CN-122011236-A - Method for extracting chondroitin sulfate sodium by one-step method by superimposing specific trypsin with multiple complex enzymes

Abstract

The invention relates to a method for extracting sodium chondroitin sulfate by a one-step method of overlapping specific trypsin with multiple complex enzymes, which comprises the following steps of S1, cleaning animal cartilage tissues, freeze-drying and crushing into powder, S2, adding the powder into a buffer solution of complex enzymes formed by combining specific trypsin 201, recombinant protease 017, lipase, cellulase, pancreatin and neutral protease, carrying out enzymolysis reaction for 4 hours at 40 ℃, and S3, after the enzymolysis reaction is finished, obtaining a sodium chondroitin sulfate product through centrifugal separation, ethanol precipitation, filtration, washing and drying. The extraction method of the invention can degrade cartilage tissue more fully through the synergistic effect of a plurality of complex enzymes, improves the yield of chondroitin sulfate sodium, reduces the loss of the intermediate extraction step by a one-pot method, and has the extraction rate of 17% of the final product which is 10% higher than the traditional extraction rate.

Inventors

• ZHANG MINGTAO
• Bian Jinhai
• TANG DANDAN
• Tai Xiaolong

Assignees

• 江苏泽泰制药有限公司

Dates

Publication Date

20260512

Application Date

20260211

Claims (6)

1. 1. A method for extracting chondroitin sulfate sodium by a one-step method by superposing specific trypsin with multiple complex enzymes is characterized by comprising the following steps: s1, cleaning animal cartilage tissue, freeze-drying and crushing into powder; S2, adding the powder into a buffer solution of a complex enzyme formed by combining specific trypsin 201, recombinant protease 017, lipase, cellulase, pancreatin and neutral protease, and performing enzymolysis reaction for 4 hours at 40 ℃; S3, after the enzymolysis reaction is finished, obtaining a chondroitin sulfate sodium product through centrifugal separation, ethanol precipitation, filtration, washing and drying.
2. 2. The method according to claim 1, wherein in step S1, the animal cartilage tissue comprises one or a mixture of pig cartilage tissue and bovine cartilage tissue.
3. 3. The method according to claim 1, wherein the buffer solution of the complex enzyme has a pH of 7.0 to 7.5 in step S2.
4. 4. The method of claim 1, wherein in the step S2, the buffer solution of the complex enzyme is prepared by adding the specific trypsin 201, the recombinant protease 017, the lipase, the cellulase, the pancreatin and the neutral protease into the phosphate buffer solution according to the mass ratio of 0.4-6:0.4-8:0.5:0.5:0.6:0.4, and the concentration of the specific trypsin 201 in the buffer solution of the complex enzyme is 0.08-1.2 g/L.
5. 5. The method according to claim 1, wherein in the step S2, the mass-to-volume ratio of the animal cartilage tissue powder obtained in the step S1 to the buffer solution of the complex enzyme is 1:5 g/mL.
6. 6. The method according to claim 1, wherein in step S3, the temperature of the filtrate obtained by the centrifugal separation is maintained at 50-60 ℃ during the ethanol precipitation, the ethanol is added while stirring, the precipitation degree reaches 65 ℃ first, the ethanol is added to the precipitate for 8 hours, the precipitation degree reaches 80 ℃ and the ethanol is added to the precipitate for 6 hours.

Description

Method for extracting chondroitin sulfate sodium by one-step method by superimposing specific trypsin with multiple complex enzymes Technical Field The invention relates to the technical field of biological enzymolysis extraction, in particular to a method for extracting chondroitin sulfate sodium by a one-step method of superimposing specific trypsin by utilizing multiple complex enzymes. Background Sodium chondroitin sulfate is an important acidic mucopolysaccharide and is widely applied to the fields of medicines, foods, cosmetics and the like. It is found in cartilage, laryngeal bone, nasal bone, diaphragm and trachea of cattle and horses. The molecular structure of the polysaccharide chain is composed of a repeated disaccharide unit formed by beta-d-glucuronic acid and n-acetamido galactose through beta 1-3 glycosidic bond. The chondroitin sulfate sodium is mainly used as a medicament for treating rheumatism and rheumatoid diseases, has the effects of anticoagulation, anticancer, antithrombotic, blood lipid reduction and the like, and can also be used for treating symptoms such as headache, migraine, coronary heart disease, angina and the like. The traditional method for extracting the sodium chondroitin sulfate mainly adopts an alkaline salt digestion precipitation method, and the method has the problems of long production period, large environmental pollution, low product content and purity and the like. For example, alkaline extraction can easily cause degradation of sodium chondroitin sulfate and affect product quality, and enzymatic hydrolysis is relatively mild, but generally adopts stepwise enzymatic hydrolysis, so that the operation is complex and the cost is high. Therefore, the development of an efficient, simple and environment-friendly extraction method of the chondroitin sulfate sodium has important practical significance. With the continuous development of biotechnology, the complex enzyme extraction method is gradually widely applied due to the high efficiency and specificity of the complex enzyme extraction method. The method for extracting the chondroitin sulfate sodium by adopting the compound enzyme one-step method to superimpose the specific trypsin can not only improve the purity and the yield of the product, but also shorten the production period, reduce the production cost and reduce the pollution, and has important economic and social significance. Disclosure of Invention In order to solve the problems in the prior art, the invention aims to provide a method for extracting chondroitin sulfate sodium by a one-step method by utilizing a plurality of complex enzymes to superimpose specific trypsin, so as to improve the extraction rate and purity of the chondroitin sulfate sodium, simplify the extraction process, shorten the production period, improve the production efficiency, reduce the production cost and reduce the environmental pollution. In order to achieve the above purpose, the present invention provides the following technical solutions: The invention provides a method for extracting chondroitin sulfate sodium by a one-step method by superposing specific trypsin with multiple complex enzymes, which comprises the following steps: s1, cleaning animal cartilage tissue, freeze-drying and crushing into powder; S2, adding the powder into a buffer solution of a complex enzyme formed by combining specific trypsin 201, recombinant protease 017, lipase, cellulase, pancreatin and neutral protease, and performing enzymolysis reaction for 4 hours at 40 ℃; S3, after the enzymolysis reaction is finished, obtaining a chondroitin sulfate sodium product through centrifugal separation, ethanol precipitation, filtration, washing and drying. Further, in step S1, the animal cartilage tissue includes one or a mixture of pig cartilage tissue and bovine cartilage tissue. Further, in step S2, the pH of the buffer solution of the complex enzyme is 7.0-7.5 In step S2, the buffer solution of the complex enzyme is prepared by adding the specific trypsin 201, the recombinant protease 017, the lipase, the cellulase, the pancreatin and the neutral protease into the phosphate buffer solution according to the mass ratio of 0.4-6:0.4-8:0.5:0.5:0.6:0.4, and mixing, wherein the concentration of the specific trypsin 201 in the buffer solution of the complex enzyme is 0.08-1.2 g/L. Preferably, the mass ratio of the specific trypsin 201 to the recombinant protease 017 to the lipase to the cellulase to the pancreatin to the neutral protease is 4.0:4.0:0.5:0.5:0.6:0.4, and the concentration of the specific trypsin 201 in the buffer solution of the complex enzyme is 1.0 g/L. Further, in the step S2, the mass-volume ratio of the animal cartilage tissue powder obtained in the step S1 to the buffer solution of the complex enzyme is 1:5 g/mL. The enzymes in the buffer solution of the complex enzyme have higher enzyme activity, and the enzyme activity is 8-35 mu/g so as to ensure the high efficiency and the specificity of