Enzymatic assisted extraction process of high-activity components from deer antler powder

Abstract

The present application relates to the technical field of deep processing of deer products, and discloses an enzyme-assisted high-activity ingredient extraction process of deer horn powder, which comprises the following steps: raw material pretreatment, composite enzymolysis, preparation of extraction liquid, ultrasonic-assisted extraction, separation and primary purification, adsorption purification, concentration and drying; the compact keratinization matrix structure of the deer horn powder is destroyed through defatting and decalcification treatment in the pretreatment step, which creates conditions for the release of subsequent active ingredients; the step of composite enzymolysis adopts step-by-step enzymolysis treatment, and the papain, acid protease, trypsin and cellulase are used for step-by-step enzymolysis in a controllable temperature oscillation reactor, which can hydrolyze the complex protein-polysaccharide composite structure in the deer horn, promote the dissolution of the internal high-value components, the multi-enzyme synergistic effect overcomes the limitations of single enzyme action, and thus the extraction sufficiency of the target ingredients and the breadth of the product spectrum are improved.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology for deer products, specifically to an enzymatically assisted extraction process for highly active components from deer antler powder. Background Technology

[0002] Deer antler powder is an important traditional Chinese medicine, mainly made from the antlers of sika deer or red deer, obtained by crushing and grinding the antlers. Deer antler powder is salty in taste and warm in nature, and enters the spleen and kidney meridians; it has the effects of warming kidney yang, strengthening tendons and bones, promoting blood circulation and reducing swelling.

[0003] Currently, in the field of extracting highly active ingredients from deer antler, conventional techniques mostly rely on traditional physicochemical methods such as water extraction and alcohol extraction. These methods have limited ability to damage the dense keratinized matrix of deer antler, making it difficult to release tightly bound active substances such as peptides and glycosaminoglycans, resulting in low extraction efficiency. Furthermore, prolonged exposure to high temperatures and strong acid / alkali environments can easily lead to the degradation and inactivation of heat-sensitive and acid-sensitive active ingredients.

[0004] Therefore, an enzymatically assisted extraction process for highly active components from deer antler powder is proposed to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an enzymatically assisted extraction process for highly active components from deer antler powder, which solves the problem of limited ability of existing methods to destroy the dense keratinized matrix of deer antler, resulting in low extraction efficiency.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an enzymatically assisted extraction process for highly active components from deer antler powder, comprising the following steps: Step 1: Raw material pretreatment. Select antlers from sika deer or red deer, and after cleaning, cutting and crushing, obtain coarse antler powder. Then, the coarse antler powder is pretreated by degreasing and decalcification. Step 2: Compound enzymatic hydrolysis. The pretreated antler powder is mixed with the compound enzymatic hydrolysis buffer at a predetermined solid-liquid ratio and placed in a temperature-controlled shaking reactor for stepwise enzymatic hydrolysis. Step 3: Preparation of extract: Add low molecular weight chitosan acid solution, natural penetration enhancer and food-grade propylene glycol to the composite enzymatic hydrolysis buffer, mix well to prepare composite extract; Step 4: Ultrasonic-assisted extraction. The enzymatically hydrolyzed deer antler powder mixture is combined with the composite extract and transferred to an ultrasonic-microwave synergistic extraction device for enhanced extraction. Step 5: Separation and primary purification. After extraction, the mixture is subjected to solid-liquid separation, the supernatant is collected, and then subjected to primary purification through microfiltration and ultrafiltration membrane systems to obtain the primary purified solution. Step 6: Adsorption purification. The primary purified solution is pumped into an adsorption column packed with composite chromatography media for dynamic adsorption. Gradient elution is then performed with eluent, and the target elution peak is collected. Step 7: Concentration and drying. The collected target eluent is concentrated under reduced pressure to obtain a concentrated extract. Finally, the concentrated extract is prepared into a freeze-dried powder of high-activity ingredients in deer antler using vacuum freeze-drying technology.

[0007] Preferably, step one includes the following specific steps: S11. Cleaning and pulverizing: Clean the surface of the antlers, cut them into 3-5cm segments, freeze them with liquid nitrogen, and then pulverize them in an ultra-fine pulverizer. Pass them through an 80-120 mesh sieve to obtain coarse antler powder. S12. Degreasing treatment: Mix the crude deer antler powder with petroleum ether at a mass-volume ratio of 1g:8-12mL, reflux extract in a Soxhlet extractor at 60-70℃ for 4-6 hours, remove the deer antler powder, and let the solvent evaporate naturally in a fume hood. S13. Decalcification treatment: Mix the defatted deer antler powder with 0.5 mol / L disodium ethylenediaminetetraacetate solution at a mass-to-volume ratio of 1 g: 15-25 mL, place in a constant temperature shaker, and shake for 12-18 hours at a temperature of 40-50℃ and a rotation speed of 150-200 r / min. After treatment, centrifuge to separate the precipitate, wash the precipitate with deionized water until neutral, and then dry it in a vacuum freeze dryer to constant weight to obtain pretreated deer antler powder.

[0008] Preferably, in step two, the composite enzymatic hydrolysis buffer is a citrate-sodium citrate buffer with a pH of 4.5-5.5 or a phosphate buffer with a pH of 7.0-7.5, and the composite enzymatic hydrolysis includes two steps: S21. First step of enzymatic hydrolysis: Mix the pretreated deer antler powder with the compound enzymatic hydrolysis buffer at a solid-liquid ratio of 1g:20-30mL. First, add papain and acidic protease. The amount of papain added is 0.8%-1.5% of the mass of the deer antler powder, and the amount of acidic protease added is 0.5%-1.2% of the mass of the deer antler powder. Enzymatic hydrolysis is carried out for 3-5 hours at a temperature of 50-55℃, pH 5.0-5.5, and a shaking frequency of 100-150r / min. S22. Second enzymatic hydrolysis: After completing the first enzymatic hydrolysis, adjust the pH of the mixture to 7.0-7.5 and the temperature to 45-50℃. Then add trypsin and cellulase. The amount of trypsin added is 0.3%-0.8% of the weight of the deer antler powder, and the amount of cellulase added is 1.0%-2.0% of the weight of the deer antler powder. Continue enzymatic hydrolysis for 2-4 hours under the condition of shaking frequency of 100-150r / min. S23. After enzymatic hydrolysis, rapidly heat the mixture to 90-95℃ and maintain for 10-15 minutes to inactivate enzyme activity.

[0009] Preferably, in step three, the composite extract is prepared from the following components in parts by weight: 28-48 parts of food-grade propylene glycol, 10-22 parts of low molecular weight chitosan acid solution, 3-9 parts of natural penetration enhancer, and 30-50 parts of phosphate buffer solution with a pH of 5.5-6.5; the low molecular weight chitosan has a number average molecular weight of 5kDa-20kDa and a degree of deacetylation ≥90%.

[0010] Preferably, in step three, the preparation method of the composite extract includes the following steps: S31. Preparation of low molecular weight chitosan acid solution: Slowly add low molecular weight chitosan powder to a 0.5%-1.5% v / v acetic acid solution under continuous stirring to prepare a chitosan acid solution with a mass-volume concentration of 2%-4%, for later use. S32. Preparation of composite natural penetration enhancer solution: Mix Tween-80 and ethanol at a volume ratio of 1:1 to 1:3 until homogeneous to obtain composite natural penetration enhancer; S33. Mixing and preparation: In a reaction vessel equipped with stirring and temperature control, first add a metered amount of phosphate buffer solution with a pH of 5.5-6.5, and stir at 200-400 r / min at room temperature. Then, slowly add metered amounts of food-grade propylene glycol, chitosan acid solution, and composite natural penetration enhancer in sequence. After all the ingredients are added, increase the stirring speed to 500-800 r / min and continue stirring for 30-60 minutes until the mixture is homogeneous, thus obtaining the composite extract.

[0011] Preferably, step four includes the following specific steps: S41. Mixing: Mix the deer antler powder mixture after enzymatic hydrolysis and inactivation in step two with the composite extract prepared in step three at a volume ratio of 1:0.5-1:1.5, and transfer it to the inner tank of the ultrasonic-microwave synergistic extraction device. S42. Ultrasonic-microwave synergistic extraction: Set the ultrasonic power to 300-600W, the ultrasonic frequency to 28-40kHz, the microwave power to 200-400W, and the extraction temperature to 50-65℃. Under these conditions, perform synergistic treatment for 20-40 minutes. During the treatment, the material temperature is controlled within the set range by the internal circulation cooling system of the device. S43. Post-processing: After the co-extraction is completed, the material is cooled to room temperature to obtain an extract mixture containing highly active components of deer antler.

[0012] Preferably, step five includes the following specific steps: S51. Solid-liquid separation: The extracted mixture obtained in step four is subjected to solid-liquid separation through a plate and frame filter or a high-speed tubular centrifuge, and the filtrate is collected. S52. Microfiltration: The filtrate is filtered using a ceramic membrane or organic polymer flat sheet microfiltration membrane with a pore size of 0.1-0.45μm under conditions of transmembrane pressure of 0.1-0.3MPa and temperature of 25-40℃ to remove macromolecular impurities, particles and residual small solids. S53. Ultrafiltration: The microfiltration permeate is treated with a spiral wound ultrafiltration membrane or hollow fiber ultrafiltration membrane module with a molecular weight cutoff of 5kDa-10kDa. The operating pressure is 0.3-0.6MPa and the temperature is 25-40℃. The permeate is collected. At the same time, all the retentate generated during the ultrafiltration process is collected and pumped back to the feed tank of the microfiltration step, mixed with the subsequent microfiltration permeate, and re-entered into this ultrafiltration step for treatment. The resulting ultrafiltration permeate is the primary purified solution.

[0013] Preferably, step six includes the following specific steps: S61. Preparation of the adsorption column: The composite chromatography medium is prepared by wet packing a column after mixing macroporous adsorption resin and ion exchange fiber at a dry basis mass ratio of 3:1-5:1. The column bed height-to-diameter ratio is 5:1-10:1. Before use, it is equilibrated with 3-5 column volumes of anhydrous ethanol and 2-3 column volumes of pH 7.0 phosphate buffer. S62. Dynamic adsorption: The primary purified solution obtained in step 5 is loaded onto the equilibrated adsorption column at a flow rate of 1-3 BV / h, and the effluent is collected to monitor the breakthrough point of the target component. S63. Gradient elution: After adsorption, the column is first rinsed with 2-3 column volumes of deionized water at a flow rate of 2-4 BV / h. Then, a stepwise gradient elution is performed with aqueous solutions containing 5%-10% ethanol, 20%-30% ethanol, and 50%-60% ethanol. The amount of eluent used for each gradient is 3-5 column volumes, and the flow rate is 1-2 BV / h. S64. Combined Collection: The elution process was monitored by an online ultraviolet detector at wavelengths of 214 nm and 280 nm. The main target component eluents were collected from the elution stage containing 20%-30% ethanol to the elution stage containing 50%-60% ethanol to obtain the combined antler high-activity component eluent.

[0014] Preferably, in step six, the macroporous adsorption resin is one of HPD-100, AB-8, D-101 or X-5, all of which are non-polar or weakly polar adsorption resins; the ion exchange fiber is a strong acid cation exchange fiber or a weak acid cation exchange fiber, with an exchange capacity of 4.0-5.0 mmol / g.

[0015] Preferably, step seven includes the following specific steps: S71. Vacuum Concentration: The combined eluent of highly active antler ingredients collected in step six is ​​introduced into a rotary evaporator and concentrated under reduced pressure at a temperature of 40-50℃ and a vacuum degree of -0.08 to -0.1MPa until the volume of the concentrated liquid is reduced to 1 / 10-1 / 15 of the original volume, to obtain a concentrated extract. S72. Pre-freezing: Transfer the concentrated extract to the material tray of the vacuum freeze dryer, spread it into a thin layer with a thickness of no more than 1.5 cm, and place it in a quick-freezing chamber at -40℃ to -50℃ for 4-8 hours to pre-freeze the material completely. S73. Vacuum freeze drying: Transfer the pre-frozen material trays to the freeze drying chamber, start the vacuum system, reduce the pressure inside the chamber to 10-30 Pa, and then start the heating program to carry out sublimation drying and desorption drying in stages: First, sublimation drying is carried out at -20℃ to -10℃ for 10-20 hours, and then the shelf temperature is gradually raised to 20-30℃ and maintained for 8-12 hours for desorption drying until the residual moisture content of the material is less than 5%; S74. Discharge and Packaging: After freeze-drying, the material is removed under nitrogen protection and immediately crushed and passed through an 80-100 mesh sieve in a drying room with a relative humidity of less than 20% to obtain light yellow to light brown freeze-dried powder of antler high-activity ingredients, which is then vacuum-packed in aluminum foil bags.

[0016] Compared with the prior art, the present invention provides an enzymatically assisted extraction process for highly active ingredients from deer antler powder, which has the following beneficial effects: 1. In this invention, the pretreatment step destroys the dense keratinized matrix structure of deer antler powder through defatting and decalcification, creating conditions for the subsequent release of active ingredients; the compound enzymatic hydrolysis step adopts a stepwise enzymatic hydrolysis treatment, which uses papain, acidic protease, trypsin and cellulase to perform enzymatic hydrolysis stepwise in a temperature-controlled shaking reactor, which can hydrolyze the complex protein-polysaccharide complex structure in deer antler, promoting the dissolution of high-value components inside. This multi-enzyme synergistic effect overcomes the limitations of single enzyme action, thereby improving the sufficiency of target component extraction and the breadth of product spectrum.

[0017] 2. In this invention, the composite extract is composed of food-grade propylene glycol, low molecular weight chitosan acid solution, natural penetration enhancer and phosphate buffer in a specific weight ratio. This composite extract is mixed with the material after stepwise enzymatic hydrolysis and subjected to enhanced extraction in an ultrasonic-microwave synergistic extraction device. The components can work synergistically to change the physicochemical environment of the extraction system, stabilize the released active ingredients, and further promote the dissolution and diffusion of active ingredients by utilizing the synergistic effect of ultrasound and microwave, thereby achieving efficient and gentle extraction of highly active ingredients from deer antler powder.

[0018] 3. In this invention, the separation and primary purification steps are performed sequentially by a microfiltration and ultrafiltration membrane system, which removes macromolecular impurities, particles, and residual solids from the extract, achieving preliminary purification and concentration. The subsequent adsorption purification step utilizes a composite chromatography medium composed of macroporous adsorption resin and ion exchange fibers for dynamic adsorption and gradient elution, which can selectively enrich the target active ingredient in the primary purified solution based on differences in molecular polarity and size. Finally, the product is concentrated under reduced pressure and freeze-dried under vacuum to produce a lyophilized powder. This integrated purification strategy improves the purity of the highly active ingredient in the final product while maintaining its biological activity. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: An enzymatically assisted extraction process for highly active components from deer antler powder, comprising the following steps: Step 1: Raw material pretreatment. Sika deer antlers are selected, cleaned, cut, and crushed to obtain coarse antler powder. The coarse antler powder then undergoes degreasing and decalcification pretreatment; this includes the following specific steps: S11. Cleaning and pulverizing: The surface of the deer antlers is brushed clean, cut into 3cm segments, then quick-frozen with liquid nitrogen and pulverized in an ultra-fine pulverizer. The powder is then passed through an 80-mesh sieve to obtain coarse deer antler powder. S12. Degreasing treatment: Mix crude deer antler powder with petroleum ether at a mass-volume ratio of 1g:8mL, reflux extract in a Soxhlet extractor at 60℃ for 4 hours, remove the deer antler powder, and let the solvent evaporate naturally in a fume hood. S13. Decalcification treatment: The defatted deer antler powder was mixed with 0.5 mol / L disodium ethylenediaminetetraacetate solution at a mass-volume ratio of 1 g: 15 mL and placed in a constant temperature shaker. The mixture was shaken for 12 hours at 40℃ and 150 r / min. After treatment, the mixture was centrifuged, the precipitate was collected, and the precipitate was washed with deionized water until neutral. Then, it was dried in a vacuum freeze dryer to constant weight to obtain pretreated deer antler powder.

[0021] Step 2: Compound enzymatic hydrolysis. The pretreated antler powder is mixed with a compound enzymatic hydrolysis buffer at a predetermined solid-liquid ratio and placed in a temperature-controlled shaking reactor for stepwise enzymatic hydrolysis. The compound enzymatic hydrolysis buffer is a pH 4.5 citrate-sodium citrate buffer. The compound enzymatic hydrolysis includes two steps: S21. First step of enzymatic hydrolysis: Mix pretreated deer antler powder with compound enzymatic hydrolysis buffer at a solid-liquid ratio of 1g:20mL. First add papain and acidic protease. The amount of papain added is 0.8% of the mass of deer antler powder, and the amount of acidic protease added is 0.5% of the mass of deer antler powder. Enzymatic hydrolysis is carried out for 3 hours at a temperature of 50℃, pH 5.0, and a shaking frequency of 100r / min. S22. Second enzymatic hydrolysis: After completing the first enzymatic hydrolysis, adjust the pH of the mixture to 7.0 and the temperature to 45℃. Then add trypsin and cellulase. The amount of trypsin added is 0.3% of the mass of deer antler powder, and the amount of cellulase added is 1.0% of the mass of deer antler powder. Continue enzymatic hydrolysis for 2 hours under the condition of shaking frequency of 100r / min. S23. After enzymatic hydrolysis, rapidly heat the mixture to 90°C and maintain for 10 minutes to inactivate the enzyme.

[0022] Step 3: Preparation of the extract. Add low molecular weight chitosan acid solution, natural penetration enhancer, and food-grade propylene glycol to the complex enzymatic hydrolysis buffer, mix thoroughly, and prepare the complex extract. The complex extract is prepared from the following components by weight: 28 parts food-grade propylene glycol, 10 parts low molecular weight chitosan acid solution, 3 parts natural penetration enhancer, and 30 parts phosphate buffer solution with a pH of 5.5; the number average molecular weight of the low molecular weight chitosan is 5 kDa, and the degree of deacetylation is ≥90%. The preparation method of the compound extract includes the following steps: S31. Preparation of low molecular weight chitosan acid solution: Slowly add low molecular weight chitosan powder to a continuously stirred 0.5% v / v acetic acid solution to prepare a chitosan acid solution with a mass-volume concentration of 2% for later use. S32. Preparation of composite natural penetration enhancer solution: Mix Tween-80 and ethanol at a volume ratio of 1:1 until homogeneous to obtain composite natural penetration enhancer; S33. Mixing preparation: In a reaction vessel equipped with stirring and temperature control, first add a metered amount of phosphate buffer solution with pH 5.5, and stir at 200 r / min at room temperature. Then, slowly add metered amounts of food-grade propylene glycol, chitosan acid solution, and composite natural penetration enhancer in sequence. After all the ingredients are added, increase the stirring speed to 500 r / min and continue stirring for 30 minutes until the mixture is homogeneous to obtain the composite extract.

[0023] Step 4: Ultrasonic-assisted extraction. The enzymatically hydrolyzed deer antler powder mixture is combined with the composite extract and transferred to an ultrasonic-microwave synergistic extraction device for enhanced extraction; this includes the following specific steps: S41. Mixing: Mix the deer antler powder mixture after enzymatic hydrolysis and inactivation in step two with the composite extract prepared in step three at a volume ratio of 1:0.5, and transfer it to the inner tank of the ultrasonic-microwave synergistic extraction device. S42, Ultrasonic-Microwave Co-extraction: Set the ultrasonic power to 300W, the ultrasonic frequency to 28kHz, the microwave power to 200W, and the extraction temperature to 50℃. Under these conditions, co-process for 20 minutes. During the process, the material temperature is controlled within the set range by the internal circulation cooling system of the device. S43. Post-processing: After the co-extraction is completed, the material is cooled to room temperature to obtain an extract mixture containing highly active components of deer antler.

[0024] Step 5: Separation and primary purification. The extracted mixture undergoes solid-liquid separation, the supernatant is collected, and then subjected to primary purification via microfiltration and ultrafiltration membrane systems to obtain the primary purified solution. This includes the following specific steps: S51. Solid-liquid separation: The extraction mixture obtained in step four is passed through a plate and frame filter press for solid-liquid separation, and the filtrate is collected. S52. Microfiltration: The filtrate is filtered through a ceramic membrane with a pore size of 0.1 μm at a transmembrane pressure of 0.1 MPa and a temperature of 25 °C to remove macromolecular impurities, particles and residual small solids. S53. Ultrafiltration: The microfiltration permeate is processed using a spiral wound ultrafiltration membrane module with a molecular weight cutoff of 5 kDa. The operating pressure is 0.3 MPa and the temperature is 25°C. The permeate is collected. At the same time, all the retentate generated during the ultrafiltration process is collected and pumped back to the feed tank of the microfiltration step. It is then mixed with the subsequent microfiltration permeate and re-entered into this ultrafiltration step for processing. The resulting ultrafiltration permeate is the primary purified solution.

[0025] Step Six: Adsorption Purification. The primary purified solution is pumped into an adsorption column packed with composite chromatography media for dynamic adsorption, followed by gradient elution with eluent, and the target elution peak is collected. This includes the following specific steps: S61. Adsorption column preparation: The composite chromatography medium is prepared by wet packing a column with a dry weight ratio of 3:1 of macroporous adsorption resin and ion exchange fiber. The column bed height-to-diameter ratio is 5:1. Before use, the column is equilibrated with 3 column volumes of anhydrous ethanol and 2 column volumes of pH 7.0 phosphate buffer. The macroporous adsorption resin is HPD-100 type nonpolar macroporous adsorption resin. The ion exchange fiber is a strongly acidic cation exchange fiber with an exchange capacity of 4.0 mmol / g. S62. Dynamic adsorption: The primary purified solution obtained in step 5 is loaded onto the equilibrated adsorption column at a flow rate of 1 BV / h, and the effluent is collected to monitor the breakthrough point of the target component. S63. Gradient elution: After adsorption, the column is first rinsed with 2 column volumes of deionized water at a flow rate of 2 BV / h. Then, a stepwise gradient elution is performed with aqueous solutions containing 5% ethanol, 20% ethanol, and 50% ethanol. The amount of eluent used in each gradient is 3 column volumes, and the flow rate is 1 BV / h. S64. Combined Collection: The elution process was monitored by an online ultraviolet detector at wavelengths of 214 nm and 280 nm. The main target component eluents were collected from the elution stage containing 20% ​​ethanol to the elution stage containing 50% ethanol to obtain the combined antler high-activity component eluent.

[0026] Step 7: Concentration and Drying. The collected target eluent is concentrated under reduced pressure to obtain a concentrated extract. Finally, the concentrated extract is prepared into a freeze-dried powder of high-activity deer antler ingredients using vacuum freeze-drying technology. This includes the following specific steps: S71. Vacuum Concentration: The combined eluent of highly active antler ingredients collected in step six is ​​introduced into a rotary evaporator and concentrated under reduced pressure at a temperature of 40°C and a vacuum of -0.08MPa until the volume of the concentrated liquid is reduced to 1 / 10 of the original volume, thus obtaining a concentrated extract. S72. Pre-freezing: Transfer the concentrated extract to the material tray of the vacuum freeze dryer, spread it into a thin layer with a thickness of no more than 1.5 cm, and place it in a quick-freezing chamber at -40℃ for 4 hours to pre-freeze the material completely. S73. Vacuum freeze drying: Transfer the pre-frozen material tray to the freeze drying chamber, start the vacuum system, reduce the pressure inside the chamber to 10Pa, and then start the heating program to carry out sublimation drying and desorption drying in stages: First, sublimation drying is carried out at -20℃ for 10 hours, and then the shelf temperature is gradually raised to 20℃ and maintained for 8 hours for desorption drying until the residual moisture content of the material is less than 5%; S74. Discharge and Packaging: After freeze-drying, the material is removed under nitrogen protection and immediately crushed and passed through an 80-mesh sieve in a drying room with a relative humidity of less than 20% to obtain light yellow to light brown freeze-dried powder of antler high-activity ingredients, which is then vacuum-packed in aluminum foil bags.

[0027] Example 2: An enzymatically assisted extraction process for highly active components from deer antler powder, comprising the following steps: Step 1: Raw material pretreatment. Sika deer antlers are selected, cleaned, cut, and crushed to obtain coarse antler powder. The coarse antler powder then undergoes degreasing and decalcification pretreatment; this includes the following specific steps: S11. Cleaning and pulverizing: The surface of the antlers is brushed clean, cut into 4cm segments, then quick-frozen with liquid nitrogen and pulverized in an ultra-fine pulverizer. The powder is then passed through a 100-mesh sieve to obtain coarse antler powder. S12. Degreasing treatment: Mix crude deer antler powder with petroleum ether at a mass-volume ratio of 1g:10mL, reflux extract in a Soxhlet extractor at 65℃ for 5 hours, remove the deer antler powder, and let the solvent evaporate naturally in a fume hood. S13. Decalcification treatment: The defatted deer antler powder was mixed with 0.5 mol / L disodium ethylenediaminetetraacetate solution at a mass-volume ratio of 1 g: 20 mL and placed in a constant temperature shaker. The mixture was shaken for 16 hours at a temperature of 45℃ and a rotation speed of 170 r / min. After the treatment was completed, the mixture was centrifuged, the precipitate was collected, and the precipitate was washed with deionized water until neutral. Then, it was dried in a vacuum freeze dryer to constant weight to obtain pretreated deer antler powder.

[0028] Step 2: Compound enzymatic hydrolysis. The pretreated antler powder is mixed with a compound enzymatic hydrolysis buffer at a predetermined solid-liquid ratio and placed in a temperature-controlled shaking reactor for stepwise enzymatic hydrolysis. The compound enzymatic hydrolysis buffer is a pH 5.0 citrate-sodium citrate buffer. The compound enzymatic hydrolysis includes two steps: S21. First step of enzymatic hydrolysis: Mix pretreated deer antler powder with compound enzymatic hydrolysis buffer at a solid-liquid ratio of 1g:25mL. First add papain and acidic protease. The amount of papain added is 1.2% of the mass of deer antler powder, and the amount of acidic protease added is 0.8% of the mass of deer antler powder. Enzymatic hydrolysis is carried out for 4 hours at a temperature of 52℃, pH 5.3, and a shaking frequency of 120r / min. S22. Second enzymatic hydrolysis: After completing the first enzymatic hydrolysis, adjust the pH of the mixture to 7.2 and the temperature to 47℃. Then add trypsin and cellulase. The amount of trypsin added is 0.5% of the mass of deer antler powder, and the amount of cellulase added is 1.5% of the mass of deer antler powder. Continue enzymatic hydrolysis for 3 hours under the condition of shaking frequency of 120r / min. S23. After enzymatic hydrolysis, rapidly heat the mixture to 92°C and maintain for 12 minutes to inactivate enzyme activity.

[0029] Step 3: Preparation of the extract. Add low molecular weight chitosan acid solution, natural penetration enhancer, and food-grade propylene glycol to the complex enzymatic hydrolysis buffer, mix thoroughly, and prepare the complex extract. The complex extract is prepared from the following components by weight: 36 parts food-grade propylene glycol, 15 parts low molecular weight chitosan acid solution, 5 parts natural penetration enhancer, and 40 parts phosphate buffer at pH 6.0; the number average molecular weight of the low molecular weight chitosan is 15 kDa, and the degree of deacetylation is ≥90%. The preparation method of the compound extract includes the following steps: S31. Preparation of low molecular weight chitosan acid solution: Slowly add low molecular weight chitosan powder to a 1.0% v / v acetic acid solution under continuous stirring to prepare a chitosan acid solution with a mass-volume concentration of 3% for later use. S32. Preparation of composite natural penetration enhancer solution: Mix Tween-80 and ethanol at a volume ratio of 1:2 to obtain composite natural penetration enhancer. S33. Mixing and preparation: In a reaction vessel equipped with stirring and temperature control, first add a metered amount of phosphate buffer solution with pH 6.0, and stir at 300 r / min at room temperature. Then, slowly add metered amounts of food-grade propylene glycol, chitosan acid solution, and composite natural penetration enhancer in sequence. After all the ingredients are added, increase the stirring speed to 650 r / min and continue stirring for 45 minutes until the mixture is homogeneous to obtain the composite extract.

[0030] Step 4: Ultrasonic-assisted extraction. The enzymatically hydrolyzed deer antler powder mixture is combined with the composite extract and transferred to an ultrasonic-microwave synergistic extraction device for enhanced extraction; this includes the following specific steps: S41. Mixing: Mix the deer antler powder mixture after enzymatic hydrolysis and inactivation in step two with the composite extract prepared in step three at a volume ratio of 1:1, and transfer it to the inner tank of the ultrasonic-microwave synergistic extraction device. S42, Ultrasonic-Microwave Co-extraction: Set the ultrasonic power to 450W, the ultrasonic frequency to 35kHz, the microwave power to 300W, and the extraction temperature to 60℃. Under these conditions, co-process for 30 minutes. During the process, the material temperature is controlled within the set range by the internal circulation cooling system of the device. S43. Post-processing: After the co-extraction is completed, the material is cooled to room temperature to obtain an extract mixture containing highly active components of deer antler.

[0031] Step 5: Separation and primary purification. The extracted mixture undergoes solid-liquid separation, the supernatant is collected, and then subjected to primary purification via microfiltration and ultrafiltration membrane systems to obtain the primary purified solution. This includes the following specific steps: S51. Solid-liquid separation: The extraction mixture obtained in step four is passed through a plate and frame filter press for solid-liquid separation, and the filtrate is collected. S52. Microfiltration: The filtrate is filtered through a ceramic membrane with a pore size of 0.3 μm at a transmembrane pressure of 0.2 MPa and a temperature of 30 °C to remove macromolecular impurities, particles and residual small solids. S53. Ultrafiltration: The microfiltration permeate is processed using a spiral wound ultrafiltration membrane module with a molecular weight cutoff of 7 kDa. The operating pressure is 0.4 MPa and the temperature is 30°C. The permeate is collected. At the same time, all the retentate generated during the ultrafiltration process is collected and pumped back to the feed tank of the microfiltration step. It is then mixed with the subsequent microfiltration permeate and re-entered into this ultrafiltration step for processing. The resulting ultrafiltration permeate is the primary purified solution.

[0032] Step Six: Adsorption Purification. The primary purified solution is pumped into an adsorption column packed with composite chromatography media for dynamic adsorption, followed by gradient elution with eluent, and the target elution peak is collected. This includes the following specific steps: S61. Adsorption Column Preparation: The composite chromatography medium was prepared by wet packing a column with a dry weight ratio of 4:1 of macroporous adsorption resin and ion exchange fiber. The column bed height-to-diameter ratio was 7:1. Before use, the column was equilibrated sequentially with 4 column volumes of anhydrous ethanol and 2.5 column volumes of pH 7.0 phosphate buffer. The macroporous adsorption resin was AB-8 type weakly polar macroporous adsorption resin; the ion exchange fiber was a strongly acidic cation exchange fiber with an exchange capacity of 4.5 mmol / g. S62, Dynamic Adsorption: The primary purified solution obtained in step 5 is loaded onto the equilibrated adsorption column at a flow rate of 2 BV / h, and the effluent is collected to monitor the breakthrough point of the target component. S63. Gradient elution: After adsorption, the column is first rinsed with 2.5 column volumes of deionized water at a flow rate of 3 BV / h. Then, a stepwise gradient elution is performed with aqueous solutions containing 7% ethanol, 25% ethanol, and 55% ethanol. The amount of eluent used for each gradient is 4 column volumes, and the flow rate is 1.5 BV / h. S64. Combined Collection: The elution process was monitored by an online ultraviolet detector at wavelengths of 214 nm and 280 nm. The main target component eluents were collected from the elution stage containing 25% ethanol to the elution stage containing 55% ethanol to obtain the combined eluent of highly active antler components.

[0033] Step 7: Concentration and Drying. The collected target eluent is concentrated under reduced pressure to obtain a concentrated extract. Finally, the concentrated extract is prepared into a freeze-dried powder of high-activity deer antler ingredients using vacuum freeze-drying technology. This includes the following specific steps: S71. Vacuum Concentration: The combined eluent of highly active deer antler components collected in step six is ​​introduced into a rotary evaporator and concentrated under reduced pressure at a temperature of 45°C and a vacuum of -0.09MPa until the volume of the concentrated liquid is reduced to 1 / 12 of the original volume, thus obtaining a concentrated extract. S72. Pre-freezing: Transfer the concentrated extract to the material tray of the vacuum freeze dryer, spread it into a thin layer with a thickness of no more than 1.5 cm, and place it in a quick-freezing chamber at -45℃ for 6 hours to pre-freeze the material completely. S73. Vacuum freeze drying: Transfer the pre-frozen material tray to the freeze drying chamber, start the vacuum system, reduce the pressure inside the chamber to 20Pa, and then start the heating program to carry out sublimation drying and desorption drying in stages: First, sublimation drying is carried out at -15℃ for 15 hours, and then the shelf temperature is gradually raised to 25℃ and maintained for 10 hours for desorption drying until the residual moisture content of the material is less than 5%; S74. Discharge and Packaging: After freeze-drying, the material is removed under nitrogen protection and immediately crushed and passed through a 90-mesh sieve in a drying room with a relative humidity of less than 20% to obtain light yellow to light brown freeze-dried powder of antler high-activity ingredients, which is then vacuum-packed in aluminum foil bags.

[0034] Example 3: An enzymatically assisted extraction process for highly active components from deer antler powder, comprising the following steps: Step 1: Raw material pretreatment. Sika deer antlers are selected, cleaned, cut, and crushed to obtain coarse antler powder. The coarse antler powder then undergoes degreasing and decalcification pretreatment; this includes the following specific steps: S11. Cleaning and pulverizing: The surface of the antlers is brushed clean, cut into 5cm segments, then quick-frozen with liquid nitrogen and pulverized in an ultra-fine pulverizer. The powder is then passed through a 120-mesh sieve to obtain coarse antler powder. S12. Degreasing treatment: Mix crude deer antler powder with petroleum ether at a mass-volume ratio of 1g:12mL, reflux extract in a Soxhlet extractor at 70℃ for 6 hours, remove the deer antler powder, and let the solvent evaporate naturally in a fume hood. S13. Decalcification treatment: The defatted deer antler powder was mixed with 0.5 mol / L disodium ethylenediaminetetraacetate solution at a mass-volume ratio of 1 g: 25 mL and placed in a constant temperature shaker. The mixture was shaken for 18 hours at 50℃ and 200 r / min. After treatment, the mixture was centrifuged, the precipitate was collected, and the precipitate was washed with deionized water until neutral. Then, it was dried in a vacuum freeze dryer to constant weight to obtain pretreated deer antler powder.

[0035] Step 2: Compound enzymatic hydrolysis. The pretreated antler powder is mixed with a compound enzymatic hydrolysis buffer at a predetermined solid-liquid ratio and placed in a temperature-controlled shaking reactor for stepwise enzymatic hydrolysis. The compound enzymatic hydrolysis buffer is a pH 5.5 citrate-sodium citrate buffer. The compound enzymatic hydrolysis includes two steps: S21. First step of enzymatic hydrolysis: Mix pretreated deer antler powder with compound enzymatic hydrolysis buffer at a solid-liquid ratio of 1g:30mL. First add papain and acidic protease. The amount of papain added is 1.5% of the mass of deer antler powder, and the amount of acidic protease added is 1.2% of the mass of deer antler powder. Enzymatic hydrolysis is carried out for 5 hours at a temperature of 55℃, pH 5.5, and a shaking frequency of 150r / min. S22. Second enzymatic hydrolysis: After completing the first enzymatic hydrolysis, adjust the pH of the mixture to 7.5 and the temperature to 50℃. Then add trypsin and cellulase. The amount of trypsin added is 0.8% of the weight of the deer antler powder, and the amount of cellulase added is 2.0% of the weight of the deer antler powder. Continue enzymatic hydrolysis for 4 hours under the condition of shaking frequency of 150r / min. S23. After enzymatic hydrolysis, rapidly heat the mixture to 95°C and maintain for 15 minutes to inactivate enzyme activity.

[0036] Step 3: Preparation of the extract. Add low molecular weight chitosan acid solution, natural penetration enhancer, and food-grade propylene glycol to the complex enzymatic hydrolysis buffer, mix thoroughly, and prepare the complex extract. The complex extract is prepared from the following components by weight: 48 parts food-grade propylene glycol, 22 parts low molecular weight chitosan acid solution, 9 parts natural penetration enhancer, and 50 parts phosphate buffer solution with a pH of 6.5; the number average molecular weight of the low molecular weight chitosan is 20 kDa, and the degree of deacetylation is ≥90%. The preparation method of the compound extract includes the following steps: S31. Preparation of low molecular weight chitosan acid solution: Slowly add low molecular weight chitosan powder to a 1.5% v / v acetic acid solution under continuous stirring to prepare a chitosan acid solution with a mass-volume concentration of 4% for later use. S32. Preparation of composite natural penetration enhancer solution: Mix Tween-80 and ethanol at a volume ratio of 1:3 to obtain composite natural penetration enhancer. S33. Mixing and preparation: In a reaction vessel equipped with stirring and temperature control, first add a metered amount of phosphate buffer solution with pH 6.5, and stir at 400 r / min at room temperature. Then, slowly add metered amounts of food-grade propylene glycol, chitosan acid solution, and composite natural penetration enhancer in sequence. After all the ingredients are added, increase the stirring speed to 800 r / min and continue stirring for 60 minutes until the mixture is homogeneous to obtain the composite extract.

[0037] Step 4: Ultrasonic-assisted extraction. The enzymatically hydrolyzed deer antler powder mixture is combined with the composite extract and transferred to an ultrasonic-microwave synergistic extraction device for enhanced extraction; this includes the following specific steps: S41. Mixing: Mix the deer antler powder mixture after enzymatic hydrolysis and inactivation in step two with the composite extract prepared in step three at a volume ratio of 1:1.5, and transfer it to the inner tank of the ultrasonic-microwave synergistic extraction device. S42. Ultrasonic-microwave synergistic extraction: Set the ultrasonic power to 600W, the ultrasonic frequency to 40kHz, the microwave power to 400W, and the extraction temperature to 65℃. Under these conditions, perform synergistic treatment for 40 minutes. During the treatment, the material temperature is controlled within the set range by the internal circulation cooling system of the device. S43. Post-processing: After the co-extraction is completed, the material is cooled to room temperature to obtain an extract mixture containing highly active components of deer antler.

[0038] Step 5: Separation and primary purification. The extracted mixture undergoes solid-liquid separation, the supernatant is collected, and then subjected to primary purification via microfiltration and ultrafiltration membrane systems to obtain the primary purified solution. This includes the following specific steps: S51. Solid-liquid separation: The extraction mixture obtained in step four is passed through a plate and frame filter press for solid-liquid separation, and the filtrate is collected. S52. Microfiltration: The filtrate is filtered through a ceramic membrane with a pore size of 0.45 μm at a transmembrane pressure of 0.3 MPa and a temperature of 40 °C to remove macromolecular impurities, particles and residual small solids. S53. Ultrafiltration: The microfiltration permeate is processed using a spiral wound ultrafiltration membrane module with a molecular weight cutoff of 10 kDa. The operating pressure is 0.6 MPa and the temperature is 40°C. The permeate is collected. At the same time, all the retentate generated during the ultrafiltration process is collected and pumped back to the feed tank of the microfiltration step. It is then mixed with the subsequent microfiltration permeate and re-entered into this ultrafiltration step for processing. The resulting ultrafiltration permeate is the primary purified solution.

[0039] Step Six: Adsorption Purification. The primary purified solution is pumped into an adsorption column packed with composite chromatography media for dynamic adsorption, followed by gradient elution with eluent, and the target elution peak is collected. This includes the following specific steps: S61. Adsorption column preparation: The composite chromatography medium is prepared by wet packing a column with a dry weight ratio of 5:1 of macroporous adsorption resin and ion exchange fiber. The column bed height-to-diameter ratio is 10:1. Before use, the column is equilibrated with 5 column volumes of anhydrous ethanol and 3 column volumes of pH 7.0 phosphate buffer. The macroporous adsorption resin is D-101 type nonpolar macroporous adsorption resin. The ion exchange fiber is a strongly acidic cation exchange fiber with an exchange capacity of 5.0 mmol / g. S62, Dynamic Adsorption: The primary purified solution obtained in step 5 is loaded onto the equilibrated adsorption column at a flow rate of 3 BV / h, and the effluent is collected to monitor the breakthrough point of the target component. S63. Gradient elution: After adsorption, the column is first rinsed with 3 column volumes of deionized water at a flow rate of 4 BV / h. Then, a stepwise gradient elution is performed with aqueous solutions containing 10% ethanol, 30% ethanol, and 60% ethanol. The amount of eluent used for each gradient is 5 column volumes, and the flow rate is 2 BV / h. S64. Combined collection: The elution process was monitored by an online ultraviolet detector at wavelengths of 214 nm and 280 nm. The main target component eluents were collected from the elution stage containing 30% ethanol to the elution stage containing 60% ethanol to obtain the combined antler high-activity component eluent.

[0040] Step 7: Concentration and Drying. The collected target eluent is concentrated under reduced pressure to obtain a concentrated extract. Finally, the concentrated extract is prepared into a freeze-dried powder of high-activity deer antler ingredients using vacuum freeze-drying technology. This includes the following specific steps: S71. Vacuum Concentration: The combined eluent of highly active deer antler components collected in step six is ​​introduced into a rotary evaporator and concentrated under reduced pressure at a temperature of 50°C and a vacuum of -0.1MPa until the volume of the concentrated liquid is reduced to 1 / 15 of the original volume, thus obtaining a concentrated extract. S72. Pre-freezing: Transfer the concentrated extract to the material tray of the vacuum freeze dryer, spread it into a thin layer with a thickness of no more than 1.5 cm, and place it in a quick-freezing chamber at -50℃ for 8 hours to pre-freeze the material completely. S73. Vacuum freeze drying: Transfer the pre-frozen material tray to the freeze drying chamber, start the vacuum system, reduce the pressure inside the chamber to 30Pa, and then start the heating program to carry out sublimation drying and desorption drying in stages: First, sublimation drying is carried out at -10℃ for 20 hours, and then the shelf temperature is gradually raised to 30℃ and maintained for 12 hours for desorption drying until the residual moisture content of the material is less than 5%; S74. Discharge and Packaging: After freeze-drying, the material is removed under nitrogen protection and immediately crushed and passed through a 100-mesh sieve in a drying room with a relative humidity of less than 20% to obtain light yellow to light brown freeze-dried powder of antler high-activity ingredients, which is then vacuum-packed in aluminum foil bags.

[0041] Comparative Example 1: The difference between this comparative example and Example 1 is that no compound enzymatic hydrolysis was performed during the extraction process. Instead, the pretreated antler powder was directly mixed with phosphate buffer and then subjected to ultrasonic-microwave synergistic extraction. All other aspects were the same as in Example 1.

[0042] Comparative Example 2 differs from Example 1 in that: in preparing the composite extract, no low molecular weight chitosan acid solution and natural penetration enhancer were added; only food-grade propylene glycol and phosphate buffer were used. All other aspects are the same as in Example 1.

[0043] Comparative Example 3 differs from Example 1 in that: this comparative example did not use an ultrasonic-microwave synergistic extraction device during the extraction process, but only used a conventional constant temperature stirring method for extraction, while the rest was the same as Example 1.

[0044] Comparative Example 4 differs from Example 1 in that: in the separation and purification process, no adsorption column was used for dynamic adsorption and gradient elution; only the permeate after ultrafiltration was concentrated and dried. The rest was the same as in Example 1.

[0045] Comparative Example 5 differs from Example 1 in that: in this comparative example, the deer antler powder was not pretreated by defatting and decalcification before extraction. The raw deer antler powder was directly mixed with phosphate buffer and then subjected to ultrasonic-microwave synergistic extraction. All other aspects are the same as in Example 1.

[0046] The performance of the deer antler extracts with high active ingredients prepared in Examples 1-3 and Comparative Examples 1-5 was tested. The test items and test methods are as follows: To determine the total solids content in the extract, 1.0 g of freeze-dried deer antler powder extract was weighed and placed in a pre-weighed weighing bottle. The extract was dried in a forced-air drying oven at 105℃ until constant weight was achieved. The percentage of residue after drying was calculated to characterize the overall extraction efficiency of the extraction process. The content of characteristic small molecule peptides was determined by high performance liquid chromatography (HPLC) with a C18 column and an acetonitrile-water solution containing 0.1% trifluoroacetic acid as the mobile phase. Gradient elution mode was used, and the detection wavelength was 214 nm. The total peak area integral of peptides with a molecular weight less than 10 kDa was determined by external standard method to characterize the enrichment effect of enzymatic hydrolysis and extraction process on small molecule active peptides. The total glycosaminoglycan content was determined by the 1,9-dimethylmethylene blue staining method. An appropriate amount of extract solution was taken, reacted with the staining solution, and the absorbance was measured at a wavelength of 525 nm. A standard curve was plotted using chondroitin sulfate as a standard, and the total glycosaminoglycan content in the sample was calculated to evaluate the extraction ability of the process for polysaccharide active ingredients. To determine antioxidant activity, a 0.1 mmol / L DPPH ethanol solution was prepared and mixed with sample extracts of different dilution concentrations. After reacting in the dark for 30 minutes, the absorbance was measured at 517 nm. The scavenging rate of DPPH free radicals was calculated and expressed as the half-maximal scavenging concentration (MCC) to comprehensively evaluate the bioactivity intensity of the active ingredients in the extract.

[0047] The test data of the extracts obtained by the enzymatic hydrolysis-assisted extraction process of high-activity components from deer antler powder in Examples 1-3 and Comparative Examples 1-5 are recorded in the table below: By comparing and analyzing the data in the table, it can be seen that the antler extract obtained using the process described in Examples 1-3 is superior to that of Comparative Examples 1-5 in terms of total solids, characteristic small molecule peptides, total glycosaminoglycan content, and antioxidant activity. This indicates that the complete extraction process system provided by the present invention has a synergistic effect. This shows that the pretreatment step, through defatting and decalcification, destroys the dense keratinized matrix structure of antler powder, creating conditions for the subsequent release of active ingredients. The stepwise enzymatic hydrolysis step, using papain, acidic protease, trypsin, and cellulase in a temperature-controlled oscillating reactor, can hydrolyze the complex protein-polysaccharide complex structure in antler, promoting the dissolution of high-value components. This multi-enzyme synergistic effect overcomes the limitations of single enzyme action, thereby improving the extraction sufficiency of target components and the breadth of product spectrum. The composite extract used is composed of food-grade propylene glycol, low molecular weight chitosan acid solution, natural penetration enhancer, and phosphate buffer in a specific weight ratio. This composite extract is mixed with the material after stepwise enzymatic hydrolysis and subjected to enhanced extraction in an ultrasonic-microwave synergistic extraction device. The components work synergistically to alter the physicochemical environment of the extraction system, stabilize the released active ingredients, and further promote the dissolution and diffusion of active ingredients by utilizing the synergistic effect of ultrasound and microwave, thereby achieving efficient and gentle extraction of highly active ingredients from deer antler powder. The separation and primary purification steps are performed through sequential microfiltration and ultrafiltration membrane systems to remove macromolecular impurities, particles, and residual solids from the extract, achieving preliminary purification and concentration. The subsequent adsorption purification step utilizes a composite chromatography medium composed of macroporous adsorption resin and ion exchange fibers for dynamic adsorption and gradient elution, which can selectively enrich the target active ingredients in the primary purified solution based on differences in molecular polarity and size. Finally, the product is concentrated under reduced pressure and freeze-dried in vacuum to produce lyophilized powder. This integrated purification strategy improves the purity of highly active ingredients in the final product while maintaining their biological activity.

[0048] By comparing and analyzing the relevant data in the table, it can be seen that the enzymatic hydrolysis-assisted extraction process provided by this invention can improve the efficiency of extracting active ingredients from deer antler powder and obtain an extract rich in small molecule peptides and glycosaminoglycans with high antioxidant activity. This indicates that the integrated pretreatment-synergistic enzymatic hydrolysis-enhanced extraction-gradual purification process constructed in this invention has excellent comprehensive performance.

[0049] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0050] 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. An enzymatically assisted extraction process for highly active components from deer antler powder, characterized in that: Includes the following steps: Step 1: Raw material pretreatment. Select antlers from sika deer or red deer, and after cleaning, cutting and crushing, obtain coarse antler powder. Then, the coarse antler powder is pretreated by degreasing and decalcification. Step 2: Compound enzymatic hydrolysis. The pretreated antler powder is mixed with the compound enzymatic hydrolysis buffer at a predetermined solid-liquid ratio and placed in a temperature-controlled shaking reactor for stepwise enzymatic hydrolysis. Step 3: Preparation of extract: Add low molecular weight chitosan acid solution, natural penetration enhancer and food-grade propylene glycol to the composite enzymatic hydrolysis buffer, mix well to prepare composite extract; Step 4: Ultrasonic-assisted extraction. The enzymatically hydrolyzed deer antler powder mixture is combined with the composite extract and transferred to an ultrasonic-microwave synergistic extraction device for enhanced extraction. Step 5: Separation and primary purification. After extraction, the mixture is subjected to solid-liquid separation, the supernatant is collected, and then subjected to primary purification through microfiltration and ultrafiltration membrane systems to obtain the primary purified solution. Step 6: Adsorption purification. The primary purified solution is pumped into an adsorption column packed with composite chromatography media for dynamic adsorption. Gradient elution is then performed with eluent, and the target elution peak is collected. Step 7: Concentration and drying. The collected target eluent is concentrated under reduced pressure to obtain a concentrated extract. Finally, the concentrated extract is prepared into a freeze-dried powder of high-activity ingredients in deer antler using vacuum freeze-drying technology.

2. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: Step one includes the following specific steps: S11. Cleaning and pulverizing: Clean the surface of the antlers, cut them into 3-5cm segments, freeze them with liquid nitrogen, and then pulverize them in an ultra-fine pulverizer. Pass them through an 80-120 mesh sieve to obtain coarse antler powder. S12. Degreasing treatment: Mix the crude deer antler powder with petroleum ether at a mass-volume ratio of 1g:8-12mL, reflux extract in a Soxhlet extractor at 60-70℃ for 4-6 hours, remove the deer antler powder, and let the solvent evaporate naturally in a fume hood. S13. Decalcification treatment: Mix the defatted deer antler powder with 0.5 mol / L disodium ethylenediaminetetraacetate solution at a mass-to-volume ratio of 1 g: 15-25 mL, place in a constant temperature shaker, and shake for 12-18 hours at a temperature of 40-50℃ and a rotation speed of 150-200 r / min. After treatment, centrifuge to separate the precipitate, wash the precipitate with deionized water until neutral, and then dry it in a vacuum freeze dryer to constant weight to obtain pretreated deer antler powder.

3. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: In step two, the complex enzymatic hydrolysis buffer is a citrate-sodium citrate buffer with a pH of 4.5-5.5 or a phosphate buffer with a pH of 7.0-7.

5. The complex enzymatic hydrolysis includes two steps: S21. First step of enzymatic hydrolysis: Mix the pretreated deer antler powder with the compound enzymatic hydrolysis buffer at a solid-liquid ratio of 1g:20-30mL. First, add papain and acidic protease. The amount of papain added is 0.8%-1.5% of the mass of the deer antler powder, and the amount of acidic protease added is 0.5%-1.2% of the mass of the deer antler powder. Enzymatic hydrolysis is carried out for 3-5 hours at a temperature of 50-55℃, pH 5.0-5.5, and a shaking frequency of 100-150r / min. S22. Second enzymatic hydrolysis: After completing the first enzymatic hydrolysis, adjust the pH of the mixture to 7.0-7.5 and the temperature to 45-50℃. Then add trypsin and cellulase. The amount of trypsin added is 0.3%-0.8% of the weight of the deer antler powder, and the amount of cellulase added is 1.0%-2.0% of the weight of the deer antler powder. Continue enzymatic hydrolysis for 2-4 hours under the condition of shaking frequency of 100-150r / min. S23. After enzymatic hydrolysis, rapidly heat the mixture to 90-95℃ and maintain for 10-15 minutes to inactivate enzyme activity.

4. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: In step three, the composite extract is prepared from the following components in parts by weight: 28-48 parts of food-grade propylene glycol, 10-22 parts of low molecular weight chitosan acid solution, 3-9 parts of natural penetration enhancer, and 30-50 parts of phosphate buffer solution with a pH of 5.5-6.5; the low molecular weight chitosan has a number average molecular weight of 5kDa-20kDa and a degree of deacetylation ≥90%.

5. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 4, characterized in that: In step three, the preparation method of the composite extract includes the following steps: S31. Preparation of low molecular weight chitosan acid solution: Slowly add low molecular weight chitosan powder to a 0.5%-1.5% v / v acetic acid solution under continuous stirring to prepare a chitosan acid solution with a mass-volume concentration of 2%-4%, for later use. S32. Preparation of composite natural penetration enhancer solution: Mix Tween-80 and ethanol at a volume ratio of 1:1 to 1:3 until homogeneous to obtain composite natural penetration enhancer; S33. Mixing and preparation: In a reaction vessel equipped with stirring and temperature control, first add a metered amount of phosphate buffer solution with a pH of 5.5-6.5, and stir at 200-400 r / min at room temperature. Then, slowly add metered amounts of food-grade propylene glycol, chitosan acid solution, and composite natural penetration enhancer in sequence. After all the ingredients are added, increase the stirring speed to 500-800 r / min and continue stirring for 30-60 minutes until the mixture is homogeneous, thus obtaining the composite extract.

6. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: Step four includes the following specific steps: S41. Mixing: Mix the deer antler powder mixture after enzymatic hydrolysis and inactivation in step two with the composite extract prepared in step three at a volume ratio of 1:0.5-1:1.5, and transfer it to the inner tank of the ultrasonic-microwave synergistic extraction device. S42. Ultrasonic-microwave synergistic extraction: Set the ultrasonic power to 300-600W, the ultrasonic frequency to 28-40kHz, the microwave power to 200-400W, and the extraction temperature to 50-65℃. Under these conditions, perform synergistic treatment for 20-40 minutes. During the treatment, the material temperature is controlled within the set range by the internal circulation cooling system of the device. S43. Post-processing: After the co-extraction is completed, the material is cooled to room temperature to obtain an extract mixture containing highly active components of deer antler.

7. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: Step five includes the following specific steps: S51. Solid-liquid separation: The extracted mixture obtained in step four is subjected to solid-liquid separation through a plate and frame filter or a high-speed tubular centrifuge, and the filtrate is collected. S52. Microfiltration: The filtrate is filtered using a ceramic membrane or organic polymer flat sheet microfiltration membrane with a pore size of 0.1-0.45μm under conditions of transmembrane pressure of 0.1-0.3MPa and temperature of 25-40℃ to remove macromolecular impurities, particles and residual small solids. S53. Ultrafiltration: The microfiltration permeate is treated with a spiral wound ultrafiltration membrane or hollow fiber ultrafiltration membrane module with a molecular weight cutoff of 5kDa-10kDa. The operating pressure is 0.3-0.6MPa and the temperature is 25-40℃. The permeate is collected. At the same time, all the retentate generated during the ultrafiltration process is collected and pumped back to the feed tank of the microfiltration step, mixed with the subsequent microfiltration permeate, and re-entered into this ultrafiltration step for treatment. The resulting ultrafiltration permeate is the primary purified solution.

8. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: Step six includes the following specific steps: S61. Preparation of the adsorption column: The composite chromatography medium is prepared by wet packing a column after mixing macroporous adsorption resin and ion exchange fiber at a dry basis mass ratio of 3:1-5:

1. The column bed height-to-diameter ratio is 5:1-10:

1. Before use, it is equilibrated with 3-5 column volumes of anhydrous ethanol and 2-3 column volumes of pH 7.0 phosphate buffer. S62, Dynamic Adsorption: The primary purified solution obtained in step 5 is loaded onto the equilibrated adsorption column at a flow rate of 1-3 BV / h, and the effluent is collected to monitor the breakthrough point of the target component. S63. Gradient elution: After adsorption, the column is first rinsed with 2-3 column volumes of deionized water at a flow rate of 2-4 BV / h. Then, a stepwise gradient elution is performed with aqueous solutions containing 5%-10% ethanol, 20%-30% ethanol, and 50%-60% ethanol. The amount of eluent used for each gradient is 3-5 column volumes, and the flow rate is 1-2 BV / h. S64. Combined Collection: The elution process was monitored by an online ultraviolet detector at wavelengths of 214 nm and 280 nm. The main target component eluents were collected from the elution stage containing 20%-30% ethanol to the elution stage containing 50%-60% ethanol to obtain the combined antler high-activity component eluent.

9. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 8, characterized in that: In step six, the macroporous adsorption resin is one of HPD-100, AB-8, D-101 or X-5, and all of these types are non-polar or weakly polar adsorption resins; the ion exchange fiber is a strong acid cation exchange fiber or a weak acid cation exchange fiber, with an exchange capacity of 4.0-5.0 mmol / g.

10. The enzymatically assisted extraction process for highly active components from deer antler powder according to claim 1, characterized in that: Step seven includes the following specific steps: S71. Vacuum Concentration: The combined eluent of highly active antler ingredients collected in step six is ​​introduced into a rotary evaporator and concentrated under reduced pressure at a temperature of 40-50℃ and a vacuum degree of -0.08 to -0.1MPa until the volume of the concentrated liquid is reduced to 1 / 10-1 / 15 of the original volume, to obtain a concentrated extract. S72. Pre-freezing: Transfer the concentrated extract to the material tray of the vacuum freeze dryer, spread it into a thin layer with a thickness of no more than 1.5 cm, and place it in a quick-freezing chamber at -40℃ to -50℃ for 4-8 hours to pre-freeze the material completely. S73. Vacuum freeze drying: Transfer the pre-frozen material trays to the freeze drying chamber, start the vacuum system, reduce the pressure inside the chamber to 10-30 Pa, and then start the heating program to carry out sublimation drying and desorption drying in stages: First, sublimation drying is carried out at -20℃ to -10℃ for 10-20 hours, and then the shelf temperature is gradually raised to 20-30℃ and maintained for 8-12 hours for desorption drying until the residual moisture content of the material is less than 5%; S74. Discharge and Packaging: After freeze-drying, the material is removed under nitrogen protection and immediately crushed and passed through an 80-100 mesh sieve in a drying room with a relative humidity of less than 20% to obtain light yellow to light brown freeze-dried powder of antler high-activity ingredients, which is then vacuum-packed in aluminum foil bags.

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