阅读说明：本技术 一种诱变菌种以及微生物发酵制备熊去氧胆酸的方法 (Mutagenic strain and method for preparing ursodeoxycholic acid by microbial fermentation ) 是由 蒋红平 唐杰 李斌 郭志军 于 2021-02-03 设计创作，主要内容包括：本发明提供了一种微生物发酵制备熊去氧胆酸的方法,将新月弯孢菌ATCC 13432的诱变菌种经培养后接种至转化培养基,对石胆酸进行发酵转化,得到熊去氧胆酸。本发明提供了一种新的以石胆酸为原料,通过生物发酵制备熊去氧胆酸的方法,该方法未使用动物源提取,绿色环保,成本低,工艺步骤简单,反应条件温和,反应收率高,试剂简单易得,适合工业化大规模生产。(The invention provides a method for preparing ursodeoxycholic acid by microbial fermentation, which comprises the steps of culturing a mutagenic strain of curvularia lunata ATCC 13432, inoculating the mutagenic strain to a transformation culture medium, and performing fermentation transformation on lithocholic acid to obtain the ursodeoxycholic acid. The invention provides a novel method for preparing ursodeoxycholic acid by taking lithocholic acid as a raw material through biological fermentation, which is free of animal source extraction, green, environment-friendly, low in cost, simple in process steps, mild in reaction conditions, high in reaction yield, simple and easily available in reagents, and suitable for industrial large-scale production.)

1. A method for preparing ursodeoxycholic acid by microbial fermentation is characterized in that a mutagenic strain of Curvularia lunata ATCC 13432 is cultured and then inoculated to a transformation medium, and lithocholic acid is fermented and transformed to obtain the ursodeoxycholic acid.

2. The method according to claim 1, wherein the mutagenic strain of Curvularia lunata ATCC 13432 is obtained by the method comprising the steps of: curvularia lunata ATCC 13432 is used as a starting strain, slant culture is carried out, spores are collected to prepare spore suspension, nitrosoguanidine is added for mutagenesis treatment, the mutagenized bacterial suspension is diluted and coated on a solid plate culture medium, a single colony is selected for lithocholic acid fermentation, the yield of ursodeoxycholic acid is detected, and a target mutagenized strain capable of producing the ursodeoxycholic acid is obtained.

3. The method of claim 1, wherein the fermentative conversion process is: inducing Curvularia lunata ATCC 13432The strain is added into a transformation medium after slant culture, primary seed culture and secondary seed culture, transformation is started after the pre-culture of thalli, the inoculation amount of the thalli is 1-10%, the temperature is 30-32 ℃, the rotating speed is 100-400 rpm, and the air flow is 0.1-0.4 Nm³The pressure is 0.01-0.1 MPa, and the conversion time is 24-72 h.

4. The method of claim 3, wherein the slant culture medium is potato dextrose agar.

5. The method of claim 3, wherein the primary seed culture and the secondary seed culture are formulated in a medium comprising: 1-20 g/L of corn steep liquor, 10-50 g/L of glucose and 6.0 of pH.

6. The method of claim 3, wherein the transformation medium is formulated as: 1-10 g/L of corn steep liquor, 10-30 g/L of glucose, 1-10 g/L of disodium hydrogen phosphate, and adjusting the pH value to 7.0; the transformation culture method is that after the thalli are pre-cultured, 2g/L lithocholic acid is added to start transformation.

7. The method of claim 1, further comprising, after said fermentative conversion: post-treatment;

the post-treatment comprises the following steps:

heating to 80 deg.C for inactivation, cooling to room temperature, adjusting pH to 12 with liquid alkali, vacuum filtering, and mixing filtrates; adjusting the pH value of the filtrate to 3.0, extracting with dichloromethane, standing and layering; filtering the lower dichloromethane layer, removing solvent, adding water, stirring for crystallization, and separating to obtain solid, i.e. ursodeoxycholic acid.

Technical Field

The invention relates to the technical field of chemical pharmacy, in particular to a method for preparing ursodeoxycholic acid by mutation strains and microbial fermentation.

Background

Ursodeoxycholic Acid (UDCA) is a main effective component of traditional precious traditional Chinese medicine bear gall, has a chemical name of 3a,7 beta-dihydroxy-5 beta-cholestane-24-Acid, has a strong effect of promoting fat and fatty Acid hydrolysis, is used for increasing bile Acid secretion in medicine, changing bile components, reducing cholesterol and cholesterol ester in bile, and facilitating gradual dissolution of cholesterol in gallstones, and is mainly used for clinically treating various liver and gall diseases and digestive tract diseases, such as cholelithiasis, cholestatic liver disease, fatty liver, various types of hepatitis, toxic liver disorder, cholecystitis, cholangitis and bile dyspepsia, bile reflux gastritis, eye diseases and the like.

At present, the preparation method of ursodeoxycholic acid generally comprises the following steps that firstly, the ursodeoxycholic acid is obtained by extracting live bear bile, and the method is gradually eliminated due to limited sources or the limitation of animal protection methods; secondly, animal cholic acids such as hyocholic acid, taurocholic acid, chenodeoxycholic acid and the like are used as raw materials and are prepared by a synthesis method, and the methods have the problems of limited raw material sources, long reaction steps, low yield and the like; thirdly, a synthetic method using androstenedione as a raw material, wherein the synthesis method uses androstenedione as a raw material and adopts the Helvetica, Chimica acta, 1984.67(2)612 to report that the androstenedione raw material is used for preparing ursodeoxycholic acid through nine-step reaction, although the method belongs to a method for preparing a raw material of non-animal cholic acid, the price is high, and the difficulty of introducing a side chain in the synthesis is high; fourthly, the preparation is carried out by an enzyme method, and Chinese patent CN109154016A reports that lithocholic acid is taken as a substrate, and oxidative or reductive coenzyme, alcohol dehydrogenase and hydroxylase are added for reaction to prepare ursodeoxycholic acid. Coenzyme is needed in the reaction, the coenzyme is expensive and high in cost, and in addition, the hydroxylase of the technology is separated from the thallus, the price is high, the technology is only suitable for experimental stage research, and the industrial feasibility is not large.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing ursodeoxycholic acid by fermentation of a mutagenic strain and a microorganism, which has a high reaction yield.

In order to achieve the purpose, the invention provides a method for preparing ursodeoxycholic acid by microbial fermentation, which comprises the steps of culturing a mutagenic strain of curvularia lunata ATCC 13432, inoculating the mutagenic strain to a transformation culture medium, and performing fermentation transformation on lithocholic acid to obtain the ursodeoxycholic acid.

The equation for the above reaction is as follows:

according to the invention, lithocholic acid is taken as a substrate, beta-configuration hydroxyl is introduced to the 7 th position of lithocholic acid by a mutagenic strain biological fermentation method of Curvularia lunata ATCC 13432, and ursodeoxycholic acid with high yield and high purity is obtained after simple post-treatment.

Preferably, the mutagenesis method of the mutagenic strain of curvularia lunata ATCC 13432 comprises the following steps: curvularia lunata ATCC 13432 is used as a starting strain, slant culture is carried out, spores are collected to prepare spore suspension, nitrosoguanidine is added for mutagenesis treatment, the mutagenized bacterial suspension is diluted and coated on a solid plate culture medium, a single colony is selected for lithocholic acid fermentation, the yield of ursodeoxycholic acid is detected, and a target mutagenized strain capable of producing the ursodeoxycholic acid is obtained.

The above Curvularia lunata ATCC 13432 is derived from the American type culture Collection.

Preferably, the fermentation conversion method comprises the following steps: the method comprises the steps of performing slant culture, primary seed culture and secondary seed culture on a mutagenic strain of curvularia lunata ATCC 13432, adding the mutagenic strain into a transformation medium, performing thallus pre-culture, and then beginning transformation, wherein the inoculum size of the thallus is 1-10%, the temperature is 30-32 ℃, the rotating speed is 100-400 rpm, and the air flow is 0.1-0.4 Nm³The pressure is 0.01-0.1 MPa, and the conversion time is 24-72 h.

In a preferred embodiment of the present invention, the culture medium for slant culture is potato dextrose agar.

In a preferred embodiment of the present invention, the culture medium formula for the first-stage seed culture and the second-stage seed culture is: 1-20 g/L of corn steep liquor, 10-50 g/L of glucose and 6.0 of pH.

According to the invention, the formula of the transformation medium is preferably as follows: 1-10 g/L of corn steep liquor, 10-30 g/L of glucose, 1-10 g/L of disodium hydrogen phosphate, and adjusting the pH value to 7.0; the transformation culture method is that after the thalli are pre-cultured, 2g/L lithocholic acid is added to start transformation.

Preferably, after the fermentation conversion, the method further comprises the following steps: post-treatment;

the post-treatment comprises the following steps:

heating to 80 deg.C for inactivation, cooling to room temperature, adjusting pH to 12 with liquid alkali, vacuum filtering, and mixing filtrates; adjusting the pH value of the filtrate to 3.0, extracting with dichloromethane, standing and layering; filtering the lower dichloromethane layer, removing solvent, adding water, stirring for crystallization, and separating to obtain solid, i.e. ursodeoxycholic acid.

Compared with the prior art, the invention provides a method for preparing ursodeoxycholic acid by microbial fermentation, which comprises the steps of culturing a mutagenic strain of curvularia lunata ATCC 13432, inoculating the mutagenic strain to a conversion culture medium, and performing fermentation conversion on lithocholic acid to obtain the ursodeoxycholic acid. The invention provides a novel method for preparing ursodeoxycholic acid by taking lithocholic acid as a raw material through biological fermentation, which is free of animal source extraction, green, environment-friendly, low in cost, simple in process steps, mild in reaction conditions, high in reaction yield, simple and easily available in reagents, and suitable for industrial large-scale production.

Drawings

FIG. 1 is a nuclear magnetic carbon spectrum of ursodeoxycholic acid prepared in the present invention;

FIG. 2 is a nuclear magnetic hydrogen spectrum of ursodeoxycholic acid prepared by the present invention.

Detailed Description

In order to further illustrate the present invention, the following examples are provided to describe the mutagenized bacteria and the method for preparing ursodeoxycholic acid by microbial fermentation.

The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

The raw materials and other chemical reagents adopted in the embodiment of the invention are all commercial products.

EXAMPLE 1 preparation of ursodeoxycholic acid

1. Bacterial strain

Original starting strains: curvularia lunata ATCC 13432.

2. And (3) mutation breeding of thalli:

transferring Curvularia lunata ATCC 13432 strain spores to a PDA slant culture medium, culturing for 5-7 days at 28 ℃, adding 0.02% Tween 80 aqueous solution to wash down the spores, transferring the spore suspension into a shake flask with glass beads, putting the shake flask into a shaker, shaking at 220rpm for 30 minutes, scattering the spores, filtering, and separating the spores from residual hypha tissues. Diluting spore suspension with sterile water to adjust spore concentration to 10⁸And (5) germinating the Erlenmeyer flask in a shaking table at 220rpm for 5-8 hours. 966. mu.l of the germinated spore suspension were taken into an EP tube, and 34. mu.l of 3% Nitrosoguanidine (NTG) solution was added to the spore suspension and mixed well.

NTG treatment time is respectively 10min,20min,30min,40min,50min and 60min, after treatment, centrifugation is carried out for 8 min at 5000rpm, precipitates are washed for three times by using 0.85% sterile normal saline, finally, the volume is fixed to 1 ml by using the sterile normal saline, the shaking is uniform, 100 microliters of spore suspension is taken for gradient dilution and coated on a flat plate, the coated flat plate is placed at 28 ℃ for culture for 7-10 days, the growth condition of bacterial colonies is observed, and the number of the bacterial colonies is recorded to calculate the lethality rate. After the incubation, the lethality rates at the above different times were 31.5%, 45.2%, 53.1%, 68.3%, 81.6%, and 96.2%, respectively, thus indicating that 50 minutes of treatment was the optimum time.

The colonies obtained by mutagenesis and the original starting strain were selected and cultured according to the following 3 "transformation procedure" and subjected to biotransformation, and the effect of the mutagenized strain was evaluated. After a large amount of mutagenesis and screening tests, a strain is obtained, the feeding amount in a shake flask is 0.2%, and the conversion rate is 79.2%.

3 transformation Process

3.1 slant culture

The formula of the culture medium is as follows: and (3) sterilizing the PDA at 121 ℃ for 30 minutes, cooling and forming, inoculating under an aseptic condition, and culturing at 29 ℃ for 5-7 days.

3.2 first order seed culture

The culture medium formula comprises 10g/L of corn steep liquor, 30g/L of glucose and 6.0,100 ml of culture medium with pH value, is filled into a 500 ml shake flask, is sterilized at 121 ℃ for 30 minutes, and is cooled to room temperature. Scraping a certain amount of colony tissue from the cultured inclined plane under the aseptic condition, inoculating the colony tissue into a liquid culture medium, and starting to culture under the culture condition of 30 ℃, 180rpm and 24-48 hours.

3.3 formula of culture medium for secondary seed: corn steep liquor 10g/L, glucose 30g/L, pH 6.0,350 ml culture medium in 2000 ml shake flask, 121 ℃ sterilization for 30 minutes, cooling to room temperature. Under aseptic conditions, 35 ml of seeds are sucked from the cultured primary seeds, and the culture is started, wherein the culture conditions are as follows: 30 ℃ at 180rpm for 24 hours.

3.4 transformation

(1) The formula of the culture medium is as follows: 10g/L of corn steep liquor, 30g/L of glucose, 3g/L of disodium hydrogen phosphate and pH 7.0, sterilizing at 121 ℃ for 30 minutes, and cooling to room temperature.

(2) Pre-culturing thalli: 10L fermentation tank, 7L fermentation liquor volume, 121 deg.C sterilization for 30 minutes, cooling to room temperature. Inoculating with 5% of inoculum size, and culturing for 16 hr.

(3) And (3) transformation: under the protection of a fire circle, 14 g of the lithocholic acid which is pretreated is added into the cultured bacterial liquid, and the transformation is started. Conversion parameters: air flow rate 0.2m³The rotation speed is 200rpm, and the tank pressure is 0.05 MPa. The conversion time is 24-72 hours, and the conversion is finished when the lithocholic acid residue is less than 5%.

The pretreatment method of the substrate lithocholic acid comprises the following steps: 14 g lithocholic acid, 200 ml sterile water, heating to 80 ℃ and inactivating for 1 hour.

3.5 extraction

After the conversion is finished, heating to 80 ℃, stirring for 30 minutes for inactivation, cooling to 30 ℃, adjusting the pH to 12 by using liquid alkali, performing suction filtration, and washing a filter cake by using a small amount of water. Mixing the filtrates, adjusting the pH to 3.0, adding dichloromethane with the volume of 0.5 times of that of the water phase, stirring and extracting at normal temperature for 1 hour, standing for 8-10 hours, and layering. Filtering the lower dichloromethane layer, concentrating the dried solvent under reduced pressure, adding a small amount of water, stirring, washing and filtering the product, performing solid-liquid separation to obtain a yellow solid, drying at 70 ℃, and performing liquid phase detection to obtain a product with about 85.75% of UDCA normalization content and about 4.19% of LCA normalization content.

Weighing the obtained crude product, adding 5V toluene, heating to 80 ℃, heating and stirring for 1 hour, cooling to 40 ℃, growing the crystals for 1 hour, performing suction filtration to obtain 9.5 g of light yellow refined product, and performing liquid phase detection to obtain about 95 percent of UDCA normalized content.

The carbon spectrum and hydrogen spectrum of ursodeoxycholic acid are shown in FIGS. 1 and 2.

13C NMR(101MHz,DMSO)δ175.36,70.20,69.92,56.32,55.15,43.52,42.65,38.20,37.74,35.31,34.23,31.24,30.72,28.64,27.18,23.78,21.33,18.78.

1H NMR(400MHz,DMSO)δ11.92(s,1H),4.43(d,J＝4.4Hz,1H),3.86(d,J＝6.8Hz,1H),2.62–2.45(m,1H),2.23(ddd,J＝15.2,9.7,5.3Hz,1H),2.09(ddd,J＝12.4,9.2,6.8Hz,1H),1.96(t,J＝17.7Hz,1H),1.84(ddd,J＝19.4,13.6,8.2Hz,1H),1.80–1.59(m,4H),1.55–1.39(m,4H),1.41–1.26(m,7H),1.27–0.93(m,8H),0.88(d,J＝6.7Hz,6H),0.62(s,3H).

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.