阅读说明：本技术 从硫酸软骨素发酵液中提纯硫酸软骨素的方法 (Method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor ) 是由 张国银 何永进 于 2019-05-10 设计创作，主要内容包括：本发明公开了一种从硫酸软骨素发酵液中提纯硫酸软骨素的方法,它包括发酵液灭活、固液分离、滤液浓缩洗涤、树脂吸附、树脂洗脱、洗脱液浓缩洗涤和洗脱浓相干燥步骤。它是将硫酸软骨素发酵液经过灭活、固液分离和滤液浓缩洗涤步骤,制得滤液浓相；将滤液浓相上阴离子交换树脂吸附,再用氢氧化钠或者氯化钠溶液洗脱,将洗脱液进行浓缩过滤和洗涤,制得洗脱浓相,将洗脱浓相进行干燥制得本发明的硫酸软骨素产品。该方法操作简便、低污染、回收率高,适合大批量工业化生产。(The invention discloses a method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor, which comprises the steps of fermentation liquor inactivation, solid-liquid separation, filtrate concentration and washing, resin adsorption, resin elution, eluent concentration and washing, and elution concentrated phase drying. Carrying out inactivation, solid-liquid separation and filtrate concentration and washing on chondroitin sulfate fermentation liquor to obtain a filtrate concentrated phase; adsorbing the filtrate concentrated phase with anion exchange resin, eluting with sodium hydroxide or sodium chloride solution, concentrating, filtering and washing the eluent to obtain an eluted concentrated phase, and drying the eluted concentrated phase to obtain the chondroitin sulfate product. The method has the advantages of simple operation, low pollution, high recovery rate, and suitability for large-scale industrial production.)

1. A method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor comprises the following steps:

s1, inactivation of fermentation liquor: adding acid into chondroitin sulfate fermentation liquor to adjust pH to 2.0-4.5 or adding alkali to adjust pH to 8.5-11.0, heating to 60-90 deg.C, and maintaining for 3-7 hr to obtain inactivated chondroitin sulfate fermentation liquor;

s2, solid-liquid separation: performing solid-liquid separation on the inactivated chondroitin sulfate fermentation liquor by adopting filtering equipment to prepare fermentation filtrate;

s3, concentrating and washing the filtrate: performing ultrafiltration concentration on the fermentation filtrate by using an ultrafiltration membrane, concentrating to 1/8-1/4 of the volume of the fermentation filtrate, and washing by using water with the volume 1-3 times that of the fermentation filtrate to obtain a filtrate concentrated phase;

s4, resin adsorption: adsorbing the filtrate concentrated phase with anion exchange resin with adsorption capacity of 20-80g/L (g chondroitin sulfate/resin volume);

s5, resin elution: eluting the anion exchange resin adsorbed with the chondroitin sulfate by using sodium hydroxide or sodium chloride solution, and collecting eluent;

s6, concentrating and washing eluent: concentrating the eluate with nanofiltration membrane or ultrafiltration membrane to 1/12-1/4 volume of the fermentation filtrate, and washing with 1-3 times volume of water to obtain concentrated eluate;

s7, eluting, concentrating and drying: drying the elution concentrated phase by adopting an oven, freeze drying or spray drying to obtain the chondroitin sulfate product.

2. The method of claim 1, wherein the step of purifying chondroitin sulfate from the chondroitin sulfate fermentation broth comprises: in step S1, the acid is hydrochloric acid or sulfuric acid, and the alkali is ammonia water or sodium hydroxide.

3. The method of claim 2, wherein the step of purifying chondroitin sulfate from the chondroitin sulfate fermentation broth comprises: in step S2, the filtering device is a ceramic membrane filter, a disc centrifuge or a plate and frame filter press; adopting a ceramic membrane filter, using a 50-500nm ceramic membrane, and operating at 60-90 ℃ and 0.1-0.3 MPa; performing solid-liquid separation at 15-30 deg.C by using disk centrifuge; solid-liquid separation is carried out by adopting a plate frame at the temperature of 15-30 ℃.

4. The method of claim 3, wherein the step of purifying chondroitin sulfate from the chondroitin sulfate fermentation broth comprises: in step S3, filtration is performed using an ultrafiltration membrane with a molecular weight cut-off of 10000-50000 Da.

5. The method of claim 4, wherein the step of purifying chondroitin sulfate from the chondroitin sulfate fermentation broth comprises: in step S4, the anion exchange resin is one of Mitsubishi resin DIAION SA12A and DIAION SA21A, Rohm and Haas IRA400 and IRA 900.

6. The method of claim 5, wherein the step of purifying chondroitin sulfate from the chondroitin sulfate fermentation broth comprises: in step S6, when the nanofiltration membrane is used for concentration, the nanofiltration membrane with the cut-off molecular weight of 150-.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor.

Background

Chondroitin Sulfate (CS) is a polysaccharide composed of repeating disaccharide units formed by connecting D-glucuronic acid and N-acetylgalactosamine by β -1, 4 glycosidic bonds, and is formed by carrying out sulfation on hydroxyl at C-4 and C-6 positions.

Chondroitin sulfate in the market at present is mainly extracted from animal bones such as pigs, cattle, sheep, chickens and sharks by an extraction method, a large amount of acid and alkali are used in the production process, the extraction route is complex, the quality is not easy to control, meanwhile, the chondroitin sulfate cannot be applied to some special crowds (such as animal protection organizations and religious factors), and with the increasing shortage of animal bone sources, a non-animal-source product is urgently needed in the market to replace the existing chondroitin sulfate product. With the rise of microbial strain screening and fermentation technologies, the problem of producing chondroitin sulfate by microbial fermentation is widely researched, but because the fermentation yield is low and the extraction process is difficult, no green, environment-friendly and large-scale extraction process is available for producing chondroitin sulfate.

Therefore, a method for purifying chondroitin sulfate from fermentation broth with low pollution, simple operation and high yield needs to be explored, so that the industrialization of producing the chondroitin sulfate by a fermentation method is realized.

Disclosure of Invention

The invention aims to provide a method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor, which is simple and convenient to operate, low in pollution and high in recovery rate.

The technical scheme of the invention is realized as follows: a method for purifying chondroitin sulfate from chondroitin sulfate fermentation liquor comprises the following steps:

s1, inactivation of fermentation liquor: adding acid into chondroitin sulfate fermentation liquor to adjust pH to 2.0-4.5 or adding alkali to adjust pH to 8.5-11.0, heating to 60-90 deg.C, and maintaining for 3-7 hr to obtain inactivated chondroitin sulfate fermentation liquor;

s2, solid-liquid separation: performing solid-liquid separation on the inactivated chondroitin sulfate fermentation liquor by adopting filtering equipment to prepare fermentation filtrate;

s3, concentrating and washing the filtrate: performing ultrafiltration concentration on the fermentation filtrate by using an ultrafiltration membrane, concentrating to 1/8-1/4 of the volume of the fermentation filtrate, and washing by using water with the volume 1-3 times that of the fermentation filtrate to obtain a filtrate concentrated phase;

s4, resin adsorption: adsorbing the filtrate concentrated phase with anion exchange resin with adsorption capacity of 20-80g/L (g chondroitin sulfate/resin volume);

s5, resin elution: eluting the anion exchange resin adsorbed with the chondroitin sulfate by using sodium hydroxide or sodium chloride solution, and collecting eluent;

s6, concentrating and washing eluent: concentrating the eluate with nanofiltration membrane or ultrafiltration membrane to 1/12-1/4 volume of the fermentation filtrate, and washing with 1-3 times volume of water to obtain concentrated eluate;

s7, eluting, concentrating and drying: drying the elution concentrated phase by adopting an oven, freeze drying or spray drying to obtain the chondroitin sulfate product.

Preferably, in step S1, the acid is hydrochloric acid or sulfuric acid, and the base is ammonia water or sodium hydroxide.

Preferably, in step S2, the filtering device is a ceramic membrane filter, a disc centrifuge or a plate and frame filter press; adopting a ceramic membrane filter, using a 50-500nm ceramic membrane, and operating at 60-90 ℃ and 0.1-0.3 MPa; performing solid-liquid separation at 15-30 deg.C by using disk centrifuge; solid-liquid separation is carried out by adopting a plate frame at the temperature of 15-30 ℃ and the operating pressure of 0.1-0.4 MPa.

Preferably, in step S3, an ultrafiltration membrane with molecular weight cut-off of 10000-50000Da is used for filtration; preferably, a roll-type ultrafiltration membrane core is used, the material of the membrane core is polyacrylonitrile, polyethersulfone or polyvinylidene fluoride, the operating pressure is 0.2-1.0MPa, and the temperature is 10-40 ℃.

Preferably, in step S4, the anion exchange resin is one of mitsubishi resins DIAION SA12A and DIAION SA21A, and rochons IRA400 and IRA 900;

preferably, in step S6, when the nanofiltration membrane is used for concentration, the nanofiltration membrane with the molecular weight cut-off of 150-300Da is used, and the membrane core material is preferably selected to be polysulfone and polyamide, the operating pressure is 1.0-2.0MP, and the temperature is 15-35 ℃; when the ultrafiltration membrane is used for concentration, the ultrafiltration membrane with the cut-off molecular weight of 5000-10000Da is used, and the membrane core material is preferably selected from polyethersulfone and polyvinylidene fluoride, the operating pressure is 0.5-1.5MPa, and the temperature is 15-35 ℃.

Compared with the prior art, the invention has the advantages that:

(1) the extraction process does not involve organic solvent, and the process has high automation degree and strong industrialization.

(2) The chondroitin sulfate extracted by the process has high content and purity, and has advantages in quality compared with products extracted from animal bones in the current market.

Detailed Description

In order to make the technical solution of the present invention clearer, the present invention is further described in detail by the following specific examples.