Extraction and preparation process of chondroitin sulfate
By using bovine cartilage, alkali leach and compound enzymatic methods in the chondroitin sulfate extraction process, and adding modified soybean phospholipids and sipan 20, the problems of high purity and large-scale extraction in the prior art are solved, and high efficiency is achieved , economical chondroitin sulfate extraction effect.
Patent Information
- Application Number
- CN202411310363.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to achieve high purity and large-scale extraction of chondroitin sulfate, and the enzymatic process control is relatively sensitive, making it easy to see batches of high protein content.
Using bovine cartilage as raw material, combined with alkaline leaching and complex enzymatic lysis, the collagen is prevented from dissolution and promote the enzymatic lysis effect by adding modified soybean phospholipids and span 20 in the leaching and enzymatic lysis steps.
Chondroitin sulfate extraction without special purification methods is achieved, which improves product quality and reduces the cost of subsequent treatment.
Smart Images
Figure CN119390873A_ABST
Abstract
Description
Field of the Invention
[0001] This application belongs to the technical field of polysaccharide / glycosaminoglycan extracts. Specifically, this application provides an extraction and preparation process for chondroitin sulfate. Background Technology
[0002] Chondroitin sulfate is widely distributed in the extracellular matrix of animal tissues. It belongs to glycosaminoglycans, and its sugar chain structure is composed of alternating glucuronic acid and N-acetylgalactosamine.
[0003] Chondroitin sulfate has wide applications in the treatment of joint and cardiovascular diseases, and can also be used to treat corneal damage. It is a major ingredient in many drugs and health products, and the demand is huge. Currently, the main extraction methods for chondroitin sulfate are based on the cartilage of livestock such as pigs and cattle, using methods such as alkaline extraction, enzymatic hydrolysis, and chromatography.
[0004] The protein in chondroitin sulfate products mainly comes from collagen and other miscellaneous proteins dissolved during alkaline extraction. Current technologies have explored various enzymes and combinations of enzymes in different proportions to enzymatically hydrolyze the proteins, reducing their molecular weight for easier removal in subsequent processes such as ultrafiltration. The proteases used in these methods vary, but their nitrogen content is generally above 3.1% (purity around 95%). For preparations requiring higher purity, such as injection solutions, low-throughput chromatography or similar methods are necessary. Furthermore, because enzymatic hydrolysis is a highly sensitive biological process, the probability of batches with high protein content is relatively high.
[0005] In summary, there is a need in this field to improve the extraction method of chondroitin sulfate in order to achieve large-scale, high-purity extraction. Summary of the Invention
[0006] On the one hand, this application provides a process for extracting and preparing chondroitin sulfate, which uses bovine cartilage as raw material and employs alkaline extraction and compound enzymatic hydrolysis to extract chondroitin sulfate.
[0007] Furthermore, the chondroitin sulfate extraction and preparation process includes the following steps:
[0008] (1) Cut the cleaned beef cartilage into pieces;
[0009] (2) Add the beef cartilage pieces to 2-3 times the weight of the beef cartilage pieces in water at 80-90℃ and treat for 2-3 hours to remove the upper layer of fat;
[0010] (3) Cool down to 40-50℃, add 5-7 times the weight of bovine cartilage blocks of 3-4% w / v NaOH solution and 5-10% the weight of bovine cartilage blocks of modified soybean lecithin, and extract at 40-50℃ for 10 hours.
[0011] (4) Adjust the pH to 8.0-8.5, add 270g of alkaline protease to 800-1000U / mL, add papain to 800-1000U / mL, add Span 20 to 0.05-0.1% w / v, and enzymatically hydrolyze at 45-50℃ for 2-3 hours;
[0012] (5) Adjust the pH to 8.0-8.5, add trypsin to 200-300 U / mL, and continue enzymatic hydrolysis at 45-50℃ for 2-3 hours;
[0013] (6) Inactivate enzymes;
[0014] (7) Filter the filtrate, ultrafilter the filtrate with a filter membrane of 5000-10000 molecular weight, concentrate it to a dry matter concentration of 5-10% w / v, and take the retentate.
[0015] (8) Add hydrogen peroxide to the retentate obtained in step (7) for oxidation and decolorization, and then filter;
[0016] (9) Add 95-97% v / v ethanol to the filtrate obtained in step (8) until the ethanol concentration in the filtrate is 70-75% v / v, stir for 1-2 hours, let stand for 1-2 hours, and then centrifuge to collect the precipitate.
[0017] (10) Wash the precipitate obtained in step (9) with anhydrous ethanol and dry to obtain the product.
[0018] Further, step (2) is as follows: cool down to 40°C, add 3-4% w / v NaOH solution (6 times the weight of bovine cartilage blocks) and 5% modified soybean lecithin (5% the weight of bovine cartilage blocks), and extract at 40°C for 10 hours.
[0019] Further, step (4) is as follows: adjust the pH to 8.5, add alkaline protease 2709 to 1000 U / mL, add papain to 1000 U / mL, add Span 20 to 0.075% w / v, and enzymatically hydrolyze at 50°C for 2 hours.
[0020] Further, step (5) is as follows: adjust the pH to 8.0, add trypsin to 200 U / mL, and continue enzymatic hydrolysis at 50°C for 3 hours.
[0021] Further, step (9) is as follows: add 95% v / v ethanol to the filtrate until the ethanol concentration in the filtrate is 70% v / v, stir for 2 hours, let stand for 2 hours, and then centrifuge to collect the precipitate.
[0022] Furthermore, the bovine cartilage is the larynx, nasal bone, crescent bone, or rib cartilage.
[0023] Furthermore, in step (7), filter cloth and diatomaceous earth are used for the filtration.
[0024] Further, in step (7), ultrafiltration is performed using a 5000 molecular weight filter membrane, and the product is concentrated to a dry matter concentration of 6-7% w / v.
[0025] Further, the drying process in step (10) is carried out using a drying or freeze-drying method.
[0026] Based on the optimized combination of alkali extraction and enzymatic extraction methods, this application adds modified soybean lecithin and Span 20 to the extraction and enzymatic hydrolysis steps, effectively preventing the dissolution of collagen and other impurities, and promoting enzymatic hydrolysis by affecting the conformation of peptide and glycan chains. Even without using special purification methods, the product purity reaches injection-grade levels, effectively improving product quality and reducing subsequent processing costs. Attached Figure Description
[0027] Figure 1 This refers to the changes in the state of materials during the preparation process.
[0028] Figure 2 The effect of adding different amounts of modified soybean phospholipids in step (3) on nitrogen content. Detailed Implementation
[0029] Example 1: Main Materials and Testing Methods
[0030] raw material:
[0031] Fresh non-fattened cattle trachea, nasal bones, crescent bones, and rib bones can all be used. To ensure uniformity of conditions during the experiment, nasal bones from the same beef cattle farm (ensuring that the cattle breed, age, etc., are basically consistent) were selected. Impurities and blood were washed away before use. Unless otherwise stated, the examples in this application are all small-scale studies using 250g of raw materials.
[0032] Main reagents:
[0033] Alkaline protease 2709: Summer peak, 200,000 U / g;
[0034] Papain: Xia Sheng, 150,000 U / g;
[0035] Trypsin: Dongheng Huadao: 4000 U / g;
[0036] Modified soybean lecithin, sucrose fatty acid ester, Span, and Tween are all domestically produced food-grade ingredients.
[0037] Detection of chondroitin sulfate:
[0038] (1) Prepare a series of concentration solutions using dry glucosamine hydrochloride; take 5 mL of the solution, add 1 mL of 4% v / v acetylacetone solution, and place in a boiling water bath for 5 min; cool, add 3 mL of anhydrous ethanol, and place in a 70℃ water bath for 5 min; add 1 mL of colorimetric reagent (1 g of p-dimethylaminobenzaldehyde, 15 mL of anhydrous ethanol, and 15 mL of dilute hydrochloric acid), shake, and place in a 70℃ water bath for 30 min; after cooling, determine A. 525nm Draw the standard curve;
[0039] (2) Add 10 mg of sample to 5 mL of 5 M hydrochloric acid and place in a boiling water bath for 30 min; adjust the pH to about 7 and bring the volume to 20 mL; take 5 mL of the diluted solution and proceed as shown in step (1), and measure A after cooling. 525nm The chondroitin sulfate content was calculated using a standard curve, and then the yield and purity were calculated.
[0040] Nitrogen content detection:
[0041] The determination was performed according to the semi-micro method described in the 2020 edition of the Pharmacopoeia.
[0042] Example 2: Basic preparation method of chondroitin sulfate
[0043] Based on the applicant's preliminary research on the enzymatic and alkaline hydrolysis steps, the basic preparation method of chondroitin sulfate has been determined as follows:
[0044] (1) Cut the cleaned beef cartilage into small pieces of about 1cm;
[0045] (2) Add the beef cartilage pieces to water at 90°C with a volume of 2 times the weight of the beef cartilage pieces and treat for 2 hours to remove the top layer of grease.
[0046] (3) Cool down to 40°C, add 3% w / v NaOH solution with 6 times the mass of bovine cartilage blocks, and extract at 40°C for 10 hours;
[0047] (4) Adjust the pH to 8.5, add alkaline protease 2709 to 1000 U / mL, add papain to 1000 U / mL, and enzymatically hydrolyze at 50℃ for 2 hours;
[0048] (5) Adjust the pH to 8.0, add trypsin to 200 U / mL, and continue enzymatic hydrolysis at 50°C for 3 hours;
[0049] (6) Heat to 85℃ for 10 minutes to inactivate the enzyme;
[0050] (7) Filter the filtrate and ultrafilter it through a 5000 molecular weight membrane to concentrate it to a dry matter concentration of 6-
[0051] 7% w / v, take the retentate;
[0052] (8) Add 5% hydrogen peroxide (by weight of bovine cartilage blocks) to the retentate, oxidize and decolorize at 30°C for 10 hours, and then filter.
[0053] (9) Add 95% v / v ethanol to the filtrate until the ethanol concentration in the filtrate is 70% v / v, stir for 2 hours, let stand for 2 hours, and then centrifuge to collect the precipitate.
[0054] (10) Wash the precipitate twice with anhydrous ethanol and dry it to obtain the product.
[0055] Pilot-scale results showed that, depending on the batch, the purity was approximately 92-93%, the yield 33-35%, the nitrogen content 3.4-3.6%, the clarity approximately 0.03, and the acidity approximately 6.3-6.5. E. coli, mold, yeast, and Salmonella were not detected. The product meets national standards. The material state changes during the process are as follows: Figure 1 As shown.
[0056] Example 3: Modified soybean lecithin was added during the extraction step.
[0057] The nitrogen content index in Example 2 already meets the pharmacopoeia requirements for oral administration. However, for the purpose of process stability and to meet the higher purity requirements of injectable drugs, the applicant further studied the process of reducing nitrogen content.
[0058] The applicant found that adding modified soybean lecithin during the extraction process (3) could interfere with the dissolution of collagen by utilizing the properties of its lipids and had virtually no effect on chondroitin sulfate. The microparticles and dispersed molecules formed by the modified soybean lecithin could be removed during subsequent filtration and ultrafiltration processes.
[0059] like Figure 2 As shown, adding 5-10% of modified soybean lecithin by weight of bovine cartilage blocks can effectively reduce nitrogen content without significantly affecting the yield (around 34%). Correspondingly, the purity is also slightly improved (around 93.5%).
[0060] Example 4: Adding a surfactant to the enzymatic hydrolysis step
[0061] The applicant further attempted to add a small amount of surfactant to the enzymatic hydrolysis step (4) to alter the spatial morphology of collagen, thereby facilitating complete enzymatic hydrolysis. The results are shown in Tables 1-3, where the percentage of surfactant added represents the percentage of the enzymatic hydrolysis system in step (4) (w / v):
[0062] Table 1. Effects of sucrose fatty acid esters on nitrogen content, yield, and purity.
[0063] Surfactant dosage % Nitrogen content % Yield % purity% 0.05 3.44±0.04 34.2±0.8 94.1±0.6 0.075 3.48±0.08 35.5±0.4 94.8±1.3 0.1 3.51±0.11 34.7±1.2 95.1±0.3 0.2 3.35±0.03 33.8±0.6 93.4±0.6
[0064] Table 2. Effects of Span 20 on nitrogen content, yield, and purity.
[0065] Surfactant dosage % Nitrogen content % Yield % purity% 0.05 3.37±0.03 34.8±0.7 94.9±0.2 0.075 3.17±0.05 36.2±0.3 95.8±0.8 0.1 3.08±0.07 34.4±0.7 96.1±0.1 0.2 3.26±0.01 35.9±1.6 95.1±1.0
[0066] Table 3. Effects of Tween 20 on nitrogen content, yield, and purity.
[0067]
[0068]
[0069] The results showed that the addition of the nonionic surfactant Span 20 had significant beneficial effects on purity and nitrogen content, which may be attributed to its influence on the conformation of collagen peptide chains and certain polysaccharide chains.
[0070] Example 5: Combination of modified soybean lecithin and Span 20
[0071] The applicant further combined modified soybean lecithin and Span 20, setting the following conditions:
[0072] A1: Modified soybean lecithin at the mass of 5% bovine cartilage blocks;
[0073] A2: Modified soybean lecithin with 10% bovine cartilage mass;
[0074] B1: 0.075% Span 20 in the enzymatic hydrolysis system;
[0075] B2: 0.1% Span 20 in the enzymatic hydrolysis system;
[0076] Table 4. Effects of combined conditions on nitrogen content, yield, and purity.
[0077] Nitrogen content % Yield % purity% A1B1 2.65±0.04 34.9±0.5 98.5±0.3 A1B2 2.88±0.08 35.3±0.4 96.7±0.6 A2B1 3.04±0.11 34.7±0.7 95.9±0.9 A2B2 2.91±0.06 35.5±0.2 96.5±0.4
[0078] The results showed that the two improvement methods acting on different stages did not have any adverse interaction. Moreover, the A1B1 condition (5% modified soybean lecithin, 0.075% Span) showed significant improvement in purity and nitrogen content compared to other combinations. The nitrogen content of 2.65% can be considered to have basically removed collagen (chondroitin sulfate itself contains about 2.4% nitrogen).
[0079] After scaling up the optimized conditions of the above combination to a pilot-scale, the nitrogen content remained consistently below 2.75%, and the purity was maintained above 97.5%. This is superior to methods reported domestically that do not employ special purification steps (such as chromatography).
Claims
1. A chondroitin sulfate extraction and preparation process, characterized in that: The chondroitin sulfate extraction and preparation process uses bovine cartilage as a raw material and adopts an alkali solution extraction and a composite enzymatic hydrolysis method to extract the chondroitin sulfate.
2. The chondroitin sulfate extraction and preparation process according to claim 1, wherein the chondroitin sulfate extraction and preparation process comprises the following steps: (1) Cut the cleaned bovine cartilage into pieces; (2) adding the bovine cartilage block to 80-90° C. water with a volume of 2-3 times the mass of the bovine cartilage block, treating for 2-3 hours, and removing the upper layer of fat; (3) cooling to 40-50° C., adding 3-4% w / v NaOH solution of 5-7 times the mass of the bovine cartilage block and modified soybean lecithin of 5-10% the mass of the bovine cartilage block, and extracting at 40-50° C. for 10 hours; (4) adjusting the pH to 8.0-8.5, adding alkaline protease 2709 to 800-1000 U / mL, adding papain to 800-1000 U / mL, adding Span 20 to 0.05-0.1% w / v, and performing enzymolysis at 45-50°C for 2-3 hours; (5) Adjust the pH to 8.0-8.5, add trypsin to 200-300 U / mL, and continue enzymatic hydrolysis at 45-50°C for 2-3 hours; (6) enzyme inactivation; (7) filtering and collecting the filtrate, ultrafiltering the filtrate through a 5000-10000 molecular weight filter membrane, concentrating the filtrate to a dry matter concentration of 5-10% w / v, and collecting the retentate; (8) adding hydrogen peroxide to the retentate obtained in step (7) for oxidative decolorization and filtering; (9) adding 95-97% v / v ethanol to the filtrate obtained in step (8) until the ethanol concentration in the filtrate is 70-75% v / v, stirring for 1-2 hours, standing for 1-2 hours, and centrifuging to obtain a precipitate; (10) Wash the precipitate obtained in step (9) with anhydrous ethanol and dry it to obtain a product.
3. The chondroitin sulfate extraction and preparation process according to claim 2, wherein step (2) is: cooling to 40°C, adding 3-4% w / v NaOH solution of 6 times the mass of bovine cartilage blocks and modified soybean lecithin of 5% the mass of bovine cartilage blocks, and extracting at 40°C for 10 hours.
4. The chondroitin sulfate extraction and preparation process according to claim 3, wherein step (4) comprises: adjusting the pH to 8.5, adding alkaline protease 2709 to 1000 U / mL, adding papain to 1000 U / mL, adding Span 20 to 0.075% w / v, and performing enzymolysis at 50°C for 2 hours.
5. The chondroitin sulfate extraction and preparation process according to claim 1, wherein step (5) is: adjusting the pH to 8.0, adding trypsin to 200 U / mL, and continuing enzymolysis at 50°C for 3 hours.
6. The chondroitin sulfate extraction and preparation process according to claim 1, wherein step (9) comprises: adding 95% v / v ethanol to the filtrate until the ethanol concentration in the filtrate is 70% v / v, stirring for 2 hours, standing for 2 hours, and then centrifuging to obtain a precipitate.
7. The process for extracting and preparing chondroitin sulfate according to claim 1, wherein the bovine cartilage is bovine laryngeal tube, nasal bone, crescent bone or soft rib.
8. The chondroitin sulfate extraction and preparation process according to claim 1, wherein filter cloth and diatomaceous earth are used for the filtration in step (7).
9. The chondroitin sulfate extraction and preparation process according to claim 1, wherein in step (7), ultrafiltration is performed using a 5000 molecular weight filter membrane and concentrated to a dry matter concentration of 6-7% w / v.
10. The chondroitin sulfate extraction and preparation process according to claim 1, wherein the drying is performed by drying or freeze-drying in step (10).