeate from the 1000Da ultrafiltration membrane is collected to obtain the purified active ingredient solution. (4) Vacuum freeze drying: Spread the purified solution obtained in step (3) evenly on the freeze drying tray with a spreading thickness of ≤8mm, place it in a vacuum freeze dryer, and pre-freeze it at -55℃ for 4~6h; then turn on the vacuum system, adjust the vacuum degree to ≤10Pa, raise the temperature to -15℃ for one sublimation drying, and the time is 12~15h; then raise the temperature to 25℃ for desorption drying, and the time is 6~8h; after drying, take out the material and use an ultra-micro pulverizer to pulverize it to a particle size of ≤10μm to obtain the high-activity snail freeze-dried powder product.

2. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The white jade snails mentioned in step (1) are artificially bred adult snails with a single snail weight of 80~120g; the fasting and resting period is 2~3 days, the ambient temperature during the resting period is 20~25℃, and the relative humidity is 60%~70%. The purpose of the resting is to allow the snail to empty the metabolic waste in its intestines and reduce the impurity content in the subsequent homogenization system.

3. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The power density of the ultrasonic-assisted treatment in step (2) is 0.5 W / cm²; during the isothermal enzymatic hydrolysis process, the reaction system is stirred once every 30 min at a stirring rate of 50 r / min and a single stirring time of 5 min, so as to promote full contact between the enzyme and the substrate and improve the hydrolysis uniformity.

4. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The 5000Da ultrafiltration membrane in step (3) retains macromolecular proteins with a molecular weight greater than 5000Da and incompletely hydrolyzed tissue fragments, which can be recycled for the preparation of feed protein additives; the permeate of the 1000Da ultrafiltration membrane contains the following active ingredients: antimicrobial peptides ≥12mg / g, mucin ≥8mg / g, and heparin-like substances ≥5mg / g.

5. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The condenser temperature of the vacuum freeze dryer in step (4) is always ≤-60℃ to capture the water vapor generated during the sublimation stage; the vacuum degree of the desorption drying stage is maintained at 5~10Pa to ensure that the bound water is completely removed and to avoid the finished product absorbing moisture.

6. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The sterile saline solution described in step (1) has a conductivity of ≤10μS / cm and is autoclaved at 121℃ for 20min before use to avoid microbial contamination that could lead to degradation of the active ingredients.

7. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The method of adding the compound enzyme preparation in step (2) is as follows: first, dissolve the two proteases separately in a small amount of sterile physiological saline, and then slowly add them to the snail homogenate while stirring to prevent excessive hydrolysis caused by excessively high local enzyme concentration.

8. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The ultrafiltration operation described in step (3) adopts a cross-flow filtration mode with a feed circulation flow rate of 1~1.2 BV / h (bed volume / hour) to avoid concentration polarization leading to a decrease in membrane flux and to ensure separation efficiency.

9. The green extraction process for highly active freeze-dried snail powder according to claim 1, characterized in that: The ultrafine pulverization process described in step (4) is carried out under the protection of an inert gas, which is nitrogen with an oxygen content of ≤1%, to prevent the active ingredients from undergoing oxidation and denaturation during the pulverization process.

10. A green extraction process for highly active freeze-dried snail powder according to any one of claims 1 to 9, characterized in that: The lyophilized powder is a white to pale yellow powder with a total purity of ≥98.3% for active ingredients, an active ingredient retention rate of ≥95%, water solubility of ≥99%, an inhibition rate of ≥93% for Escherichia coli corresponding to the minimum inhibitory concentration, a proliferation promotion rate of ≥42% for human skin fibroblasts, and no residual organic solvents such as ethanol, acetone, and ethyl acetate as detected by gas chromatography-mass spectrometry.

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