CN1624004A

CN1624004A - Process for extracting chondroitin sulfate

Info

Publication number: CN1624004A
Application number: CN 200410072346
Authority: CN
Inventors: 陈宁; 刘淑云; 王振声; 徐庆阳; 黎兴荣
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2004-10-20
Filing date: 2004-10-20
Publication date: 2005-06-08
Anticipated expiration: 2024-10-20
Also published as: CN1279063C

Abstract

A process for extracting chonsurid includes such steps as enzymolyzing cartilage, removing protein, depositing and drying. Its advantages are high output rate and purity, short reaction time, less consumption of alkali, and low environmental pollution.

Description

Extraction method of chondroitin sulfate

[ technical field]: the invention relates to a biochemical medicament, in particular to a production process for directly extracting chondroitin sulfate from animal cartilage.

[ background Art]A method of: chondroitin sulfate is a viscous polysaccharide extracted from animal cartilage, is mostly present in animal cartilage, larynx bone, nasal bone (41% of pig), middle diaphragm of cattle and horse and trachea (36% -39%), is an acidic mucopolysaccharide-glycosaminoglycan, and can be dissociated from protein in cartilage tissue under the action of trypsin. Can be used for treating headache, migraine, coronary heart disease, angina pectoris, arthritis pain, etc., and the pharmacological research on chondroitin sulfate has reached molecular level in foreign countries.

At present, dilute alkali or concentrated alkali and enzymolysis are generally adopted at home and abroad to extract chondroitin sulfate, and a dilute alkali and dilute salt comprehensive extraction method, an enzymolysis-resin extraction method, an ultrasonic extraction method and an acetic acid extraction method are reported. The separation and purification of chondroitin sulfate include ethanol precipitation, ion exchange chromatography, quaternary ammonium salt compounding, adsorption, and cellulose separation. In the above extraction methods, the yield can reach a high level, for example, the yield can reach 32.6% by adopting concentrated alkali-complex enzyme enzymolysis method (new process research of chondroitin sulfate production, university of Qingdao, 2003, 18 (4): 55-58) and the like. However, the above production methods all have a great problem that the amount of diluted alkaline solution used in the diluted alkaline enzymolysis process is too large, which is more than 6 times of the weight of cartilage, and in the subsequent alcohol precipitation, the amount of alcohol used is larger, such as concentrated stock solution, which consumes much energy. The concentrated alkali enzymolysis process uses a large amount of alkali, such as Gaohua, which is 8% of the weight of the cartilage, so that inconvenience is brought to subsequent purification, and if the reaction time is prolonged, the molecular weight of chondroitin sulfate is reduced, and the problem of complicated steps also exists. In addition, the patent application No. 96116057.8, entitled chondroitin sulfate and its extraction method, published by the national intellectual Property office in 1998, 5/27 is a dilute salt and dilute alkali method, which has the disadvantages of large salt and alkali consumption, long extraction time, insufficient extraction and low yield.

In summary, the problems with the current production process are: large amount of alkali salt, high energy consumption, complicated production steps, low yield and the like.

[ summary of the invention]: the invention aims to provide a novel chondroitin sulfate extraction method from the aspects of energy conservation, reagent saving, production time shortening, process simplification and the like, and the method is favorable for industrial production.

Because chondroitin sulfate and the protein in the cartilage are covalently bonded together by glycopeptide, the glycosaminoglycan can be dissociated from the collagen in the cartilage tissue under alkaline conditions and with trypsin as a catalyst. According to the principle, the process directly adds trypsin, adjusts the conditions of pH, temperature and the like of the reaction, and directly produces the chondroitin sulfate. Therefore, the process flow is shortened, the reaction time is only 4-8 hours, the alkali consumption is saved by more than 80% compared with a dilute alkali enzymolysis method, the yield is not reduced, and the yield is 3-5% higher than the original yield.

The specific extraction method sequentially comprises the following steps:

(1) putting the clean and white cartilage blocks into a reaction kettle, adding a proper amount of water, adding trypsin accounting for 0.1-1.5% of the weight of the cartilage blocks, adjusting the pH to be 8.0-9.0, controlling the temperature to be 45-55 ℃, and stirring for 3-10 hours;

(2) after the cartilage block is completely reacted, adjusting the pH value of the enzymolysis liquid to be 5-7 by using hydrochloric acid or acetic acid, heating to 70-85 ℃, and preserving the heat for 10-40 minutes;

(3) cooling the enzymolysis liquid, filtering or centrifugally removing slag to obtain cleaner filtered enzymolysis liquid, adding active carbon which is 0.2-2.0% of the weight of the enzymolysis liquid, stirring for 20-60 minutes under the condition that the pH value is 3-5, and filtering or centrifugally removing slag;

(4) adding industrial alcohol which is 2.5-4 times of the volume of the enzymolysis liquid into the enzymolysis liquid obtained in the last step to ensure that the concentration of the ethanol reaches 55-80%, and precipitating;

(5) washing the precipitate with 95% alcohol for 2-3 times, and drying to obtain the chondroitin sulfate product.

The method has the advantages and effects that: the method adopts a one-step enzymolysis method to produce Chondroitin Sulfate (CS) by improving and innovating the prior art, and finds out that the optimized process conditions for CS production are as follows through an orthogonal test: the hydrolysis time is 6h, the hydrolysis temperature is 48 ℃, the pancreatin dosage is 0.7 percent of the weight of the fresh cartilage, and the influence degree on the hydrolysis is mainly the hydrolysis temperature, and is secondly the enzyme dosage and the hydrolysis time. The pH value in the whole reaction process is 8.5-9.0. This combination of optimum levels can result in chondroitin sulfate yields of 42% or greater. After the new process is adopted, the production flow can be simplified, the production time is greatly shortened, the alkali consumption is reduced, the difficulty of downstream purification is reduced, the pollution to the environment is reduced, the original production equipment can be adopted, and the industrial production is easy to realize.

[ embodiments]of the present invention:

the technological process for extracting chondroitin sulfate is as follows:

example 1:

(1) putting the clean cartilage blocks into a reaction kettle, adding a proper amount of water, adding trypsin accounting for 0.7 percent of the weight of the cartilage blocks, adjusting the pH to be between 8.5 and 9.0, controlling the temperature to be 48 ℃, and stirring for 6 hours;

(2) when the cartilage block is completely reacted, adjusting the pH value of the enzymolysis solution to be between 5 and 6 by using acetic acid, heating to 80 ℃, and preserving heat for 15 minutes;

(3) cooling the enzymolysis solution, filtering or centrifuging to remove residue to obtain clear filtered enzymolysis solution, adding 0.5% active carbon, adjusting pH to 4, stirring for 30 min, and filtering or centrifuging to remove residue;

(4) adding 3 times of industrial alcohol into the enzymatic hydrolysate obtained in the previous step to enable the concentration of the ethanol to reach 70%, and adding 0.5% of sodium acetate for salting out;

(5) washing the precipitate with 95% alcohol for 3 times, and drying to obtain the chondroitin sulfate finished product.

The chondroitin sulfate produced by the above method was analyzed, and the results were as follows:

project results

Water content/% 6.9

Nitrogen content/% 3.12

Purity/% 88.6

Chloride is less than 0.14 percent

Inorganic sulfates (in SO)₄Calculated) is less than 0.24 percent

Heavy metals (in terms of Pb)<40ppm

50g/L CS solution OD₆₄₀Value 0.03

Other examples reference values (in order of reaction according to example 1) are given in the following table:

TABLE 1,

Pancreatic egg White enzyme	Temperature of	Stirring the mixture Time of day	Enzymolysis Liquid for treating urinary tract infection pH	Heating of Temperature of	Heat preservation Time of day	Activity of Carbon (C)	pH value	Stirring the mixture Time of day	Adding wine Extract of Chinese medicinal materials	Ethanol Concentration of	Acetic acid Sodium salt
Pancreatic egg White enzyme	Temperature of	Stirring the mixture Time of day	Enzymolysis Liquid for treating urinary tract infection pH	Heating of Temperature of	Heat preservation Time of day	Activity of Carbon (C)	pH value	Stirring the mixture Time of day	Adding wine Extract of Chinese medicinal materials	Ethanol Concentration of	Acetic acid Sodium salt	0.3％	45℃	8h	8.5-9. 0	℃	15min	0.4％	3.5	20h	2.5 times of	65％	1.0％
1.5％	55℃	4h	8.5-9. 0	℃1	30min	1.5％	5.0	50h	4 times of	74％	0.3％	0.3％	45℃	8h	8.5-9. 0	℃	15min	0.4％	3.5	20h	2.5 times of	65％	1.0％

Claims

1. A method for extracting chondroitin sulfate is characterized by sequentially comprising the following steps:

2. The method for extracting chondroitin sulfate as claimed in claim 1, wherein sodium acetate in an amount of 0.2-2.0% by weight of the enzymolysis solution is added in the step (4) for salting out to enhance the precipitation effect of chondroitin sulfate.

3. The method for extracting chondroitin sulfate as claimed in claim 1 or 2, wherein the optimal hydrolysis temperature is 48 ℃.

4. The method for extracting chondroitin sulfate as claimed in claim 1 or 2, wherein the optimal hydrolysis time is 6 hours, and the optimal pancreatin dosage is 0.7% of the weight of fresh cartilage.