CN109929896B - Production process of ursodeoxycholic acid - Google Patents
Production process of ursodeoxycholic acid Download PDFInfo
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Abstract
The invention discloses a production process of ursodeoxycholic acid, which takes chenodeoxycholic acid as a substrate, and carries out catalytic reaction through 7 alpha-steroid dehydrogenase and/or thalli containing 7 alpha-steroid dehydrogenase, the obtained product is filtered through a ceramic membrane, and enzyme protein or thalli are removed, so as to obtain 7-ketolithocholic acid; and then carrying out catalytic reaction on the obtained 7-ketolithocholic acid serving as a substrate through 7 beta-steroid dehydrogenase and/or thallus containing 7 beta-steroid dehydrogenase, filtering the obtained product through a ceramic membrane to remove enzyme protein or thallus to obtain a crude ursodeoxycholic acid product, removing small molecular protein impurities from the crude product through an ultrafiltration membrane to obtain a clear solution of the ursodeoxycholic acid ultrafiltration membrane, concentrating through a nanofiltration membrane, decolorizing with resin, concentrating, and drying. Compared with the prior art, the invention can improve the quality and yield of ursodeoxycholic acid and reduce the production cost.
Description
Technical Field
The invention belongs to the field of chemistry, and particularly relates to a production process of ursodeoxycholic acid.
Background
Ursodeoxycholic acid, the main component is 3a, 7 beta-dihydroxy-5 beta-cholestane-24-acid, is an organic compound, odorless, bitter, soluble in ethanol, and insoluble in chloroform; it is easily soluble in glacial acetic acid, and is soluble in sodium hydroxide solution. The medicine is used for increasing bile acid secretion, changing bile components, reducing cholesterol and cholesterol ester in bile and being beneficial to gradually dissolving cholesterol in gallstone. Ursodeoxycholic acid is the main effective component of rare traditional Chinese medicine fel Ursi, which is classified as a cholelithiasis dissolving medicine in the second part of the Chinese pharmacopoeia edition, and classified as a liver and gall disease medicine in the 2009 edition of the national basic medicine catalog (the part for use in the basic medical and health institution), which is a common tablet.
Ursodeoxycholic acid is one of animal bile acids, and other common cholic acid compounds include cholic acid lithocholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, and the like. Wherein, ursodeoxycholic acid and chenodeoxycholic acid are epimers, and the only difference between the two is that the 7-hydroxyl structure configuration is different. The 7-hydroxyl of ursodeoxycholic acid is in beta configuration, while the 7-hydroxyl of chenodeoxycholic acid is in alpha configuration. The 7-hydroxyl of chenodeoxycholic acid can be oxidized into keto, namely 7-ketolithocholic acid, and the 7-keto is converted into beta hydroxyl, namely ursodeoxycholic acid, through reduction reaction. The molecular structural formula of ursodeoxycholic acid, chenodeoxycholic acid and 7-ketolithocholic acid is shown in figure 1.
At present, ursodeoxycholic acid is mainly applied to treating various liver, gall and digestive tract diseases clinically. With the continuous progress of molecular biology, ursodeoxycholic acid base and clinical research, people find that ursodeoxycholic acid also has an active effect on the aspects of promoting immunoregulation, treating coronary heart disease and the like. Therefore, with the progress of research, the utility value of ursodeoxycholic acid is more and more recognized and paid attention to, and the demand for ursodeoxycholic acid is also increasing year by year.
However, the sources of natural bear's gall are decreasing and efforts are constantly being made to find alternative methods to obtain ursodeoxycholic acid from natural bear's gall. In the early days, ursodeoxycholic acid was produced by using cholic acid separated and extracted from bovine and ovine bile as a starting material and then by a complicated chemical method, wherein after chenodeoxycholic acid is prepared, the ursodeoxycholic acid is generated by oxidation reduction. However, this method has many steps, a long production time and high cost. In the last 80 s, enzymology and microbial fermentation methods developed. The chenodeoxycholic acid is isomerized by an enzymology and microbial fermentation method or the ursodeoxycholic acid is produced by beta-hydroxylation of lithocholic acid, but the method still has the defects of multiple steps, long production time and high cost.
The literature reports that the transformation routes are mainly 3. Firstly, using chenodeoxycholic acid as a raw material, and obtaining ursodeoxycholic acid by methyl esterification, selective hydroxyl protection, Jones reagent oxidation and metal sodium and nickel dichloride reduction with the total yield of 42%. Secondly, the ursodeoxycholic acid is prepared by taking the chenodeoxycholic acid as a raw material, selecting the high-specificity compound Ni-alpha-Al 2O3 and a catalyst at 800 ℃ and carrying out hydrogenation reduction under high pressure, wherein the total yield is 65 percent. ③ using 7-ketolithocholic acid as raw material, using hydrogen-Raney nickel high pressure reduction or electrochemical reduction to obtain ursodeoxycholic acid, the yield is more than 90%. Wherein 7-ketolithocholic acid can be synthesized by using chenodeoxycholic acid as a raw material and acetone and water as media and oxidizing by N-bromosuccinimide (NBS), and the yield reaches 89%. The method has the advantages of more reaction steps, long route and lower yield; the reaction conditions of the route II and the route III are harsh, high pressure is needed, the requirement on equipment is high, and the purity of the product in the route III is only 92.5 percent.
The prior chemical method for producing ursodeoxycholic acid mainly has the following defects:
(1) the investment cost is high, and the production cost is high;
(2) the chemical synthesis needs conditions of high temperature, high pressure and the like, and a large amount of organic solvent is used, so the production process is unsafe;
(3) the reaction steps are multiple, the route is long, and the yield of the product is not high;
(4) serious pollution and serious environmental protection problem.
At present, related scientific research institutions propose to synthesize ursodeoxycholic acid by using whole-cell catalysis, but in the catalytic synthesis, the separation of a target product and catalyst whole cells is a difficult obstacle, because the viscosity of the catalytic product is very high, the catalytic product is not suitable for traditional plate-and-frame filter pressing, more sewage can be brought, and the automation degree is low; and an ultrafiltration membrane is not used for removing protein, so that a large amount of impurities are brought to later purification, the product quality and purity are not high, and a large amount of organic solvent is consumed.
In view of this, a new process with low production cost, safe and reliable process, low investment, and high product quality and yield is still to be provided.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a process for extracting ursodeoxycholic acid by using a membrane separation technology aiming at the defects of the prior art, thereby providing a production process of the ursodeoxycholic acid with high purity and high yield.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a production process of ursodeoxycholic acid comprises the following steps:
(1) catalyzing chenodeoxycholic acid by 7 alpha-steroid dehydrogenase or thallus containing 7 alpha-steroid dehydrogenase to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) by a microfiltration membrane to obtain a permeate containing 7-ketolithocholic acid;
(3) adding 7 beta-steroid dehydrogenase or thallus containing 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2) for catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) through an ultrafiltration membrane to obtain an ultrafiltration membrane permeate containing the ursodeoxycholic acid;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using ion exchange resin to obtain a resin eluent containing the ursodeoxycholic acid;
(8) and (4) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
Specifically, in the step (1), the volume ratio of the dosage of the 7 alpha-steroid dehydrogenase or the thallus containing the 7 alpha-steroid dehydrogenase to the chenodeoxycholic acid is 0.001-2%, the temperature for catalysis is 10-60 ℃, and the time is 0.5-5 h. The 7 alpha-steroid dehydrogenase functions to catalyze the conversion of chenodeoxycholic acid to 7-ketolithocholic acid.
In the step (2), the microfiltration membrane is a ceramic membrane, the filtration precision is 500nm, the filtration temperature is 80 ℃, and the filtration pressure is 0.1 MPa. The microfiltration membrane is used for removing the 7 alpha-steroid dehydrogenase enzyme protein or the bacterial cells containing the 7 alpha-steroid dehydrogenase. In the microfiltration process, when the filtration precision of the microfiltration membrane is 5nm, the flux of the microfiltration membrane is only 40% of that of the microfiltration membrane with the filtration precision of 500nm, and 0.8MPa pressure is required as the driving force for the operation of the membrane equipment; when the filtration precision of the microfiltration membrane is 500nm, the initial flux is 25 percent larger than 50nm and 20 percent larger than that of the microfiltration membrane with the filtration precision of 200nm, but the flux attenuation is faster, and about 3 percent of macromolecular protein and pigment can permeate the microfiltration membrane, so that the quality of the filtrate is reduced. The filtering temperature is 10-80 ℃, the temperature is preferably 50-70 ℃, and the filtering pressure is 0.1-0.8 MPa, preferably 0.25-0.4 MPa. Furthermore, when the temperature is 60 ℃, the pressure is 0.35MPa, and the filtration precision of the microfiltration membrane is 50-200 nm, the filtration flux is large, the flux is slowly reduced, the concentration can be nearly 5 times, the removal rate of the enzyme protein or the thallus is more than 99.9%, and the quality of the filtrate is good.
In the step (3), the volume ratio of the 7 beta-steroid dehydrogenase or the thallus containing the 7 beta-steroid dehydrogenase to the permeate containing the 7-ketolithocholic acid is 0.001-2%, the temperature for catalysis is 10-60 ℃, and the time is 0.5-5 h. The 7 beta-steroid dehydrogenase functions to catalyze the conversion of 7-ketolithocholic acid to ursodeoxycholic acid.
In the step (4), the microfiltration membrane is a ceramic membrane, the filtration precision is 5-500 nm, the filtration temperature is 10-80 ℃, and the filtration pressure is 0.1-0.8 MPa. The microfiltration membrane is used for removing 7 beta-steroid dehydrogenase enzyme protein or bacteria containing 7 beta-steroid dehydrogenase.
In the step (5), the ultrafiltration membrane is a roll-type ultrafiltration membrane, the molecular weight cutoff is 1000-40000 Da, the temperature is 10-60 ℃, and the pressure is 0.5-2.5 MPa. The ultrafiltration membrane is used for removing macromolecular proteins, pigments and other impurities in the catalytic product and improving the purity of the ursodeoxycholic acid. In the ultrafiltration process, when the ultrafiltration cut-off molecular weight is 1000Da, the flux is only 68% of that of 20000Da, 1.8MPa pressure is required as the driving force for the operation of the membrane equipment, and 30% of ursodeoxycholic acid product can be cut off; when the ultrafiltration cut-off molecular weight is 40000Da, about 2.5 percent of small molecular protein and pigment permeate the microfiltration membrane, and the quality of the filtrate is reduced. The filtering temperature is 10-60 ℃, the temperature is preferably 30-50 ℃, and the filtering pressure is 0.5-2.5 MPa, preferably 0.6-1.0 MPa. Furthermore, when the temperature is 35 ℃, the pressure is 0.7MPa, and the cut-off molecular weight of the ultrafiltration membrane is 8000-10000 Da, the filtration flux is stable, the concentration can be nearly 20 times, the removal rate of the small molecular protein is over 99.9 percent, the quality of filtrate is good, and the product recovery rate can reach 96.7 percent.
In the step (6), the nanofiltration membrane is a roll-type ultrafiltration membrane, the molecular weight cutoff is 100-800 Da, the temperature is 10-60 ℃, and the pressure is 0.5-2.5 MPa. The nanofiltration membrane is used for filtering to improve the concentration of ursodeoxycholic acid entering the ion exchange resin, reduce the feeding amount of the resin and improve the working efficiency of the resin. In the nanofiltration process, when the molecular weight cut-off of the nanofiltration membrane is 100Da, the flux of the nanofiltration membrane is only 40 percent of that of the nanofiltration membrane with the molecular weight of 800Da, and 2.5MPa pressure is required as the driving force for the operation of membrane equipment; when the molecular weight of the nanofiltration membrane is 800Da, the flux is 25% larger than 300Da and 40% larger than that of the nanofiltration membrane with the molecular weight of 150Da, but about 5% of products penetrate through the nanofiltration membrane, and the product yield is reduced. The temperature is 10-50 ℃, the temperature is preferably 30-50 ℃, the pressure is 0.5-2.5 MPa, and preferably 1.0-2.0 MPa. Furthermore, when the temperature is 30 ℃, the pressure is 1.5MPa, and the molecular weight cut-off of the nanofiltration membrane is 150-300 Da, the filtration flux is stable, the concentration can be nearly 10 times, and the cut-off rate of the product is more than 99.5%.
In the step (7), the ion exchange resin is acrylic acid type strong-base anion exchange resin, the flow rate is 2-6 BV/h, the preferable flow rate is 3-5 BV/h, the temperature is 20-80 ℃, and the preferable temperature is 40-50 ℃. When the flow rate is 4BV/h and the temperature is 50 ℃, the viscosity of the feed liquid is small, the decoloring effect is optimal, the decoloring effect and the production efficiency can be ensured, the energy consumption is relatively low, and the yield of the ursodeoxycholic acid is over 99.6 percent.
Has the advantages that:
1. the invention adopts ceramic membrane filtration in the production process of ursodeoxycholic acid, can effectively remove macromolecular protein or thallus cells, and improves the product quality. The ceramic membrane can resist high temperature, high pressure and chemical corrosion and has longer service life; on the other hand, the pollution of solid waste to the environment is also avoided.
2. The invention adopts ultrafiltration membrane filtration in the production process of ursodeoxycholic acid, can effectively remove macromolecular protein, greatly improves the purity of the product, reduces the feeding load of the following ion exchange resin, and simultaneously reduces the dosage of organic solvent.
3. The production process of ursodeoxycholic acid adopts a nanofiltration membrane for preconcentration, greatly reduces the using amount of resin, reduces more than 80% of evaporated water, reduces production energy consumption, and reduces production cost. The nanofiltration membrane has high precision and can improve the yield of the ursodeoxycholic acid.
4. The process of the invention adopts the membrane separation equipment and the ion exchange equipment, reduces the floor area of the equipment, lowers the capital construction cost, performs a large amount of optimization work on the parameters of new equipment and the traditional process, obtains the optimal production process parameters, ensures the high-efficiency and energy-saving operation of production, and simultaneously has higher product quality. The production process is energy-saving, has high automation degree compared with the traditional production process, can save 50 percent of labor cost, and has remarkable economic benefit.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 shows the molecular structural formulas of ursodeoxycholic acid, chenodeoxycholic acid and 7-ketolithocholic acid.
FIG. 2 is a flow chart of the production process of ursodeoxycholic acid of the present invention.
Detailed Description
The invention will be better understood from the following examples.
In the following examples, the enzyme activity of 7. alpha. -steroid dehydrogenase was measured using chenodeoxycholic acid as a substrate, and 2.89mL of 0.2mM NAD + (50mM potassium phosphate buffer, prepared at pH 8.0), 10. mu.L of 150mM chenodeoxycholic acid, 100. mu.L of diluted enzyme solution were contained in a 3mL reaction mixture, and the increase in absorbance was measured at 340 nm. The unit of enzyme activity (unit/mL) is calculated as: [. DELTA.A 340/min. times.3 (mL). times.crude enzyme dilution ]/[ 6.22. times.0.1 (mL) ]. The enzyme activity was measured to be 205 u/mg.
The enzyme activity of 7 beta-steroid dehydrogenase was measured using ursodeoxycholic acid as a substrate, and in a 3mL reaction mixture, 2.89mL of 0.2mM ADP + (50mM potassium phosphate buffer, pH 8.0), 10. mu.L of 150mM ursodeoxycholic acid, and 100. mu.L of diluted enzyme solution, the increase in absorbance was measured at 340 nm. The enzyme activity unit (unit/mL) is calculated as: [. DELTA.A 340/min. times.3 (mL). times.crude enzyme dilution ]/[ 6.22X 0.1(mL) ]. The enzyme activity was measured to be 205 u/mg.
Example 1
Ursodeoxycholic acid was prepared according to the scheme shown in figure 1:
(1) catalyzing chenodeoxycholic acid by 7 alpha-steroid dehydrogenase, wherein the addition amount of the chenodeoxycholic acid is 0.001% of the volume of the chenodeoxycholic acid, and the catalysis temperature is 20 ℃ to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) through a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 5nm, the filtering temperature is 20 ℃, and the filtering pressure is 0.8MPa), so as to obtain a permeate containing 7-ketolithocholic acid;
(3) adding 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2), wherein the addition amount of the 7 beta-steroid dehydrogenase is 0.001% of the volume of the permeate containing 7-ketolithocholic acid, the catalytic temperature is 20 ℃, and carrying out catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 5nm, the filtering temperature is 20 ℃, and the filtering pressure is 0.8MPa) to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) through an ultrafiltration membrane (the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 40000Da, the temperature is 10-60 ℃, and the pressure is 0.5MPa), so as to obtain the permeate containing the ursodeoxycholic acid through the ultrafiltration membrane;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane (the nanofiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 800Da, the temperature is 10 ℃, and the pressure is 2.5MPa), so as to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using Rohm and Haas Amberlite FPA98Cl ion exchange resin (acrylic acid type strong-base anion exchange resin, the flow rate is 2BV/h, and the temperature is 20 ℃) to obtain a resin eluent containing the ursodeoxycholic acid;
(8) and (4) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
The yield of the ursodeoxycholic acid obtained in the embodiment is 98.6%, the purity of the ursodeoxycholic acid is 96.6%, the content of protein in the product is 3.1%, the content of pigment in the product is 0.3%, and the ceramic membrane has high filtration precision, low temperature, small flux and long filtration time. The flux of the nanofiltration membrane is smaller due to higher protein content.
Example 2
Ursodeoxycholic acid was prepared according to the scheme shown in figure 1:
(1) catalyzing chenodeoxycholic acid by thalli containing 7 alpha-steroid dehydrogenase, wherein the addition amount of the chenodeoxycholic acid is 2% of the volume of the chenodeoxycholic acid, and the catalysis temperature is 60 ℃ to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) by using a microfiltration membrane (the filtration precision is 500nm, the filtration temperature is 80 ℃, and the filtration pressure is 0.1MPa) to obtain a permeate containing 7-ketolithocholic acid;
(3) adding thallus containing 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2), wherein the addition amount of the thallus is 2% of the volume of the permeate containing 7-ketolithocholic acid, the catalytic temperature is 60 ℃, and carrying out catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 50nm, the filtering temperature is 80 ℃, and the filtering pressure is 0.1MPa) to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) by using an ultrafiltration membrane (the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 1000Da, the temperature is 60 ℃, and the pressure is 2.5MPa), so as to obtain the permeate containing the ursodeoxycholic acid ultrafiltration membrane;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane (the nanofiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 100Da, the temperature is 10 ℃, and the pressure is 2.5MPa), so as to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using Rohm and Haas Amberlite FPA98Cl ion exchange resin (acrylic acid type strong-base anion exchange resin, the flow rate is 6BV/h, and the temperature is 60 ℃) to obtain a resin eluent containing the ursodeoxycholic acid;
(8) and (4) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
The yield of ursodeoxycholic acid obtained in this example is 90.1%, the purity of ursodeoxycholic acid is 99.6%, the content of protein in the product is 0.2%, and the content of pigment is 0.2%; the flux of the ceramic membrane is larger, but the molecular weight cut-off of the ultrafiltration membrane is smaller, so that a part of ursodeoxycholic acid is cut-off, and the yield is lower.
Example 3
Ursodeoxycholic acid was prepared according to the scheme shown in figure 1:
(1) catalyzing chenodeoxycholic acid by 7 alpha-steroid dehydrogenase, wherein the addition amount of the chenodeoxycholic acid is 0.05 percent of the volume of the chenodeoxycholic acid, and the catalysis temperature is 20 ℃ to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) through a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 200nm, the filtering temperature is 20 ℃, and the filtering pressure is 0.2MPa), so as to obtain a permeate containing 7-ketolithocholic acid;
(3) adding 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2), wherein the addition amount of the 7 beta-steroid dehydrogenase is 0.05% of the volume of the permeate containing 7-ketolithocholic acid, the catalytic temperature is 20 ℃, and carrying out catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) through a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 200nm, the filtering temperature is 20 ℃, and the filtering pressure is 0.2MPa), so as to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) by using an ultrafiltration membrane (the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 20000Da, the temperature is 40 ℃, and the pressure is 0.8MPa), so as to obtain the permeate containing the ursodeoxycholic acid through the ultrafiltration membrane;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane (the nanofiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 300Da, the temperature is 40 ℃, and the pressure is 2.0MPa), so as to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing ursodeoxycholic acid obtained in the step (6) by using Rohm and Haas Amberlite FPA98Cl ion exchange resin (acrylic acid type strongly basic anion exchange resin, flow rate is 4BV/h, temperature is 40 ℃), and obtaining a resin eluent containing ursodeoxycholic acid;
(8) and (5) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
The yield of ursodeoxycholic acid obtained in this example is 99.3%, the purity of ursodeoxycholic acid is 98.3%, the protein content in the product is 1.5%, and the pigment content is 0.2%; the ceramic membrane has low filtering temperature and pressure, so that the filtering flux is low, the ultrafiltration membrane has high molecular weight cut-off, part of small-molecular proteins permeate, the flux of the rear-end nanofiltration membrane is low, and the protein content of the final product is high.
Example 4
Ursodeoxycholic acid was prepared according to the scheme shown in figure 1:
(1) catalyzing chenodeoxycholic acid by thalli containing 7 alpha-steroid dehydrogenase, wherein the addition amount of the chenodeoxycholic acid is 0.5 percent of the volume of the chenodeoxycholic acid, and the catalysis temperature is 50 ℃ to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) through a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 50nm, the filtering temperature is 50 ℃, and the filtering pressure is 0.6MPa), so as to obtain a permeate containing 7-ketolithocholic acid;
(3) adding thallus containing 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2), wherein the addition amount of the thallus is 0.5 percent of the volume of the permeate containing 7-ketolithocholic acid, the catalytic temperature is 50 ℃, and carrying out catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 50nm, the filtering temperature is 50 ℃, and the filtering pressure is 0.6MPa) to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) by using an ultrafiltration membrane (the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 10000Da, the temperature is 30 ℃, and the pressure is 1.0MPa), so as to obtain the permeate containing the ursodeoxycholic acid ultrafiltration membrane;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane (the nanofiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 300Da, the temperature is 30 ℃, and the pressure is 2.5MPa), so as to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using Rohm and Haas Amberlite FPA98Cl ion exchange resin (acrylic acid type strong-base anion exchange resin, the flow rate is 2BV/h, and the temperature is 50 ℃) to obtain a resin eluent containing the ursodeoxycholic acid;
(8) and (5) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
The yield of ursodeoxycholic acid obtained in this example is 99.3%, the purity of ursodeoxycholic acid is 99.7%, the content of protein in the product is 0.15%, and the content of pigment is 0.05%; the flux of the ceramic membrane of the product is larger, the protein and pigment content of the product is very low, the quality is higher, but the filtering pressure of the ceramic membrane is higher, the intercepted molecular weight of the nanofiltration membrane is lower, the filtering pressure is also high, the flow rate of the resin is low, the resin consumption of unit capacity is large, and the production energy consumption is higher.
Example 5
Ursodeoxycholic acid was prepared according to the scheme shown in figure 1:
(1) catalyzing chenodeoxycholic acid by thalli containing 7 alpha-steroid dehydrogenase, wherein the addition amount of the chenodeoxycholic acid is 1% of the volume of the chenodeoxycholic acid, and the catalysis temperature is 40 ℃ to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) through a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 50nm, the filtering temperature is 60 ℃, and the filtering pressure is 0.35MPa), so as to obtain a permeate containing 7-ketolithocholic acid;
(3) adding thallus containing 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2), wherein the addition amount of the thallus is 1% of the volume of the permeate containing 7-ketolithocholic acid, the catalytic temperature is 40 ℃, and a catalytic reaction is carried out to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane (the microfiltration membrane is a ceramic membrane, the filtering precision is 50nm, the filtering temperature is 60 ℃, and the filtering pressure is 0.35MPa), so as to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) by using an ultrafiltration membrane (the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 10000Da, the temperature is 35 ℃, and the pressure is 0.7MPa), so as to obtain the permeate containing the ursodeoxycholic acid ultrafiltration membrane;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane (the nanofiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 150Da, the temperature is 30 ℃, and the pressure is 1.5MPa), so as to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using ion exchange resin (acrylic acid type strong-base anion exchange resin, the flow rate is 4BV/h, the temperature is 50 ℃) to obtain a resin eluent containing the ursodeoxycholic acid;
(8) and (4) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid.
The yield of ursodeoxycholic acid obtained in this example is 99.7%, the purity of ursodeoxycholic acid is 99.8%, the content of protein in the product is 0.08%, and the content of pigment is 0.07%; the ceramic membrane of the product has high flux, low protein and pigment content, and high quality.
Comparative example 1
Stirring 7-ketodeoxycholic acid, an alcohol solvent and a nano Pd/C catalyst uniformly, filling hydrogen into a reaction kettle, reacting for 8 hours at the temperature of 90 ℃ under the pressure of 20MPa to obtain a reaction solution, evaporating and concentrating the reaction solution to obtain an ursodeoxycholic acid product, wherein the yield is 78.3%, the purity of the ursodeoxycholic acid is 95.2%, the content of protein in the product is 4.2%, and the content of pigment is 0.6%; . The production process needs hydrogenation catalytic reaction under 20MPa, and has the disadvantages of harsh production conditions, insecurity and serious environmental pollution.
The present invention provides a method and a concept for producing ursodeoxycholic acid, and a method and a way for implementing the technical scheme are many, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (1)
1. A production process of ursodeoxycholic acid is characterized by comprising the following steps:
(1) catalyzing chenodeoxycholic acid by 7 alpha-steroid dehydrogenase and/or thallus containing 7 alpha-steroid dehydrogenase to obtain a catalytic product A;
(2) filtering the catalytic product A obtained in the step (1) by a microfiltration membrane to obtain a permeate containing 7-ketolithocholic acid;
(3) adding 7 beta-steroid dehydrogenase and/or thallus containing 7 beta-steroid dehydrogenase into the permeate containing 7-ketolithocholic acid obtained in the step (2) for catalytic reaction to obtain a catalytic product B;
(4) filtering the catalytic product B obtained in the step (3) by a microfiltration membrane to obtain a permeate containing ursodeoxycholic acid;
(5) filtering the permeate containing the ursodeoxycholic acid obtained in the step (4) through an ultrafiltration membrane to obtain an ultrafiltration membrane permeate containing the ursodeoxycholic acid;
(6) filtering the ultrafiltration membrane permeate containing the ursodeoxycholic acid obtained in the step (5) by using a nanofiltration membrane to obtain a nanofiltration membrane concentrated solution containing the ursodeoxycholic acid;
(7) decolorizing the nanofiltration membrane concentrated solution containing the ursodeoxycholic acid obtained in the step (6) by using ion exchange resin to obtain a resin eluent containing the ursodeoxycholic acid;
(8) evaporating and crystallizing the resin eluent containing the ursodeoxycholic acid obtained in the step (7) to obtain the ursodeoxycholic acid;
in the step (1), the volume ratio of the 7 alpha-steroid dehydrogenase to the chenodeoxycholic acid is 0.001-2%, the temperature for catalysis is 10-60 ℃, and the time is 0.5-5 h;
in the step (2), the microfiltration membrane is a ceramic membrane, the filtration precision is 5-500 nm, the filtration temperature is 10-80 ℃, and the filtration pressure is 0.1-0.8 MPa;
in the step (3), the volume ratio of the 7 beta-steroid dehydrogenase to the permeation liquid containing the 7-ketolithocholic acid is 0.001-2%, the temperature for catalysis is 10-60 ℃, and the time is 0.5-5 h;
in the step (4), the microfiltration membrane is a ceramic membrane, the filtration precision is 5-500 nm, the filtration temperature is 10-80 ℃, and the filtration pressure is 0.1-0.8 MPa;
in the step (5), the ultrafiltration membrane is a roll-type ultrafiltration membrane, the cut-off molecular weight is 1000-40000 Da, the temperature is 10-60 ℃, and the pressure is 0.5-2.5 MPa;
in the step (6), the nanofiltration membrane is a roll-type ultrafiltration membrane, the molecular weight cutoff is 100-800 Da, the temperature is 10-60 ℃, and the pressure is 0.5-2.5 MPa;
in the step (7), the ion exchange resin is acrylic acid type strong-base anion exchange resin, the flow rate is 2-6 BV/h, and the temperature is 20-80 ℃.
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