Process for preparing high-activity cartilage extract by high-efficiency enzymolysis

Abstract

This invention discloses a highly efficient enzymatic hydrolysis process for preparing a highly active cartilage extract, belonging to the field of bioactive substance extraction technology. The process first involves low-temperature washing, pulverization, and fluid extraction defatting of the cartilage raw material. Then, the pretreated cartilage is suspended in a phosphate buffer solution. An MCM-41 type mesoporous silica-loaded complex enzyme combined with a soybean peptide template is used for isothermal enzymatic hydrolysis and in-situ assembly. After separating the carrier, the hydrolysate is obtained. After pH adjustment, argon-air mixed gas microbubbling, and ultrasonic treatment, the hydrolysate is filtered to obtain a crude extract. This extract is then subjected to ultrafiltration fractionation, nanofiltration desalting, pH adjustment, and enhanced stirring. Finally, it is concentrated by ultrafiltration, spray-dried, and sieved to obtain the highly active cartilage extract. This invention significantly improves enzymatic hydrolysis efficiency and active ingredient retention rate through the synergistic effect of mesoporous confined targeted enzymatic hydrolysis and in-situ assembly of peptide templates, resulting in a stable nanostructure, greatly improving water solubility and dispersion uniformity, and effectively enhancing product purity and storage stability.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction technology, specifically a process for the efficient enzymatic hydrolysis preparation of highly active cartilage extract. Background Technology

[0002] Animal cartilage is rich in chondroitin sulfate, collagen peptides, glycosaminoglycans and other bioactive substances, and has extremely high application value in joint health care, functional foods and biomedicine.

[0003] The current industrial production of cartilage extracts faces numerous insurmountable drawbacks. For example, high-temperature hydrolysis severely damages the molecular structure of active substances, leading to a significant loss of product bioactivity. Single-stage free enzymatic hydrolysis lacks spatial confinement and targeting, easily causing excessive fragmentation of active components, resulting in poor hydrolysis efficiency and controllability, and the inability to recycle enzyme preparations. The enzymatic hydrolysis products lack in-situ stabilization treatment, exhibiting high molecular surface energy that easily leads to aggregation and precipitation, resulting in extremely poor water solubility and dispersibility, failing to meet subsequent application requirements. The extraction process lacks efficient separation and enhancement methods, leading to insufficient dissolution of active ingredients, low product yield, and difficulty in removing salts and small molecule impurities, resulting in insufficient product purity. While existing technologies attempt to combine enzymatic hydrolysis with membrane separation to optimize the process, they generally suffer from problems such as disconnect between enzymatic hydrolysis and separation processes, lack of structured product assembly, poor system compatibility, and insufficient stability.

[0004] Therefore, it is necessary to provide a process for the efficient enzymatic hydrolysis preparation of highly active cartilage extract to solve the above-mentioned technical problems. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient enzymatic hydrolysis process for preparing highly active cartilage extracts, so as to solve the technical problem that existing cartilage extract preparation processes cannot simultaneously achieve both enzymatic hydrolysis efficiency and product structural stability.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a process for efficiently preparing highly active cartilage extract by enzymatic hydrolysis, comprising the following steps: (1) Raw material pretreatment: After removing impurities, washing at low temperature, and pulverizing at low temperature, the cartilage raw material is then degreased by fluid extraction to obtain pretreated cartilage; (2) Enzymatic hydrolysis and assembly: The pretreated cartilage and buffer solution were prepared into a suspension, and MCM-41 type mesoporous silica-loaded complex enzyme and polypeptide template were added. After enzymatic hydrolysis by constant temperature stirring, the MCM-41 type mesoporous silica-loaded complex enzyme was separated to obtain the enzymatic hydrolysate. (3) Ultrasonic microbubble treatment: After adjusting the pH of the enzymatic hydrolysate, a mixed gas is introduced to form microbubbles, which are then treated with ultrasound and filtered to obtain a crude extract; (4) Membrane purification: The crude extract is subjected to ultrafiltration membrane fractionation, nanofiltration membrane desalting, pH adjustment and stirring, and then ultrafiltration concentration to obtain concentrated solution; (5) Drying: The concentrate is spray-dried and sieved to obtain a highly active cartilage extract.

[0007] Preferably, in step (1): the low-temperature cleaning uses deionized water at 4-8℃ and the cleaning is performed 2-3 times; the low-temperature pulverization temperature is ≤15℃, the particle size after pulverization is 1.0-2.0mm, and then fluid extraction degreasing is performed at 15-25℃ and 15-25MPa for 0.8-1.2h.

[0008] Preferably, in step (2): the buffer solution is a phosphate buffer solution with a pH of 5.5 to 6.0, the concentration of the phosphate buffer solution is 0.05 to 0.1 mol / L, the solid-liquid ratio of the pretreated cartilage to the phosphate buffer solution is 1:20 to 25; the temperature of the isothermal stirring enzymatic hydrolysis is 35 to 40°C, the stirring rate is 400 to 600 r / min, and the enzymatic hydrolysis time is 2 to 3 h.

[0009] Preferably, in step (2): the amount of the MCM-41 type mesoporous silica-loaded complex enzyme added is 0.5% to 0.8% of the dry weight of the cartilage raw material; the polypeptide template is soybean peptide with a molecular weight of 3000 to 5000 Da, and the amount of soybean peptide added is 0.1% to 0.2% of the dry weight of the cartilage raw material; the pore size of the MCM-41 type mesoporous silica is 15 to 20 nm, and the complex enzyme is composed of papain, chondroitin sulfate ABC and neutral protease in a mass ratio of (2 to 4): 2: 2, with an enzyme fixation amount of 50 to 60 mg / g.

[0010] Preferably, in step (3): the pH of the enzymatic hydrolysate is adjusted to 5.6–6.0, the mixed gas is argon and air, the diameter of the formed microbubbles is 20–50 μm, and the flow rate of the mixed gas is 0.3–0.5 m³ / h. 3 / min; the ultrasonic treatment power is 200-300W, the frequency is 50-70kHz, and the treatment time is 8-12min.

[0011] Preferably, in step (4): the molecular weight cutoff of the ultrafiltration membrane in the ultrafiltration membrane fractionation is 3000-10000 Da, and the molecular weight cutoff of the nanofiltration membrane in the nanofiltration membrane desalination is 300-500 Da; then the pH is adjusted to 3.8-4.2 and stirred for 8-12 min; the ultrafiltration pressure of the ultrafiltration concentration is 0.3-0.4 MPa, the feed circulation rate is 25-35 L / h, and the solid content of the concentrate is 10%-15%.

[0012] Preferably, in step (4): the pH is adjusted using hydrochloric acid with a concentration of 0.08 to 0.12 mol / L, and the stirring rate is 300 to 400 r / min; the ultrafiltration membrane operates at a pressure of 0.33 to 0.37 MPa and the feed circulation rate is 28 to 32 L / h.

[0013] Preferably, in step (5): the inlet air temperature of the spray dryer is 190-200℃, the outlet air temperature is 80-85℃, the feed rate is 5-8mL / min, and the dried product is passed through an 80-100 mesh sieve.

[0014] Preferably, the preparation method of the MCM-41 type mesoporous silica-loaded complex enzyme is as follows: 1a) Take MCM-41 type mesoporous silica, ultrasonically clean it with anhydrous ethanol, and dry it at 100-110℃ for 2-3 hours; 2a) The MCM-41 type mesoporous silica was added to a toluene solution of 3wt% to 5wt% γ-aminopropyltriethoxysilane and refluxed at 60 to 70°C for 4 to 6 hours. After washing, it was vacuum dried at 80 to 90°C to obtain modified MCM-41 type mesoporous silica. 3a) Weigh papain, chondroitin sulfate ABC and neutral protease in a mass ratio of (2-4):2:2, dissolve them in phosphate buffer at pH 7.0-7.5, and prepare a 1-2 mg / mL complex enzyme solution. 4a) Add the modified MCM-41 type mesoporous silica to the composite enzyme solution, adsorb at 25-30°C for 1-2 hours, add 0.5wt%-1.0wt% of glutaraldehyde to the system, and perform cross-linking reaction for 1-2 hours; 5a) After filtration, wash with phosphate buffer at pH 5.5–6.0 and vacuum dry to constant weight to obtain MCM-41 type mesoporous silica-loaded complex enzyme.

[0015] Preferably, the soybean peptide template is prepared by: 1b) Prepare a protein solution of 8wt% to 12wt% soy protein isolate, adjust the pH to 7.0 to 7.5, and preheat it at 50 to 60°C; 2b) Add a mixture of neutral protease and alkaline protease at a concentration of 2% to 4% of the dry weight of soy protein isolate. Enzymatically hydrolyze the enzyme at 50 to 60°C for 3 to 4 hours, and then inactivate the enzyme at 90 to 95°C. 3b) The enzymatic hydrolysate is filtered through a 300-400 mesh filter cloth to remove residue; 4b) Pass the crude extract sequentially through 10000Da and 3000Da ultrafiltration membranes, and collect the 3000Da to 10000Da retentate, wherein the retentate contains soybean peptides with a molecular weight of 3000 to 5000Da; 5b) The retentate was concentrated under vacuum and then spray-dried to obtain the soybean peptide template.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a composite enzyme formed by immobilizing papain, chondroitin sulfate ABC, and neutral protease on MCM-41 mesoporous silica. Targeted enzymatic hydrolysis is achieved through the spatial confinement of the mesoporous channels, synergistically interacting with the soybean peptide template within the system to control the hydrolysis process and prevent excessive degradation of active ingredients. The composite enzyme simultaneously degrades chondroitin and the polysaccharide matrix, while the soybean peptide template simultaneously stabilizes the structure of the in-situ generated hydrolysate fragments. Both work synergistically to improve hydrolysis efficiency. The loaded enzyme can be separated and recovered to reduce production costs. Low-temperature hydrolysis conditions, combined with the structural stabilizing effect of the soybean peptide template, maximize the protection of the active structures of chondroitin sulfate and collagen peptides.

[0017] 2. This invention uses MCM-41 mesoporous silica-loaded complex enzyme as the enzymatic hydrolysis unit and soybean peptide as the in-situ assembly template, with the enzymatic hydrolysis and assembly processes occurring simultaneously. The enzymatic hydrolysis product and the soybean peptide template complete in-situ self-assembly through hydrogen bonds and electrostatic attraction to form uniform nanoscale particles, reducing molecular surface energy and inhibiting product aggregation and precipitation. The nanoscale particle structure significantly improves the water solubility and dispersion uniformity of the product, solving the problems of poor solubility and easy stratification of traditional cartilage extracts.

[0018] 3. This invention couples the enzymatic hydrolysis process of MCM-41 loaded complex enzyme with the in-situ assembly process of soybean peptide template, and with the help of microbubbles formed by argon and air and ultrasonic cavitation, achieves mild and efficient dissociation of enzymatic assembly products and substrate residues, enhances the dissolution and mass transfer efficiency of active ingredients, and improves product yield; the entire process is free from high temperature and strong mechanical shearing, and does not damage the structure of the active product formed by the enzymatic hydrolysis of loaded complex enzyme and the assembly of soybean peptide template.

[0019] 4. This invention targets nanoparticles formed by the synergistic combination of MCM-41 loaded complex enzyme and soybean peptide template. Ultrafiltration membrane is used to remove macromolecular impurities, and nanofiltration membrane is used to achieve efficient desalination. Combined with pH adjustment and stirring treatment, the nanoparticle structure is solidified, effectively removing impurities to improve product purity. The synergistic system reduces interference from free water and salt, avoids product moisture absorption, inactivation and agglomeration, and improves product storage stability and shelf life. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 This embodiment provides a highly efficient enzymatic hydrolysis process for preparing highly active cartilage extract, including the following steps: (1) Raw material pretreatment: The cartilage raw material was cleaned by removing impurities and washing twice with deionized water at 4℃. The low temperature pulverization temperature was not higher than 15℃. The particle size after pulverization was 1.0 mm. Fluid extraction and degreasing were carried out at 15℃ and 15MPa for 0.8 h to obtain pretreated cartilage. (2) Enzymatic hydrolysis and assembly: The pretreated cartilage was mixed with phosphate buffer at pH 5.5 and concentration 0.05 mol / L at a solid-liquid ratio of 1:20 to prepare a suspension. 0.5% of the dry weight of the cartilage raw material was added with MCM-41 type mesoporous silica-loaded complex enzyme and 0.1% of the dry weight of the cartilage raw material was added with soybean peptide with a molecular weight of 3000-5000 Da. The pore size of the MCM-41 type mesoporous silica was 15 nm and the enzyme immobilization amount was 50 mg / g. Enzymatic hydrolysis was carried out at 35℃ and stirring rate 400 r / min for 2 h. The MCM-41 type mesoporous silica-loaded complex enzyme was separated to obtain the enzymatic hydrolysate. (3) Ultrasonic microbubble treatment: The pH of the enzymatic hydrolysate was adjusted to 5.6, and a mixture of argon and air was introduced to form microbubbles with a diameter of 20 μm. The flow rate of the mixed gas was 0.3 m³ / s. 3 The solution was ultrasonically treated for 8 minutes at a power of 200W and a frequency of 50kHz, and then filtered to obtain the crude extract. (4) Membrane purification: The crude extract was sequentially passed through an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da and a nanofiltration membrane with a molecular weight cutoff of 300-500 Da for desalination. The pH was adjusted to 3.8 with 0.08 mol / L hydrochloric acid, stirred for 8 min at a stirring rate of 300 r / min, and concentrated to a solid content of 10% by ultrafiltration at an ultrafiltration pressure of 0.3 MPa and a feed circulation rate of 25 L / h to obtain a concentrated solution. (5) Drying: The concentrated liquid is spray-dried under the conditions of inlet air temperature of 190℃, outlet air temperature of 80℃ and feed rate of 5mL / min. The dried product is passed through an 80-mesh sieve to obtain a highly active cartilage extract.

[0022] The preparation method of the MCM-41 type mesoporous silica-loaded complex enzyme in this embodiment is as follows: 1a) Take MCM-41 type mesoporous silica, ultrasonically clean it with anhydrous ethanol, and dry it at 100℃ for 2 hours; 2a) The MCM-41 type mesoporous silica was added to a toluene solution of 3wt% γ-aminopropyltriethoxysilane and refluxed at 60°C for 4h. After washing, it was vacuum dried at 80°C to obtain modified MCM-41 type mesoporous silica. 3a) Weigh papain, chondroitin sulfate ABC and neutral protease in a mass ratio of 2:2:2, dissolve them in phosphate buffer at pH 7.0, and prepare a 1 mg / mL complex enzyme solution. 4a) The modified MCM-41 type mesoporous silica was added to the composite enzyme solution and adsorbed at 25°C for 1 h. Then, 0.5 wt% glutaraldehyde was added to the system and the cross-linking reaction was carried out for 1 h. 5a) After filtration, wash with phosphate buffer at pH 5.5 and vacuum dry to constant weight to obtain MCM-41 type mesoporous silica-loaded complex enzyme.

[0023] The method for preparing the soybean peptide template in this embodiment is as follows: 1b) Prepare an 8wt% soy protein isolate solution, adjust the pH to 7.0, and preheat it at 50°C; 2b) Add a mixture of neutral protease and alkaline protease at a concentration of 2% of the dry weight of soy protein isolate. Enzymatically hydrolyze the mixture at 50°C for 3 hours, and then inactivate the enzyme at 90°C. 3b) The enzymatic hydrolysate is filtered through a 300-mesh filter cloth to remove residue; 4b) Pass the crude extract sequentially through 10000Da and 3000Da ultrafiltration membranes, and collect the 3000Da to 10000Da retentate, wherein the retentate contains soybean peptides with a molecular weight of 3000 to 5000Da; 5b) The retentate was concentrated under vacuum and then spray-dried to obtain the soybean peptide template.

[0024] Example 2 This embodiment provides a highly efficient enzymatic hydrolysis process for preparing highly active cartilage extract, including the following steps: (1) Raw material pretreatment: The cartilage raw material was cleaned by removing impurities and washing twice with deionized water at 6℃. The low temperature pulverization temperature was not higher than 15℃. The particle size after pulverization was 1.5mm. Fluid extraction and degreasing were carried out at 20℃ and 20MPa for 1.0h to obtain pretreated cartilage. (2) Enzymatic hydrolysis and assembly: The pretreated cartilage was mixed with phosphate buffer at pH 5.8 and concentration 0.07 mol / L at a solid-liquid ratio of 1:22 to form a suspension. 0.65% of the dry weight of the cartilage raw material was added with MCM-41 type mesoporous silica-loaded complex enzyme and 0.15% of the dry weight of the cartilage raw material was added with soybean peptide with a molecular weight of 3000-5000 Da. The pore size of the MCM-41 type mesoporous silica was 18 nm and the enzyme immobilization amount was 55 mg / g. Enzymatic hydrolysis was carried out at 38℃ and stirring rate of 500 r / min for 2.5 h. The MCM-41 type mesoporous silica-loaded complex enzyme was separated to obtain the enzymatic hydrolysate. (3) Ultrasonic microbubble treatment: The pH of the enzymatic hydrolysate was adjusted to 5.8, and a mixture of argon and air was introduced to form microbubbles with a diameter of 35 μm. The flow rate of the mixed gas was 0.4 m³ / s. 3 The solution was ultrasonically treated for 10 minutes at a power of 250W and a frequency of 60kHz, and then filtered to obtain the crude extract. (4) Membrane purification: The crude extract was sequentially passed through an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da and a nanofiltration membrane with a molecular weight cutoff of 300-500 Da for desalination. The pH was adjusted to 4.0 with 0.10 mol / L hydrochloric acid, stirred for 10 min at a stirring rate of 350 r / min, and concentrated by ultrafiltration at an ultrafiltration pressure of 0.35 MPa and a feed circulation rate of 30 L / h to a solid content of 12.5% ​​to obtain a concentrate. (5) Drying: The concentrate was spray-dried at an inlet air temperature of 195°C, an outlet air temperature of 83°C, and a feed rate of 6.5 mL / min. The dried product was passed through a 90-mesh sieve to obtain a highly active cartilage extract.

[0025] The preparation method of the MCM-41 type mesoporous silica-loaded complex enzyme in this embodiment is as follows: 1a) Take MCM-41 type mesoporous silica, ultrasonically clean it with anhydrous ethanol, and dry it at 105℃ for 2.5h; 2a) The MCM-41 type mesoporous silica was added to a toluene solution of 4 wt% γ-aminopropyltriethoxysilane and refluxed at 65°C for 5 h. After washing, it was vacuum dried at 85°C to obtain modified MCM-41 type mesoporous silica. 3a) Weigh papain, chondroitin sulfate ABC and neutral protease in a mass ratio of 3:2:2, dissolve them in phosphate buffer at pH 7.2, and prepare a 1.5 mg / mL complex enzyme solution. 4a) The modified MCM-41 type mesoporous silica was added to the composite enzyme solution and adsorbed at 27°C for 1.5 h. Then, 0.75 wt% glutaraldehyde was added to the system and the cross-linking reaction was carried out for 1.5 h. 5a) After filtration, wash with phosphate buffer at pH 5.8 and vacuum dry to constant weight to obtain MCM-41 type mesoporous silica-loaded complex enzyme.

[0026] The method for preparing the soybean peptide template in this embodiment is as follows: 1b) Prepare a 10wt% soy protein isolate solution, adjust the pH to 7.2, and preheat it at 55°C; 2b) Add a mixture of neutral protease and alkaline protease at a concentration of 3% of the dry weight of soy protein isolate. Enzymatically hydrolyze the mixture at 55°C for 3.5 hours, and then inactivate the enzyme at 92°C. 3b) The enzymatic hydrolysate is filtered through a 350-mesh filter cloth to remove residue; 4b) Pass the crude extract sequentially through 10000Da and 3000Da ultrafiltration membranes, and collect the 3000Da to 10000Da retentate, wherein the retentate contains soybean peptides with a molecular weight of 3000 to 5000Da; 5b) The retentate was concentrated under vacuum and then spray-dried to obtain the soybean peptide template.

[0027] Example 3 This embodiment provides a highly efficient enzymatic hydrolysis process for preparing highly active cartilage extract, including the following steps: (1) Raw material pretreatment: The cartilage raw material was cleaned by removing impurities, washed three times with deionized water at 8℃, and the low temperature pulverization temperature was not higher than 15℃. The particle size after pulverization was 2.0 mm. The cartilage was degreased by fluid extraction at 25℃ and 25MPa for 1.2 h to obtain pretreated cartilage. (2) Enzymatic hydrolysis and assembly: The pretreated cartilage was mixed with phosphate buffer at pH 6.0 and concentration 0.1 mol / L at a solid-liquid ratio of 1:25 to form a suspension. 0.8% of the dry weight of the cartilage raw material was added with MCM-41 type mesoporous silica-loaded complex enzyme and 0.2% of the dry weight of the cartilage raw material with a molecular weight of 3000-5000 Da soybean peptide. The pore size of the MCM-41 type mesoporous silica was 20 nm and the enzyme immobilization amount was 60 mg / g. Enzymatic hydrolysis was carried out at 40 °C and a stirring rate of 600 r / min for 3 h. The MCM-41 type mesoporous silica-loaded complex enzyme was separated to obtain the enzymatic hydrolysate. (3) Ultrasonic microbubble treatment: The pH of the enzymatic hydrolysate is adjusted to 6.0, and a mixture of argon and air is introduced to form microbubbles with a diameter of 50 μm. The flow rate of the mixed gas is 0.5 m³ / s. 3 The solution was ultrasonically treated for 12 minutes at a power of 300W and a frequency of 70kHz, and then filtered to obtain the crude extract. (4) Membrane purification: The crude extract was sequentially passed through an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da and a nanofiltration membrane with a molecular weight cutoff of 300-500 Da for desalination. The pH was adjusted to 4.2 with 0.12 mol / L hydrochloric acid. The mixture was stirred for 12 min at a stirring rate of 400 r / min and then concentrated by ultrafiltration at an ultrafiltration pressure of 0.4 MPa and a feed circulation rate of 35 L / h to a solid content of 15% to obtain a concentrate. (5) Drying: The concentrate is spray-dried at an inlet air temperature of 200°C, an outlet air temperature of 85°C, and a feed rate of 8 mL / min. The dried product is then passed through a 100-mesh sieve to obtain a highly active cartilage extract.

[0028] The preparation method of the MCM-41 type mesoporous silica-loaded complex enzyme in this embodiment is as follows: 1a) Take MCM-41 type mesoporous silica, ultrasonically clean it with anhydrous ethanol, and dry it at 110℃ for 3h; 2a) The MCM-41 type mesoporous silica was added to a 5wt% toluene solution of γ-aminopropyltriethoxysilane and refluxed at 70°C for 6 hours. After washing, it was vacuum dried at 90°C to obtain modified MCM-41 type mesoporous silica. 3a) Weigh papain, chondroitin sulfate ABC and neutral protease in a mass ratio of 4:2:2, dissolve them in phosphate buffer at pH 7.5, and prepare a 2 mg / mL complex enzyme solution. 4a) The modified MCM-41 type mesoporous silica was added to the composite enzyme solution and adsorbed at 30°C for 2 hours. Then, 1.0 wt% glutaraldehyde was added to the system and the cross-linking reaction was carried out for 2 hours. 5a) After filtration, wash with phosphate buffer at pH 6.0 and vacuum dry to constant weight to obtain MCM-41 type mesoporous silica-loaded complex enzyme.

[0029] The method for preparing the soybean peptide template in this embodiment is as follows: 1b) Prepare a 12wt% soy protein isolate solution, adjust the pH to 7.5, and preheat it at 60°C; 2b) Add a mixture of neutral protease and alkaline protease at a concentration of 4% of the dry weight of soy protein isolate. Enzymatically hydrolyze the mixture at 60°C for 4 hours, and then inactivate the enzyme at 95°C. 3b) The enzymatic hydrolysate is filtered through a 400-mesh filter cloth to remove residue; 4b) Pass the crude extract sequentially through 10000Da and 3000Da ultrafiltration membranes, and collect the 3000Da to 10000Da retentate, wherein the retentate contains soybean peptides with a molecular weight of 3000 to 5000Da; 5b) The retentate was concentrated under vacuum and then spray-dried to obtain the soybean peptide template.

[0030] Comparative Example 1 The only difference between this comparative example and Example 1 is that the raw material pretreatment in step (1) did not involve fluid extraction for degreasing, while the other steps and parameters were the same as in Example 1.

[0031] Expected performance: The grease in the cartilage raw material cannot be removed. The grease encapsulates the substrate fibers, which hinders the full contact between the complex enzyme and the cartilage matrix. The enzymatic hydrolysis reaction is insufficient, the enzymatic hydrolysis efficiency is significantly reduced, the active ingredients are not fully released, and the residual grease impurities will also reduce the purity of the final cartilage extract, affecting the separation and purification effect and product quality.

[0032] Comparative Example 2 The only difference between this comparative example and Example 1 is that soybean peptide templates are not added in step (2) of enzymatic hydrolysis and assembly, while other steps and parameters are the same as in Example 1.

[0033] Expected performance: The enzymatic hydrolysate lacks the in-situ assembly and stabilization effect of soybean peptide templates, and cannot form uniform and stable nano-sized particles. The high surface energy product is prone to agglomeration and precipitation, the water solubility of the extract is greatly reduced, the dispersibility is poor, the active ingredients are prone to denaturation and inactivation, and the storage stability is significantly reduced.

[0034] Comparative Example 3 The only difference between this comparative example and Example 1 is that in step (2), the enzymatic digestion and assembly use a free complex enzyme instead of the MCM-41 type mesoporous silica-loaded complex enzyme. All other steps and parameters are the same as in Example 1.

[0035] Expected performance: Free enzymes lack the spatial confinement and targeted enzymatic hydrolysis of mesoporous silica carriers. The enzymatic hydrolysis is non-selective, which easily leads to excessive degradation and destruction of active groups, resulting in a significant decrease in the bioactivity of the extract. Furthermore, the free enzymes cannot be separated and recovered, causing waste of enzyme preparations and affecting product purity.

[0036] Comparative Example 4 The only difference between this comparative example and Example 1 is that: in step (3) ultrasonic microbubble treatment, ultrasonic microbubble treatment is not performed, but only conventional filtration is used. All other steps and parameters are the same as in Example 1.

[0037] Expected performance: Lacking the cavitation dissociation and mass transfer enhancement effects of ultrasonic microbubbles, the enzymatic hydrolysis products cannot be fully dissolved and separated from the substrate residue, the active ingredients are not completely extracted, the product yield and extraction efficiency are significantly reduced, and some effective ingredients remain in the filter residue and cannot be recovered.

[0038] Comparative Example 5 The only difference between this comparative example and Example 1 is that step (4) membrane purification does not involve nanofiltration membrane desalination, while the other steps and parameters are the same as in Example 1.

[0039] Expected performance: Inorganic salts and small molecule impurities in the system cannot be removed. Salt residue reduces the purity of cartilage extract, damages the stability of nanostructure, and subsequent spray drying is prone to moisture absorption and clumping, shortening the product's shelf life and deteriorating storage stability and performance.

[0040] Comparative Example 6 The only difference between this comparative example and Example 1 is that the pH is not adjusted and the mixture is stirred in step (4) of membrane purification, while the other steps and parameters are the same as in Example 1.

[0041] Expected performance: The lack of pH adjustment and stirring to strengthen the structure means that the assembled nanoparticles cannot be stably solidified, resulting in uneven particle size, decreased water solubility and dispersibility, reduced retention of active ingredients, and easy stratification and aggregation in the system. Ultimately, the uniformity and quality of the final product performance are significantly reduced.

[0042] To compare the performance differences between the processes for preparing highly active cartilage extracts via efficient enzymatic hydrolysis provided in Examples 1-3 and Comparative Examples 1-6, the present invention provides the following experimental methods: 1. Active ingredient retention rate test Chondroitin sulfate and collagen peptides were used as characteristic active substances, and ultraviolet spectrophotometry was used for quantitative determination. The ratio of the actual content of the target active ingredient in the sample to the theoretical maximum content was calculated to obtain the retention rate of the active ingredient.

[0043] 2. Enzymatic hydrolysis efficiency test The free amino nitrogen content in the system was determined by formaldehyde titration. The amount of amino nitrogen generated was used to characterize the degree of degradation of the cartilage substrate, and the enzymatic hydrolysis efficiency was calculated.

[0044] 3. Water solubility test Weigh an equal amount of cartilage extract sample, add excess deionized water, stir thoroughly at room temperature until dissolution equilibrium is reached, filter to separate insoluble matter, dry the insoluble matter to constant weight and weigh it, calculate the percentage of dissolved part to the total sample mass, and thus characterize water solubility.

[0045] 4. Product purity test The purity of the product was determined by high performance liquid chromatography (HPLC) and expressed as the percentage of the peak area of ​​the target active component to the total peak area, reflecting the removal efficiency of small molecule impurities, unreacted substrates, and residual proteins.

[0046] 5. Storage stability test After sealing each sample, it was placed in a constant temperature and humidity chamber at 40℃ and 75% relative humidity for accelerated aging test. After 30 days, samples were taken to measure the change rate of active ingredient retention and appearance. The smaller the change rate, the better the storage stability.

[0047] 6. Dispersion uniformity test The samples were prepared into aqueous solutions of the same concentration. The clarity and stratification of the solutions were observed. The particle dispersion was observed under a microscope to evaluate whether there was aggregation, precipitation, or stratification. The experimental data are as follows: Table 1 Performance test results of the examples and comparative examples

[0048] Based on the experimental data in Table 1, it can be seen that in Examples 1-3, due to the synergistic effect of the targeted confined enzymatic hydrolysis of the MCM-41 type mesoporous silica-loaded complex enzyme and the in-situ assembly of soybean peptide templates, coupled with the process coupling of ultrasonic microbubble mass transfer enhancement and multi-stage membrane purification, the overall performance of the products reached the optimal level. The retention rate of active ingredients was 92.4%~95.8%, the enzymatic hydrolysis efficiency was 90.1%~94.6%, the water solubility was 96.3%~98.5%, the product purity was 93.7%~96.2%, the activity decline rate after 30 days of accelerated aging was only 2.1%~3.8%, and the dispersion uniformity was good to excellent.

[0049] In contrast, Comparative Example 1 lacked a fluid extraction defatting process. The oil in the cartilage raw material formed a hydrophobic shielding layer, physically preventing effective contact between the substrate and the MCM-41-loaded complex enzyme. This disrupted the targeted enzymatic hydrolysis pathway of the mesoporous channels, resulting in insufficient enzymatic hydrolysis and a hydrolysis efficiency of only 59.3%. Simultaneously, the combination of oil impurities and active products reduced the retention rate of active ingredients to 64.2% and the product purity to only 74.8%, significantly diminishing the core performance of the product. Comparative Example 2 did not add a soybean peptide template. The system lost its in-situ assembled structural stability unit. The active fragments generated by enzymatic hydrolysis could not form a stable nanoscale structure through hydrogen bonds and electrostatic attraction. The high surface energy active product spontaneously aggregated and precipitated, causing a sharp drop in water solubility to 74.2%. Furthermore, the product without template protection was prone to inactivation during storage, with an accelerated aging activity decline rate of up to 14.9% after 30 days, and dispersion uniformity deteriorated to a poor level. Comparative Example 3 used a free complex enzyme instead of the MCM-41 loaded complex enzyme. The system lost the spatial confinement and targeted regulation of the mesoporous channels, resulting in non-selective enzymatic hydrolysis and a tendency for excessive degradation. This directly destroyed the active structures of chondroitin sulfate and collagen peptides, leading to a retention rate of only 54.6%, the lowest among all groups. Comparative Example 4 did not undergo ultrasonic microbubble treatment, missing the mass transfer enhancement effects of ultrasonic cavitation and microbubble-assisted dissociation. The enzymatic hydrolysis products could not be fully dissolved and separated from the cartilage matrix residue, resulting in incomplete substrate degradation and product release. The enzymatic hydrolysis efficiency was only 61.4%, significantly reducing the yield and extraction effect of active ingredients. Comparative Example 5 did not undergo nanofiltration desalination. Residual inorganic salt ions in the system disrupted the charge balance of the active product's nanostructure, leading to a loose product structure, impurity enrichment, and a product purity as low as 69.2%. Simultaneously, the salt caused moisture absorption and structural instability, significantly deteriorating the product's storage stability. Comparative Example 6 lacked pH adjustment and stirring processes, which prevented the structural solidification of nanoparticles formed by enzymatic hydrolysis and template assembly. As a result, the product had uneven particle size and was prone to dissociation. The water solubility decreased to 79.5%, the dispersion uniformity deteriorated, and the structural stability and retention rate of the active ingredients decreased simultaneously.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A process for efficiently preparing highly active cartilage extract via enzymatic hydrolysis, characterized in that, Includes the following steps: (1) Raw material pretreatment: After removing impurities, washing at low temperature and pulverizing at low temperature, the cartilage raw material is then degreased by fluid extraction to obtain pretreated cartilage; (2) Enzymatic hydrolysis and assembly: The pretreated cartilage and buffer solution were prepared into a suspension, and MCM-41 type mesoporous silica-loaded complex enzyme and polypeptide template were added. After enzymatic hydrolysis by constant temperature stirring, the MCM-41 type mesoporous silica-loaded complex enzyme was separated to obtain the enzymatic hydrolysate. (3) Ultrasonic microbubble treatment: After adjusting the pH of the enzymatic hydrolysate, a mixed gas is introduced to form microbubbles, which are then treated with ultrasound and filtered to obtain a crude extract; (4) Membrane purification: The crude extract is subjected to ultrafiltration membrane fractionation, nanofiltration membrane desalting, pH adjustment and stirring, and then ultrafiltration concentration to obtain concentrated solution; (5) Drying: The concentrate is spray-dried and sieved to obtain a highly active cartilage extract.

2. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 1, characterized in that, In step (1): the low-temperature cleaning uses deionized water at 4-8℃ and the cleaning is performed 2-3 times; the low-temperature pulverization temperature is ≤15℃, the particle size after pulverization is 1.0-2.0mm, and then fluid extraction degreasing is performed at 15-25℃ and 15-25MPa for 0.8-1.2h.

3. The process according to claim 1, characterized in that, In step (2): the buffer solution is a phosphate buffer solution with a pH of 5.5 to 6.0, the concentration of the phosphate buffer solution is 0.05 to 0.1 mol / L, the solid-liquid ratio of the pretreated cartilage to the phosphate buffer solution is 1:20 to 25; the temperature of the isothermal stirring enzymatic hydrolysis is 35 to 40°C, the stirring rate is 400 to 600 r / min, and the enzymatic hydrolysis time is 2 to 3 h.

4. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 3, characterized in that, In step (2): the amount of the MCM-41 type mesoporous silica-loaded complex enzyme added is 0.5% to 0.8% of the dry weight of the cartilage raw material; the polypeptide template is soybean peptide with a molecular weight of 3000 to 5000 Da, and the amount of soybean peptide added is 0.1% to 0.2% of the dry weight of the cartilage raw material; the pore size of the MCM-41 type mesoporous silica is 15 to 20 nm, and the complex enzyme is composed of papain, chondroitin sulfate ABC and neutral protease in a mass ratio of (2 to 4): 2: 2, with an enzyme fixation amount of 50 to 60 mg / g.

5. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 1, characterized in that, In step (3): the pH of the enzymatic hydrolysate is adjusted to 5.6–6.0, the mixed gas is argon and air, the diameter of the formed microbubbles is 20–50 μm, and the flow rate of the mixed gas is 0.3–0.5 m³ / h. 3 / min; the ultrasonic treatment power is 200-300W, the frequency is 50-70kHz, and the treatment time is 8-12min.

6. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 1, characterized in that, In step (4): the molecular weight cutoff of the ultrafiltration membrane in the ultrafiltration membrane fractionation is 3000-10000 Da, and the molecular weight cutoff of the nanofiltration membrane in the nanofiltration membrane desalination is 300-500 Da; then the pH is adjusted to 3.8-4.2 and stirred for 8-12 min; the ultrafiltration pressure of the ultrafiltration concentration is 0.3-0.4 MPa, the feed circulation rate is 25-35 L / h, and the solid content of the concentrate is 10%-15%.

7. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 6, characterized in that, In step (4): pH is adjusted using hydrochloric acid with a concentration of 0.08 to 0.12 mol / L, and the stirring rate is 300 to 400 r / min; the ultrafiltration membrane operates at a pressure of 0.33 to 0.37 MPa and the feed circulation rate is 28 to 32 L / h.

8. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 1, characterized in that, In step (5): the inlet air temperature of the spray dryer is 190-200℃, the outlet air temperature is 80-85℃, the feed rate is 5-8mL / min, and the dried product is passed through an 80-100 mesh sieve.

9. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 4, characterized in that, The preparation method of the MCM-41 type mesoporous silica-loaded complex enzyme is as follows: 1a) Take MCM-41 type mesoporous silica, ultrasonically clean it with anhydrous ethanol, and dry it at 100-110℃ for 2-3 hours; 2a) The MCM-41 type mesoporous silica was added to a toluene solution of 3wt% to 5wt% γ-aminopropyltriethoxysilane and refluxed at 60 to 70°C for 4 to 6 hours. After washing, it was vacuum dried at 80 to 90°C to obtain modified MCM-41 type mesoporous silica. 3a) Weigh papain, chondroitin sulfate ABC and neutral protease in a mass ratio of (2-4):2:2, dissolve them in phosphate buffer at pH 7.0-7.5, and prepare a 1-2 mg / mL complex enzyme solution. 4a) Add the modified MCM-41 type mesoporous silica to the composite enzyme solution, adsorb at 25-30°C for 1-2 hours, add 0.5wt%-1.0wt% of glutaraldehyde to the system, and perform cross-linking reaction for 1-2 hours; 5a) After filtration, wash with phosphate buffer at pH 5.5–6.0 and vacuum dry to constant weight to obtain MCM-41 type mesoporous silica-loaded complex enzyme.

10. The process for preparing highly active cartilage extract by efficient enzymatic hydrolysis according to claim 4, characterized in that, The method for preparing the soybean peptide template is as follows: 1b) Prepare a protein solution of 8wt% to 12wt% soy protein isolate, adjust the pH to 7.0 to 7.5, and preheat it at 50 to 60°C; 2b) Add a mixture of neutral protease and alkaline protease at a concentration of 2% to 4% of the dry weight of soy protein isolate. Enzymatically hydrolyze the enzyme at 50 to 60°C for 3 to 4 hours, and then inactivate the enzyme at 90 to 95°C. 3b) The enzymatic hydrolysate is filtered through a 300-400 mesh filter cloth to remove residue; 4b) Pass the crude extract sequentially through 10000Da and 3000Da ultrafiltration membranes, and collect the 3000Da to 10000Da retentate, wherein the retentate contains soybean peptides with a molecular weight of 3000 to 5000Da; 5b) The retentate was concentrated under vacuum and then spray-dried to obtain the soybean peptide template.

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