A method for extracting elastin peptides from animal cartilage

Abstract

This invention relates to the field of elastin peptide technology, specifically a method for extracting elastin peptides from animal cartilage. The specific steps are as follows: The animal cartilage is cleaned of surface connective tissue and fat, washed with physiological saline, and then ground and defatted to obtain pretreated cartilage particles. The pretreated cartilage particles are placed in a container, an ionic liquid is added, and the mixture is stirred. After stirring, the precipitate is collected by centrifugation. The precipitate is added to phosphate buffer to form a suspension. An immobilized complex enzyme is then added for enzymatic hydrolysis. After hydrolysis, the enzyme is inactivated by heating. The hydrolysate is filtered to obtain an enzymatic hydrolysate. Macroporous resin is added to the hydrolysate, and the mixture is filtered to obtain a clear solution. The clear solution is then ultrafiltered to obtain elastin peptides. The ionic liquid interacts with impurities in the animal cartilage, making them easier to remove, thus creating a purer raw material environment for subsequent elastin peptide extraction and improving extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of elastin peptide technology, specifically a method for extracting elastin peptides from animal cartilage. Background Technology

[0002] Animal cartilage is a widely available and relatively abundant biological resource, commonly found in livestock such as cattle, pigs, and sheep, as well as some marine organisms. However, the utilization of animal cartilage is currently relatively limited, with a large amount of cartilage resources being discarded in industries such as meat processing. This not only wastes resources but may also cause environmental problems. Developing efficient animal cartilage utilization technologies, such as extracting and utilizing elastin peptides, can not only achieve full utilization of resources but also create significant economic and environmental benefits.

[0003] In existing enzymatic hydrolysis processes, enzyme proteins may remain in the extracted product and mix with elastin peptides. The residual enzymes increase the content of protein impurities in the product and interfere with the improvement of elastin peptide purity. Therefore, we propose a method for extracting elastin peptides from animal cartilage. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting elastin peptides from animal cartilage, in order to solve the problem mentioned in the background art that in the existing enzymatic hydrolysis process, enzyme proteins may remain in the extraction product and mix with the elastin peptides. The residual enzymes will increase the content of protein impurities in the product and interfere with the improvement of the purity of elastin peptides.

[0005] To achieve the above objectives, the present invention provides a method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0006] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, grind and degrease it to obtain pretreated cartilage particles;

[0007] S1.2 Place the pretreated cartilage particles into a container, add ionic liquid and stir; after stirring, centrifuge, wash with deionized water 2-3 times, and collect the precipitate;

[0008] Collagen fibers and proteoglycans in cartilage form a dense network through electrostatic interactions. The cationic groups of ionic liquids (such as quaternary ammonium salts) can neutralize negatively charged glycosaminoglycans (GAGs), weakening their binding with collagen / elastin. This makes cartilage particles easier to disperse and dissociate, releasing the target elastin. In addition, the hydrophobic alkyl chains of ionic liquids can insert into the phospholipid bilayer membrane structure, disrupting its integrity. The hydrophilic ends bind with water to form micelles that encapsulate lipids, facilitating thorough removal during subsequent centrifugation and washing, and reducing interference from impurities in subsequent enzymatic hydrolysis.

[0009] Elastin is rich in hydrophobic cross-linked regions (such as desmokinesin), which are originally wrapped by glycoproteins. Ionic liquids dissolve the outer glycoprotein, making it easier for immobilized complex enzymes (such as elastase) to contact the substrate in enzymatic hydrolysis, thus shortening the hydrolysis time and increasing the peptide release rate.

[0010] S1.3 Add the precipitate to a phosphate buffer solution with a pH of 7-8 to form a suspension; then add the immobilized complex enzyme to carry out the enzymatic hydrolysis reaction; after the enzymatic hydrolysis is completed, heat to 80-85℃ and maintain for 5-10 minutes to inactivate; filter to obtain the enzymatic hydrolysate;

[0011] Immobilized complex enzymes are formed into a stable three-dimensional structure through agarose gel adsorption, glutaraldehyde cross-linking, and zinc chloride treatment. This structure not only protects the active sites of enzyme molecules from damage caused by external environments (such as pH and temperature fluctuations), but also significantly extends the enzyme's lifespan. Compared with free enzymes, immobilized enzymes can be reused multiple times, reducing enzyme preparation consumption and lowering production costs.

[0012] When immobilized enzymes come into contact with cartilage suspension, enzyme molecules on the carrier surface can efficiently recognize and act on specific peptide bonds of elastin; neutral proteases preferentially hydrolyze non-elastin impurities, while elastases specifically cleave the hydrophobic regions of elastin. The synergistic effect of the two can directionally release the target peptides; in addition, the orderly arrangement of immobilized enzymes on the carrier can reduce substrate inhibition effects and increase the rate of enzymatic hydrolysis.

[0013] S1.4 Add macroporous resin with a pore size of 30-50 nm to the enzymatic hydrolysate, stir at 200-300 rpm for 30-60 min at 50-60℃, and filter to obtain a clear solution.

[0014] S1.5. The clarified liquid is subjected to ultrafiltration at a pressure of 0.1-0.3 MPa and a temperature of 25-40℃ to obtain elastin peptides.

[0015] Preferably, in step S1.1, the grinding is performed using a grinding machine with a grinding speed of 1000-2000 rpm and a grinding time of 5-15 min.

[0016] The degreasing process uses a 70%-90% ethanol solution, assisted by ultrasound with a power of 200-300W and a processing time of 15-30 minutes.

[0017] Preferably, in S1.2, the ionic liquid is hexadecyltrimethylammonium bromide, tetrabutylammonium chloride, or trimethyloctylammonium chloride.

[0018] Preferably, the mass ratio of the pretreated cartilage particles to the ionic liquid is 1:3-5.

[0019] Preferably, in step S1.2, the stirring temperature is 25-45℃, the stirring speed is 200-400rpm, and the stirring time is 1-4h.

[0020] The centrifuge speed is 3000-4000 rpm, and the centrifugation time is 10-30 min.

[0021] Preferably, in step S1.3, the mass ratio of the immobilized complex enzyme to the precipitate is 1:20-50.

[0022] Preferably, the specific preparation steps of the immobilized complex enzyme are as follows:

[0023] The agarose gel was placed in a mixed enzyme solution containing neutral protease and elastase and allowed to stand at 4-10℃ for 1-2 hours to allow enzyme molecules to adsorb onto the surface of the agarose gel.

[0024] Add a 0.5%-1% glutaraldehyde solution to the container containing the gel and enzyme solution, and react for 2-6 hours at pH 6.0-8.0 and 4-10℃; wash 3-5 times with phosphate buffer solution at pH 7-8.

[0025] The washed agarose gel immobilized with neutral protease and elastase was placed in a zinc chloride solution with a concentration of 0.1-0.5 mol / L and stirred at 200-300 rpm for 2-4 h at 25-37 °C. After the reaction was completed, the gel was washed 3-5 times with phosphate buffer at pH 7-8 to obtain the immobilized complex enzyme.

[0026] Zinc ions (Zn²⁺) can form coordination bonds with functional groups such as hydroxyl groups on the surface of agarose gel, amino groups remaining after glutaraldehyde cross-linking, or carboxyl / thiol groups in enzyme molecules, further strengthening the binding between the enzyme and the carrier. This double cross-linking (glutaraldehyde covalent cross-linking + zinc ion chelation) makes the enzyme molecules more firmly fixed on the carrier surface, reducing the risk of leakage in subsequent enzymatic hydrolysis reactions. Some active sites of neutral proteases and elastases rely on metal ions (such as Zn²⁺ and Ca²⁺) to maintain their conformation, and zinc ions enhance the immobilization stability through chelation. In addition, after glutaraldehyde cross-linking, unreacted free aldehyde groups may attack the active site of enzyme molecules (such as the serine catalysis of elastase). Zn²⁺ can bind to the aldehyde groups, blocking the side reactions with the enzyme and preventing the destruction of the enzyme active site, thereby ensuring the efficiency of enzymatic hydrolysis.

[0027] Preferably, the neutral protease and elastase mixed enzyme solution is obtained by mixing a neutral protease solution with a concentration of 5-10 mg / mL and an elastase solution with a concentration of 3-8 mg / mL at a mass ratio of 1:2-1.

[0028] Preferably, in step S1.3, the temperature of the enzymatic hydrolysis reaction is 30-37℃, the hydrolysis time is 6-10h, and the stirring speed is 100-300rpm.

[0029] Preferably, in step S1.5, the ultrafiltration membrane has a molecular weight cutoff of 3-10 kDa.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. In this method for extracting elastin peptides from animal cartilage, the ionic liquid interacts with impurities (such as fat and non-elastin) in the animal cartilage, making them easier to remove. This creates a purer raw material environment for subsequent elastin peptide extraction, improving extraction efficiency. Compared to traditional organic solvents, ionic liquids have better biocompatibility and can protect the structure of elastin during cartilage particle processing, reducing damage to the elastin peptide chains and helping to maintain its bioactivity and functional properties.

[0032] 2. In this method for extracting elastin peptides from animal cartilage, neutral protease and elastase are adsorbed onto agarose gel, cross-linked with glutaraldehyde, and treated with zinc chloride to form a stable three-dimensional structure. This structure not only protects the active sites of the enzyme molecules from damage by the external environment but also significantly extends the enzyme's lifespan. Compared to free enzymes, immobilized enzymes can be reused multiple times, reducing enzyme consumption, lowering production costs, and maintaining stable catalytic activity. Neutral protease and elastase work together to precisely hydrolyze different structural regions of elastin, significantly improving enzymatic hydrolysis efficiency and greatly increasing the extraction rate of elastin peptides. In addition, the application of immobilized composite enzymes reduces enzyme residues in the target product and improves product purity. Detailed Implementation

[0033] 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.

[0034] In the following examples, the preparation steps of agarose gel are as follows: Weigh 0.8g of agarose into an Erlenmeyer flask, add 100mL of Tris-HCl buffer solution with pH 7-8 to the Erlenmeyer flask to fully soak the agarose; place the Erlenmeyer flask on a heated stirrer, heat to 90°C and stir at 200rpm until the agarose is completely dissolved to form a uniform and transparent solution; cool the dissolved agarose solution to about 50°C and let it cool and solidify to form agarose gel.

[0035] The specific preparation steps for the immobilized complex enzyme are as follows:

[0036] 100 g of agarose gel was placed in 500 mL of a mixed enzyme solution containing 6 mg / mL Solarbiote Neutrase 3.0 SG (Bacillus subtilis neutral protease) and 5 mg / mL Sigma Elastase from porcine pancreas (elastase), and allowed to stand at 5 °C for 2 h to allow enzyme molecules to adsorb onto the surface of the agarose gel.

[0037] The mixed enzyme solution of neutral protease and elastase was obtained by mixing neutral protease solution with a concentration of 6 mg / mL and elastase solution with a concentration of 5 mg / mL at a mass ratio of 1:2.

[0038] Add a 1% glutaraldehyde solution to a container containing gel and enzyme solution, and react at pH 7 and 10℃ for 3 hours; wash 5 times with phosphate buffer solution at pH 7.

[0039] The washed agarose gel immobilized with neutral protease and elastase was placed in a 0.3 mol / L zinc chloride solution and stirred at 200 rpm for 2 h at 25 °C. After the reaction was completed, the gel was washed 5 times with phosphate buffer at pH 8 to obtain the immobilized complex enzyme.

[0040] The ionic liquid is hexadecyltrimethylammonium bromide, tetrabutylammonium chloride, or trimethyloctylammonium chloride, and the following examples preferably use hexadecyltrimethylammonium bromide.

[0041] Example 1: A method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0042] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, and grind it with a grinder at 1500 rpm for 15 minutes. After grinding, treat it with an 80% ethanol solution using ultrasonic-assisted treatment at a power of 200W for 20 minutes to obtain pretreated cartilage particles.

[0043] S1.2. Place the pretreated cartilage particles into a container, add hexadecyltrimethylammonium bromide, with a mass ratio of pretreated cartilage particles to hexadecyltrimethylammonium bromide of 1:3, and stir at 300 rpm for 4 hours at 35°C. After stirring, centrifuge at 3000 rpm for 30 minutes. Wash three times with deionized water and collect the precipitate.

[0044] S1.3 Add the precipitate to phosphate buffer at pH 7 to form a suspension; then add the immobilized complex enzyme at a mass ratio of 1:20 to the precipitate, and stir at 300 rpm for 10 h at 37 °C; after enzymatic hydrolysis, heat to 85 °C and hold for 10 min to inactivate; obtain the enzymatic hydrolysate by filtration.

[0045] S1.4 Add macroporous resin with a pore size of 50 nm to the enzymatic hydrolysate, stir at 300 rpm for 60 min at 50 °C, and filter to obtain a clear solution.

[0046] S1.5. The clarified liquid is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5kDa at a pressure of 0.3MPa and a temperature of 35℃ to obtain elastin peptides.

[0047] Example 2: A method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0048] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, and grind it with a grinder at 1500 rpm for 15 minutes. After grinding, treat it with an 80% ethanol solution using ultrasonic-assisted treatment at a power of 200W for 20 minutes to obtain pretreated cartilage particles.

[0049] S1.2. Place the pretreated cartilage particles into a container, add hexadecyltrimethylammonium bromide, with a mass ratio of pretreated cartilage particles to hexadecyltrimethylammonium bromide of 1:4, and stir at 300 rpm for 4 hours at 35°C. After stirring, centrifuge at 3000 rpm for 30 minutes. Wash three times with deionized water and collect the precipitate.

[0050] S1.3 Add the precipitate to phosphate buffer at pH 7 to form a suspension; then add the immobilized complex enzyme at a mass ratio of 1:20 to the precipitate, and stir at 300 rpm for 10 h at 37 °C; after enzymatic hydrolysis, heat to 85 °C and hold for 10 min to inactivate; obtain the enzymatic hydrolysate by filtration.

[0051] S1.4 Add macroporous resin with a pore size of 50 nm to the enzymatic hydrolysate, stir at 300 rpm for 60 min at 50 °C, and filter to obtain a clear solution.

[0052] S1.5. The clarified liquid is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5kDa at a pressure of 0.3MPa and a temperature of 35℃ to obtain elastin peptides.

[0053] Example 3: A method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0054] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, and grind it with a grinder at 1500 rpm for 15 minutes. After grinding, treat it with an 80% ethanol solution using ultrasonic-assisted treatment at a power of 200W for 20 minutes to obtain pretreated cartilage particles.

[0055] S1.2 Place the pretreated cartilage particles into a container, add hexadecyltrimethylammonium bromide, with a mass ratio of pretreated cartilage particles to hexadecyltrimethylammonium bromide of 1:5, and stir at 300 rpm for 4 hours at 35°C; after stirring, centrifuge at 3000 rpm for 30 minutes; wash three times with deionized water and collect the precipitate;

[0056] S1.3 Add the precipitate to phosphate buffer at pH 7 to form a suspension; then add the immobilized complex enzyme at a mass ratio of 1:20 to the precipitate, and stir at 300 rpm for 10 h at 37 °C; after enzymatic hydrolysis, heat to 85 °C and hold for 10 min to inactivate; obtain the enzymatic hydrolysate by filtration.

[0057] S1.4 Add macroporous resin with a pore size of 50 nm to the enzymatic hydrolysate, stir at 300 rpm for 60 min at 50 °C, and filter to obtain a clear solution.

[0058] S1.5. The clarified liquid is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5kDa at a pressure of 0.3MPa and a temperature of 35℃ to obtain elastin peptides.

[0059] Example 4: A method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0060] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, and grind it with a grinder at 1500 rpm for 15 minutes. After grinding, treat it with an 80% ethanol solution using ultrasonic-assisted treatment at a power of 200W for 20 minutes to obtain pretreated cartilage particles.

[0061] S1.2 Place the pretreated cartilage particles into a container, add hexadecyltrimethylammonium bromide, with a mass ratio of pretreated cartilage particles to hexadecyltrimethylammonium bromide of 1:5, and stir at 300 rpm for 4 hours at 35°C; after stirring, centrifuge at 3000 rpm for 30 minutes; wash three times with deionized water and collect the precipitate;

[0062] S1.3 Add the precipitate to phosphate buffer at pH 7 to form a suspension; then add the immobilized complex enzyme at a mass ratio of 1:10 to the precipitate, and stir at 300 rpm for 10 h at 37 °C; after enzymatic hydrolysis, heat to 85 °C and hold for 10 min to inactivate; obtain the enzymatic hydrolysate by filtration.

[0063] S1.4 Add macroporous resin with a pore size of 50 nm to the enzymatic hydrolysate, stir at 300 rpm for 60 min at 50 °C, and filter to obtain a clear solution.

[0064] S1.5. The clarified liquid is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5kDa at a pressure of 0.3MPa and a temperature of 35℃ to obtain elastin peptides.

[0065] Example 5: A method for extracting elastin peptides from animal cartilage, comprising the following steps:

[0066] S1.1 Remove the surface connective tissue and fat from the animal cartilage, wash it with physiological saline, and grind it with a grinder at 1500 rpm for 15 minutes. After grinding, treat it with an 80% ethanol solution using ultrasonic-assisted treatment at a power of 200W for 20 minutes to obtain pretreated cartilage particles.

[0067] S1.2. Place the pretreated cartilage particles into a container, add hexadecyltrimethylammonium bromide, with a mass ratio of pretreated cartilage particles to hexadecyltrimethylammonium bromide of 1:3, and stir at 300 rpm for 4 hours at 35°C. After stirring, centrifuge at 3000 rpm for 30 minutes. Wash three times with deionized water and collect the precipitate.

[0068] S1.3 Add the precipitate to phosphate buffer at pH 7 to form a suspension; then add the immobilized complex enzyme at a mass ratio of 1:50 to the precipitate, and stir at 300 rpm for 10 h at 37 °C; after enzymatic hydrolysis, heat to 85 °C and hold for 10 min to inactivate; filter to obtain the enzymatic hydrolysate.

[0069] S1.4 Add macroporous resin with a pore size of 50 nm to the enzymatic hydrolysate, stir at 300 rpm for 60 min at 50 °C, and filter to obtain a clear solution.

[0070] S1.5. The clarified liquid is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 5kDa at a pressure of 0.3MPa and a temperature of 35℃ to obtain elastin peptides.

[0071] Comparative Example 1: Using the method of Example 5, in the method of extracting elastin peptides from animal cartilage, neutral protease was used directly, without immobilized complex enzyme.

[0072] Comparative Example 2: The method of Example 5 was used, in the method of extracting elastin peptides from animal cartilage, without the addition of ionic liquids.

[0073] Comparative Example 3: Using the method of Example 5, in the method of extracting elastin peptides from animal cartilage, the immobilized complex enzyme was not treated with zinc chloride.

[0074] This invention extracts elastin peptides from animal cartilage using an immobilized complex enzyme. The performance indicators and testing standards for the extracted elastin peptides are as follows:

[0075] The sample was diluted with acetonitrile to a concentration of 5-50 mg / mL and filtered through a 0.22 μm filter membrane to remove particulate matter. The sample solution was then injected into a high-performance liquid chromatograph (HPLC) for separation and detection using reversed-phase HPLC. The purity of the sample was determined by calculating the ratio of the peak area of ​​the elastin peptide main peak to the total peak area.

[0076] Weigh the elastin peptide sample and place it in a Kjeldahl flask. Add concentrated sulfuric acid and copper sulfate, and digest under heating conditions until the sample is completely decomposed and turns into a transparent blue-green solution. After cooling, transfer the digest to a distillation apparatus and add excess sodium hydroxide solution to convert the ammonium salt into ammonia gas. Introduce the ammonia gas into a receiving flask containing boric acid solution through the distillation apparatus. After distillation is complete, titrate the solution in the receiving flask with standard hydrochloric acid, using methyl red-bromocresol green as an indicator, until the solution changes from green to dark red. Record the volume of acid consumed. Calculate the nitrogen content in the sample according to the formula and multiply by the conversion factor (usually 6.25) to obtain the elastin peptide content.

[0077] The data obtained using the above testing criteria are shown in Table 1:

[0078] Table 1 Test data of Examples 1-5 and Comparative Examples 1-3

[0079]

[0080] As can be seen from Table 1, the elastin peptides extracted in Examples 1-5 all have high purity and content.

[0081] Based on the above test experiments, Example 5 is considered the optimal example.

[0082] A comparison of Example 3 and Comparative Example 1 shows that a single neutral protease has a relatively limited action site, which may not be able to fully hydrolyze elastin, resulting in limited elastin peptide production and reduced content. In addition, neutral proteases are more susceptible to inactivation by environmental factors such as temperature and pH in the reaction system, preventing the enzymatic hydrolysis reaction from proceeding fully and ultimately affecting the content of elastin peptides. In contrast, the immobilized complex enzyme, through the synergistic action of multiple enzymes, can enzymatically hydrolyze proteins in animal cartilage from different sites and in different ways, thereby more comprehensively breaking down elastin into peptides.

[0083] A comparison of Example 3 and Comparative Example 2 shows that ionic liquids possess unique dissolving properties, effectively disrupting cell structures and promoting the release of elastin from animal chondrocytes. Without ionic liquids, cell disruption may be incomplete, preventing the release of some elastin from the cells, thus reducing the substrate amount for enzymatic hydrolysis to generate elastin peptides and ultimately lowering the elastin peptide content. Furthermore, ionic liquids exhibit selective dissolution of elastin, preferentially dissolving elastin and some structurally similar peptides during extraction, while having relatively weak solubility for other impurities, facilitating the initial separation of elastin from other impurities. Without ionic liquids, more other proteins, polysaccharides, fats, and other impurities may be extracted simultaneously during extraction, mixing with the elastin peptides and reducing their purity.

[0084] A comparison of Example 3 and Comparative Example 3 shows that zinc chloride can act as an activator for certain proteases. By binding to specific sites in the enzyme molecule, it alters the enzyme's spatial conformation, allowing its active site to better bind to the substrate, thereby improving the enzymatic hydrolysis efficiency of proteases on elastin in animal cartilage. In addition, zinc chloride may inhibit peptide aggregation, preventing the aggregation of elastin peptides into large polymers during extraction, thus avoiding situations where some peptides cannot be effectively separated and collected due to aggregation. At the same time, zinc chloride may also inhibit the non-specific degradation of elastin peptides to a certain extent, thereby improving the purity of elastin peptides.

[0085] To investigate the stability of the stable three-dimensional structure formed by protease after adsorption onto agarose gel, cross-linking with glutaraldehyde, and treatment with zinc chloride, and to examine the effects of various substances on this structure, the following control experiment was designed in this invention:

[0086] Enzyme preparations:

[0087] 6 mg / mL Solarbio Neutrase 3.0SG, where 3.0 represents enzyme activity units and SG represents stabilized granule formulation;

[0088] 5mg / mL Sigma Elastase from porcine pancreas

[0089] Chemical reagents:

[0090] Glutaraldehyde (25% aqueous solution)

[0091] Zinc chloride (ZnCl2, purity 99.99%)

[0092] Phosphate-buffered saline (PBS, pH 7.4, 0.1M)

[0093] Elastin substrate (Elastin-Congo Red)

[0094] Animal cartilage (cow knee joint cartilage, freshly collected)

[0095] Deionized water

[0096] Ethanol (95%)

[0097] Hydrochloric acid (HCl, 1M)

[0098] Sodium hydroxide (NaOH, 1M).

[0099] Enzyme solution preparation:

[0100] Neutral protease solution (6 mg / mL) and elastase solution (5 mg / mL) were prepared separately using PBS (pH 7.4) as solvent.

[0101] Neutral protease solution and elastase solution were mixed at a mass ratio of 1:2 to obtain a mixed enzyme solution with a total enzyme concentration of 7.5 mg / mL.

[0102] Activation of agarose gel:

[0103] Take 100 g of agarose gel and wash it three times with 500 mL of deionized water to remove the preservation solution.

[0104] The washed gel was placed in 500 mL PBS (pH 7.4) and soaked overnight at 4°C to equilibrate pH and ionic strength.

[0105] Control group 1: only adsorption

[0106] Take 10 g of activated agarose gel, add 100 mL of mixed enzyme solution (neutral protease and elastase mass ratio 1:1, total enzyme concentration 7.5 mg / mL), and let it stand at 4℃ for 2 hours for adsorption.

[0107] After adsorption was complete, the gel was washed three times with 100 mL PBS to remove unadsorbed free enzymes, resulting in a gel containing only the adsorbed enzyme.

[0108] Control group 2: Glutaraldehyde cross-linking after adsorption

[0109] Take 10 g of activated agarose gel, add 100 mL of mixed enzyme solution (as above), and let it stand at 4℃ for 2 hours to adsorb.

[0110] After adsorption was complete, 10 mL of 25% glutaraldehyde solution (final concentration 0.5%) was added, and the pH was adjusted to 7.0 with 1 M HCl or NaOH. The reaction was carried out at 4℃ for 4 hours.

[0111] After the reaction was complete, the sample was washed five times with 100 mL PBS to remove unreacted glutaraldehyde and free enzyme.

[0112] Control group 3: Zinc chloride treatment after adsorption

[0113] Take 10 g of activated agarose gel, add 100 mL of mixed enzyme solution (as above), and let it stand at 4℃ for 2 hours to adsorb.

[0114] After adsorption was complete, the gel was transferred to 100 mL of 0.2 mol / L ZnCl2 solution and stirred at 200 rpm for 3 hours at 25 °C.

[0115] After the reaction was complete, the sample was washed five times with 100 mL PBS to remove unbound zinc ions and free enzymes.

[0116] Experimental group: glutaraldehyde cross-linking after adsorption and zinc chloride treatment

[0117] Take 10 g of activated agarose gel, add 100 mL of mixed enzyme solution (as above), and let it stand at 4℃ for 2 hours to adsorb.

[0118] After adsorption was complete, 10 mL of 25% glutaraldehyde solution (final concentration 0.5%) was added, the pH was adjusted to 7.0, and the reaction was carried out at 4℃ for 4 hours.

[0119] After cross-linking, wash three times with 100 mL PBS to remove unreacted glutaraldehyde.

[0120] Subsequently, the cross-linked gel was transferred to 100 mL of 0.2 mol / L ZnCl2 solution and stirred at 200 rpm for 3 hours at 25 °C.

[0121] Finally, the enzyme was washed five times with 100 mL PBS to obtain the immobilized complex enzyme.

[0122] Initial enzyme activity assay: Take 0.1 g of immobilized enzyme from each group, add 10 mL of 0.1% elastin-CongoRed substrate solution (dissolved in PBS, pH 7.4), and react at 37℃ for 30 minutes.

[0123] After the reaction was completed, the supernatant was collected by centrifugation (5000 rpm, 5 minutes) and the absorbance at 490 nm was measured using a spectrophotometer.

[0124] Enzyme activity (U / g) was calculated based on the Congo Red standard curve, where 1 U is defined as the amount of enzyme that releases 1 μmol of Congo Red per minute.

[0125] Thermal stability test: Take 0.1 g of each group of immobilized enzyme, place it in 10 mL PBS (pH 7.4), and incubate at 50℃ for 2 hours.

[0126] After incubation, the sample was rapidly cooled to room temperature, and the residual enzyme activity was determined according to method 2.2.1.

[0127] Calculate the percentage of residual activity: Residual activity (%) = (Activity after heat preservation / Initial activity) × 100%.

[0128] pH stability determination: Take 0.1 g of each group of immobilized enzyme and place them in 10 mL of pH 5.0 (citric acid-phosphate buffer) and pH 9.0 (Tris-HCl buffer), respectively, and treat them at 25℃ for 1 hour.

[0129] After treatment, wash three times with PBS (10 mL each time), and determine the residual enzyme activity according to method 2.2.1.

[0130] Calculate the percentage of residual activity: Residual activity (%) = (Activity after treatment / Initial activity) × 100%.

[0131] Reusability test:

[0132] Take 0.5 g of each group of immobilized enzymes, add 50 mL of 0.1% elastin-Congo Red substrate solution (pH 7.4), and react at 37℃ for 2 hours.

[0133] After the reaction was completed, the immobilized enzyme was recovered by centrifugation (5000 rpm, 5 minutes), washed three times with PBS (50 mL each time), and reused for the next round of reaction.

[0134] Repeat the experiment 5 times, measuring enzyme activity each time. Calculate the activity retention rate (%) after the 5th test = (5th test activity / initial activity) × 100%.

[0135] Enzymatic hydrolysis efficiency test: Fresh bovine knee joint cartilage was collected, and the surface connective tissue and fat were removed with a scalpel. The cartilage was then rinsed thoroughly with physiological saline. The cartilage was cut into small pieces and ground into granules using a grinder (1000 rpm, 10 minutes). The cartilage granules were immersed in 80% ethanol and subjected to ultrasonic-assisted treatment (250 W, 20 minutes), repeated three times. After defatting, the cartilage was washed three times with deionized water, centrifuged (4000 rpm, 15 minutes), and the precipitate was collected. The precipitate was then frozen at -80℃ and freeze-dried under vacuum until constant weight.

[0136] Take 5 g of pretreated cartilage powder and add 50 mL of PBS (pH 7.4) to form a homogeneous suspension.

[0137] Each group of immobilized enzymes (enzyme to cartilage mass ratio 1:30) was added separately, and the enzymes were stirred and hydrolyzed for 8 hours at 37℃ and 200 rpm.

[0138] After enzymatic hydrolysis, heat to 85℃ and maintain for 10 minutes to inactivate the enzyme. Centrifuge (10,000 rpm, 20 minutes) and collect the supernatant.

[0139] The supernatant was filtered through a 0.22 μm filter membrane, and the elastin peptide content (expressed as protein content) was determined by the Lowry method.

[0140] Extraction rate (%) = (mass of elastin peptides in supernatant / mass of cartilage powder) × 100%.

[0141] Table 2. Performance comparison of immobilized complex enzymes under different treatment conditions

[0142]

[0143] The experimental group (glutaraldehyde cross-linking and zinc chloride treatment) performed exceptionally well, with an initial enzyme activity of 96.7 U / g, thermal stability of 86.4%, pH stability between 79.2% and 81.5%, reusability of 76.2%, and enzymatic hydrolysis efficiency as high as 74.5%, all significantly superior to control group 1 (adsorption only), control group 2 (glutaraldehyde cross-linking only), and control group 3 (zinc chloride treatment only). This indicates that glutaraldehyde provides a strong enzyme-carrier bond through covalent cross-linking, while zinc ions protect the enzyme active site and enhance stability through chelation. The combined effect of these two factors allows the immobilized enzyme to maintain high activity under high temperature, extreme pH, and reusable conditions, thereby significantly improving the efficiency of extracting elastin peptides from animal cartilage.

[0144] The results showed that the experimental group was significantly superior to the three control groups in terms of initial enzyme activity, thermal stability, pH stability, reusability, and enzymatic hydrolysis efficiency (p<0.05), demonstrating the synergistic effect of agarose gel, glutaraldehyde, and zinc chloride in the preparation of immobilized composite enzymes. Glutaraldehyde provides a stable enzyme-carrier binding through covalent cross-linking, while zinc ions protect the enzyme's active site through chelation, reducing activity loss during the cross-linking process. Furthermore, glutaraldehyde restricts the thermal motion of enzyme molecules during cross-linking, and zinc ions maintain the enzyme's spatial structure, jointly resisting high-temperature denaturation.

[0145] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method of extracting elastin peptides from animal cartilage, characterized by, The method comprises the following steps: S1.1, removing the surface connective tissue and fat of the animal cartilage, washing with normal saline, grinding and defatting to obtain pretreated cartilage particles; S1.2, placing the pretreated cartilage particles into a container, adding an ionic liquid and stirring; after stirring, centrifuging by a centrifuge, washing with deionized water for 2-3 times, and collecting the precipitate; S1.3, adding the precipitate into a phosphate buffer solution with a pH of 7-8 to form a suspension; then adding an immobilized composite enzyme to perform an enzymatic reaction; after the enzymatic reaction, heating to 80-85℃ for 5-10 min to inactivate; and filtering to obtain an enzymatic hydrolysate; S1.4, adding a macroporous resin with a pore size of 30-50 nm to the enzymatic hydrolysate, stirring at a speed of 200-300 rpm at 50-60℃ for 30-60 min, and filtering to obtain a clear liquid; S1.5, performing ultrafiltration on the clear liquid under a pressure of 0.1-0.3 MPa and a temperature of 25-40℃ to obtain an elastin peptide; In S1.2, the ionic liquid is hexadecyl trimethyl ammonium bromide, tetrabutyl ammonium chloride or trimethyloctyl ammonium chloride; The specific preparation steps of the immobilized composite enzyme are as follows: placing the agarose gel into a mixed enzyme solution containing neutral protease and elastase, and standing for adsorption at 4-10℃ for 1-2 h to allow the enzyme molecules to be adsorbed to the surface of the agarose gel; adding a glutaraldehyde solution with a concentration of 0.5%-1% to the container containing the gel and the enzyme solution, and reacting at 4-10℃ under a pH of 6.0-8.0 for 2-6 h; and washing with a phosphate buffer solution with a pH of 7-8 for 3-5 times; placing the washed agarose gel with immobilized neutral protease and elastase into a zinc chloride solution with a concentration of 0.1-0.5 mol / L, and stirring at a speed of 200-300 rpm at 25-37℃ for 2-4 h; after the reaction, washing with a phosphate buffer solution with a pH of 7-8 for 3-5 times to obtain the immobilized composite enzyme; the mixed enzyme solution of the neutral protease and the elastase is obtained by mixing a neutral protease solution with a concentration of 5-10 mg / mL and an elastase solution with a concentration of 3-8 mg / mL at a mass ratio of 1:2-1; in S1.3, the temperature of the enzymatic reaction is 30-37℃, the enzymatic reaction time is 6-10 h, and the stirring speed is 100-300 rpm.

2. The method of claim 1, wherein the method is characterized by, in S1.1, the grinding is performed by a grinding machine, the grinding speed is 1000-2000 rpm, and the grinding time is 5-15 min; the defatting treatment is performed by using an ethanol solution with a concentration of 70%-90% through ultrasonic auxiliary treatment, the ultrasonic power is 200-300 W, and the treatment time is 15-30 min.

3. The method of claim 1, wherein the method is characterized by, the mass ratio of the pretreated cartilage particles to the ionic liquid is 1:3-5.

4. The method of claim 1, wherein the method is characterized by, in S1.2, the stirring temperature is 25-45℃, the stirring speed is 200-400 rpm, and the stirring time is 1-4 h; the centrifuge speed is 3000-4000 rpm, and the centrifugation time is 10-30 min.

5. The method of claim 1, wherein the method is characterized by, In the S1.3, the mass ratio of the immobilized complex enzyme and the precipitate is 1:20-50.

6. The method of claim 1, wherein the method is characterized by, In the S1.5, the molecular weight cut-off of the ultrafiltration membrane is 3-10 kDa.

Images