CN114957509A - Scalable purification method of kola acid - Google Patents
Scalable purification method of kola acid Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention is applicable to the field of purification and production of kola acid, and provides an amplifiable purification method of kola acid, which comprises the following steps: adding CaCl 2 Adding the solid-liquid separation pretreatment into the kola acid fermentation liquor for solid-liquid separation pretreatment; performing polymer reverse phase chromatography on the filtrate to obtain flow-through liquid A; carrying out composite filler series chromatography on the flow-through liquid A to obtain flow-through liquid B; carrying out ultrafiltration concentration and liquid exchange on the flow-through liquid B to obtain a solution C; and carrying out alcohol precipitation, dehydration and vacuum drying on the solution C to obtain a finished product. The hyaluronic acid fermentation liquor separation and purification method provided by the invention is a clean production process, active carbon is not used, no toxic substance is added, the minimum amount of ethanol is added after ultrafiltration and concentration, and the produced finished product is safer and more environment-friendly. The invention adopts a flow-through chromatography mode to prepare the kola acid with the purity of more than 1000 kilodaltons in a large scale and high efficiency. The product prepared by the invention has less than 0.1 percent of protein residue, less than 0.1 percent of nucleic acid residue A260 (2 g/L), less than 10ppm of heavy metal residue and endotoxin meeting the requirement of medicinal grade.
Description
Technical Field
The invention belongs to the field of purification of kola acid production, and particularly relates to an amplifiable purification method for high molecular weight kola acid.
Background
The microbial extracellular polysaccharide is a biopolymer which is generated by microbes such as bacteria, fungi, blue algae and the like in the metabolic process and has a protection effect on the microbes. Under natural conditions, most bacteria are coated with polysaccharides. Polysaccharides coated on the surface of bacteria play an important role in the survival and growth of bacteria in a competitive environment. The kola acid is one of microbial extracellular polysaccharide, the solid is white fibrous heteropolysaccharide with an amorphous structure, the solid mainly comprises D-glucuronic acid, D-glucose, D-galactose and L-fucose, the composition ratio is 1:2:2:1, and the kola acid also comprises chemical modification structures on various side chains such as O-acetyl, pyruvic acid and the like. High molecular weight polymeric heteropolysaccharides having a molecular weight of up to 1000 kilodaltons.
Microbial exopolysaccharides have unique physical and rheological properties and are widely used in the food industry as stabilizers, thickeners, gelling agents and emulsifiers. In recent years, research shows that bacterial polysaccharides show remarkable biological activity in the aspects of antitumor, antiviral, immunostimulating, anti-inflammatory activity, antioxidation, antimicrobial action and the like, and therefore, the bacterial polysaccharides attract more attention. Koalatine was first reported in 2017 in the magazine cell to fragment mitochondria and has the effect of extending the life of caenorhabditis elegans. The colanic acid is a natural hydrogel due to the porous cellulose structure and a large number of hydrophilic groups positioned on the surface of the colloid, has excellent water replenishing capacity and soft texture, and is a good candidate product for the future cosmetic and health care markets. In addition, the colanic acid as a unique active biological polymer has special biological characteristics and physiological parameters, and has wide research and application prospects in the aspects of prolonging the service life and resisting aging.
When colibacillus expresses the kola acid, impurities similar to the target object, such as monosaccharide, protein, nucleic acid, cell debris, glycoprotein and the like, can be generated correspondingly, the impurities influence the final content and purity of the kola acid and have important influence on the quality of the product. In literature, the method of centrifugation to remove ethanol precipitate of bacteria is mostly adopted to separate and purify the kola acid, and then the sevag method is used to remove nucleic acid and protein impurities. How to efficiently reduce the residual quantity of impurities such as protein, nucleic acid and the like to an extremely low level is the key of large-scale production and medical and aesthetic application of products. However, there is still no commercial clavulanic acid product and a perfect production and purification method of the clavulanic acid, which severely limits the commercial application of the clavulanic acid.
In the field of polysaccharides, ethanol precipitation is generally adopted for crude purification, salting out or sevag method for protein removal, and finally an anion chromatography method is adopted for fine purification of the polysaccharide. The Sevag process is a classical protein removal process, but suffers from large losses, is solvent unfriendly, is difficult to scale up, and does not completely remove protein impurities. DEAE anion chromatography for decolorization and impurity removal is suitable for purification of polysaccharides with molecular weights less than 2000KDa, because the pore size of the filler is not suitable for ultra-high molecular weight clavulanic acid.
In the patent CN113637620A, thalli are removed by centrifugation after boiling, then lipopolysaccharide is removed by glacial acetic acid precipitation, protein impurities are removed by chloroform and methanol, and solid is obtained by freeze drying. The reported methods use toxic organic solvents and are not suitable for scale-up production, as nucleic acid and protein impurities are not removed completely.
Patent CN111893150A discloses that crude product is obtained by boiling, centrifuging to remove thallus, adding glacial acetic acid to precipitate lipopolysaccharide, rotary evaporating supernatant to reduce water content, and freeze drying. After the crude product is re-melted, the salt is removed by ultrafiltration, and then the product of the colanic acid is obtained by freeze drying. The reported method needs boiling and rotary evaporation means, so that the efficiency is low, the amplification realization difficulty is high, meanwhile, the removal of key impurity protein and nucleic acid is not researched, and the method is not suitable for producing high-purity clavulanic acid products on a large scale.
Disclosure of Invention
The invention aims to provide a method for preparing the 1000 kilodalton molecular weight kola acid with low cost and high purity by amplification and purification.
The invention provides a separation and purification process of 1000 ten thousand dalton kola acid according to the defects of the technical background, the process method is simple, the cost is low, the process is environment-friendly, and protein, nucleic acid impurities and some impurities with relatively small molecular weight can be effectively removed.
The purification method of the invention takes the kola acid fermentation liquor as the raw material and comprises the following steps:
1. adding CaCl into the fermentation liquor 2 Carrying out solid-liquid separation pretreatment;
2. performing polymer reverse phase chromatography on the filtrate;
3. performing composite filler series chromatography on the flow-through liquid;
4. carrying out ultrafiltration concentration and liquid change on the flow-through liquid;
5. precipitating with ethanol, dehydrating, and vacuum drying.
According to the method provided by the above, 0.1-2.0 wt% of CaCl is added into the fermentation liquor in the step 1 2 Adjusting the pH value of the solution to 8.0-10.0 for flocculation pretreatment; centrifuging or plate-and-frame filter pressing to remove precipitate such as thallus to obtain clarified liquid.
And (3) purifying the clarified liquid obtained in the step (1) by flow-through mode reverse phase chromatography, adjusting the pH value of a sample to be 6.0-9.0, loading the sample to be 100-300 mg/mL, keeping the sample for 2-6 minutes, and collecting a flow-through liquid A. The polymer reverse phase filler matrix can be polymethacrylate or polystyrene, the pore size of the used filler is 500-2000A, and the particle size of the filler is 50-150 mu m.
And (3) carrying out chromatography purification on the flow-through liquid A in the step (2) by using hydroxyapatite and ion composite filler in series connection, carrying the sample loading amount to 200-400 mg/mL, keeping the sample loading amount for 2-6 minutes, and collecting the flow-through liquid B. The hydroxyapatite is CHT II type, the ion composite filler is anion and hydrophobic composite filler, and the particle size is 30-150 mu m.
And (3) carrying out hollow fiber membrane ultrafiltration concentration and liquid change on the flow-through liquid B in the step (3), adding EDTA solid into the flow-through liquid B, controlling the transmembrane pressure to be less than 1Bar, concentrating the sample by 3-5 times, then washing by using ultrapure water by 3-7 times, emptying and collecting trapped fluid after ultrafiltration is finished, and circularly washing the membrane for 5-20 min by using the ultrapure water with the dead volume of 1-3 times, and emptying and collecting. And combining the trapped fluid and the membrane washing solution to obtain a solution C. The aperture of the ultrafiltration membrane is 50-300 KD.
And (3) adding 1.5-4 times volume of 95% (V/V, the same below) ethanol into the solution C in the step (4), then slowly dropwise adding a saturated sodium chloride solution until the final concentration is 1-3 wt%, standing, and collecting precipitates. And adding 95% ethanol into the precipitate for dehydration twice, wherein the dehydration lasts for 1-3 h each time. And after dehydration, filtering off ethanol, carrying out vacuum drying at the drying temperature of 30-50 ℃ for 3-6 h, and grinding the solid and sieving with a 200-mesh sieve. And continuously drying for 8-15 h, and collecting to obtain a final product.
Effects of the invention
Compared with the prior art, the method adopts continuous purification means such as polymer reversed-phase filler, hydroxyapatite series ion composite filler and the like, can prepare 1000 ten thousand daltons of high molecular weight colanic acid, can add a chelating agent to remove heavy metal ion impurities during ultrafiltration liquid exchange, and can prepare fine granular colanic acid by adding ethanol first and then dripping sodium chloride during ethanol precipitation, so that the fine granular colanic acid is convenient to use subsequently.
The hyaluronic acid fermentation liquor separation and purification method provided by the invention is a clean production process, active carbon is not used, no toxic substance is added, the minimum amount of ethanol is added after ultrafiltration concentration, and the produced finished product is safer and more environment-friendly. The method for separating and purifying the kola acid fermentation liquor provided by the invention adopts a flow-through chromatography mode to prepare the kola acid with the purity of more than 1000 ten thousand daltons in a large scale and high efficiency manner. According to the method for separating and purifying the kola acid fermentation liquor, the protein residue of the prepared product is less than 0.1 percent, the nucleic acid residue A260 is less than 0.1 (2 g/L), the heavy metal residue is less than 10ppm, and the endotoxin meets the requirement of medicinal grade.
Drawings
FIG. 1 is a GPC chromatogram of example 1;
fig. 2 is a flow diagram of a purification process for colanic acid.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described herein are illustrative only and not limiting, and that various changes and modifications may be suggested to one skilled in the art based on the teachings herein. The starting materials used in the examples are all commercially available.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
The fermentation liquor of the kola acid used in the invention is provided by fermenting engineering strains constructed by Eichschka escherichia coli by Shenzhen arborvitae whole boundless Lianji Limited company. The protein content is measured by the Bradford method, the assay of the kola acid content is measured by the cysteine sulfate method, and the endotoxin content is measured by the limulus reagent gel method.
Example 1
500mL of kola acid fermentation liquor is taken, diluted by two times by purified water, and 0.5wt% of CaCl is added 2 The pH of the solution was adjusted to 9.0 after the solution was sufficiently dissolved, and the residue of the cells was removed by centrifugation to obtain a clear solution. Adding 5% ethanol into the clear solution, performing polyRP (50 μm, pore diameter 500A) filler chromatography with column volume of 20mL and flow rate of 5mL/min, and collecting flow-through solution A. Adjusting the pH value of the flow-through liquid A to 7.0, carrying out 10mL capto Adhere +10mL CHT II tandem column chromatography at the flow rate of 5mL/min, and collecting a flow-through liquid B. 0.05wt% EDTA solid was added to the flow-through solution B to carry out ultrafiltration with a 50KD hollow fiber membrane, followed by concentration by 3 times by volume and then washing with ultrapure water by 5 times by volume. Collecting the ultrafiltration trapped fluid and the membrane washing liquid, adding 2 times of ethanol by volume, then dropwise adding saturated sodium chloride solution until the final concentration is 2wt%, and standing for precipitation. The precipitate was dehydrated 2 times for 2h with 95% ethanol. And (4) drying the dehydrated sample at 50 ℃ for 5 hours in vacuum, grinding and sieving the sample by a 200-mesh sieve, and drying to obtain a finished product.
Example 2
Taking 500mL of kola acid fermentation broth, diluting with purified water twice, and adding 1.0wt% of CaCl 2 The pH of the solution was adjusted to 9.0 after the solution was sufficiently dissolved, and the residue of the cells was removed by centrifugation to obtain a clear solution. Adding 5% ethanol into the clear solution, performing polyRP (50 μm, pore diameter 1000A) filler chromatography with column volume of 20mL and flow rate of 5mL/min, and collecting flow-through solution A. Adjusting the pH value of the flow-through liquid A to 7.5, carrying out 10mL capto Adhere +10mL CHT II tandem column chromatography at the flow rate of 5mL/min, and collecting a flow-through liquid B. 0.05wt% EDTA solid was added to the flow-through solution B to perform ultrafiltration with a 100KD hollow fiber membrane, followed by concentration by 3 times by volume and then washing with ultrapure water by 5 times by volume. Collecting ultrafiltrationAdding 2 times volume of ethanol into the trapped solution and the membrane washing solution, then dropwise adding a saturated sodium chloride solution until the final concentration is 2wt%, and standing for precipitation. The precipitate was dehydrated 2 times for 2h with 95% ethanol. And (4) drying the dehydrated sample at 50 ℃ for 5 hours in vacuum, grinding and sieving by a 200-mesh sieve to obtain a finished product.
Example 3
500mL of kola acid fermentation liquor is taken, diluted twice by purified water, and 1.0wt% of CaCl is added 2 The pH of the solution was adjusted to 9.0 after the solution was sufficiently dissolved, and the residue of the cells was removed by centrifugation to obtain a clear solution. Adding 5% ethanol into the clear solution, performing polyRP (30 μm, pore diameter 2000A) filler chromatography with column volume of 20mL and flow rate of 5mL/min, and collecting flow-through liquid. Adjusting the pH value of the flow-through liquid A to 8.0, carrying out 10mL capto Adhere +10mL CHT II tandem column chromatography at the flow rate of 5mL/min, and collecting a flow-through liquid B. 0.05wt% EDTA solid was added to the flow-through solution B to perform 300KD hollow fiber membrane ultrafiltration, concentrating by 3 times volume, followed by washing by ultrapure water by 5 times volume. Collecting the ultrafiltration trapped fluid and the membrane washing liquid, adding 2 times of ethanol by volume, then dropwise adding saturated sodium chloride solution until the final concentration is 2wt%, and standing for precipitation. The precipitate was dehydrated 2 times for 2h with 95% ethanol. And (4) drying the dehydrated sample at 37 ℃ in vacuum for 10h, grinding and sieving by a 200-mesh sieve to obtain a finished product.
Example 4
Diluting 10L of kola acid fermentation broth with purified water twice, and adding 0.5wt% of CaCl 2 The pH of the solution was adjusted to 9.0 after the solution was sufficiently dissolved, and the residue of the cells was removed by centrifugation to obtain a clear solution. Adding 5% ethanol into the clear solution, performing polyRP (150 μm, pore diameter 1000A) filler chromatography with column volume of 500mL and flow rate of 100mL/min, and collecting flow-through solution A. Adjusting the pH value of the flow-through liquid A to 7.0, carrying out 250mL capto Adhere +250mL CHT II tandem column chromatography at the flow rate of 100mL/min, and collecting a flow-through liquid B. 0.05wt% EDTA solid was added to the flow-through solution B to perform 300KD hollow fiber membrane ultrafiltration, concentrating by 3 times volume, followed by washing by ultrapure water by 5 times volume. Collecting the ultrafiltration trapped fluid and the membrane washing liquid, adding 2 times of ethanol by volume, then dropwise adding saturated sodium chloride solution until the final concentration is 2wt%, and standing for precipitation. The precipitate was dehydrated 2 times for 2h with 95% ethanol. And (4) drying the dehydrated sample at 37 ℃ in vacuum for 10h, grinding and sieving by a 200-mesh sieve to obtain a finished product.
TABLE 1 measurement results of purity, molecular weight and protein content of finished colanic acid
As can be seen from the data in Table 1, the method for separating and purifying the kola acid fermentation liquor, provided by the invention, can be used for preparing the kola acid with the concentration of more than 1000 ten thousand daltons in a large scale and high efficiency by adopting a flow-through chromatography mode, and the lowest protein residue in the product can reach 0.075%. Therefore, the process method provided by the application is simple, low in cost and environment-friendly, can effectively remove protein, can prepare over 1000 kilodaltons of colanic acid, and is suitable for large-scale production.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A scalable purification process for kola acid, comprising the steps of:
step 1: adding CaCl into the kola acid fermentation liquor 2 Adjusting the pH value, performing flocculation pretreatment, and then performing solid-liquid separation to obtain a clarified liquid;
step 2: purifying the clarified liquid by flow-through mode reverse phase chromatography, and collecting flow-through liquid A;
and step 3: carrying out composite filler series chromatography purification on the flow-through liquid A to obtain flow-through liquid B;
and 4, step 4: carrying out ultrafiltration on the flow-through liquid B, collecting trapped fluid after the ultrafiltration is finished, washing the membrane with pure water, collecting membrane washing liquid, and combining the trapped fluid and the membrane washing liquid to obtain a solution C;
and 5: and (3) adding ethanol into the solution C, then dropwise adding a sodium chloride solution, and standing and precipitating to obtain the purified colanic acid.
2. The purification method according to claim 1, wherein in step 1: the CaCl is 2 The solid-liquid separation method is characterized by adding the raw materials in the form of aqueous solution, wherein the concentration of the aqueous solution is 0.1-2.0 wt%, the pH value ranges from 8.0-10.0, and the solid-liquid separation method is centrifugation or plate-and-frame filter pressing.
3. The purification method according to claim 1, wherein in step 2: the filler of the chromatographic column in the reverse phase chromatography is a polymer reverse phase filler matrix, and the pH value of the clear liquid in the step 1 is required to be adjusted to 6.0-9.0 before sample loading.
4. The purification method according to claim 3, wherein in step 2: the polymer reversed phase filler matrix is polymethacrylate or polystyrene.
5. The purification method according to claim 4, wherein in step 2: the polymer reversed phase filler has the pore diameter of 500-2000A and the particle diameter of 50-150 mu m.
6. The purification method according to claim 1, wherein in step 3: the composite filler is hydroxyapatite and an ionic composite filler, the hydroxyapatite is CHT II type, and the ionic composite filler is an anionic and hydrophobic composite filler.
7. The purification method according to claim 1, wherein in step 3: the particle size of the composite filler is 30-150 mu m.
8. The purification method according to claim 1, wherein in step 4: adding a chelating agent into the flow-through liquid B, and then carrying out ultrafiltration.
9. The purification method according to claim 8, wherein in step 4: the chelating agent is EDTA, and the ultrafiltration is carried out by adopting a hollow fiber ultrafiltration membrane with the aperture of 50-300 KD.
10. The purification method according to claim 1, wherein in step 5: dropwise adding a sodium chloride solution until the final concentration is 1-3 wt%; and adding ethanol into the precipitate for dehydration, filtering out the ethanol after dehydration, drying, grinding and sieving the obtained solid, and drying again to obtain the finished product.
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