CN114573655B

CN114573655B - Cholic acid derivative and preparation method thereof

Info

Publication number: CN114573655B
Application number: CN202210160175.3A
Authority: CN
Inventors: 刘双江; 刘畅; 周楠; 姜成英; 杜梦璇
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2023-04-11
Anticipated expiration: 2042-02-22
Also published as: CN114573655A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to preparation of a natural medicine/prodrug for controlling metabolic abnormalities such as blood sugar and blood fat. The invention discloses a cholic acid derivative, which has the following chemical structural formula:

wherein R is

Or

Description

Cholic acid derivatives and process for producing the same

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to preparation of a natural medicine/prodrug for controlling metabolic abnormalities such as blood sugar, blood fat and the like.

Background

In recent years, with the change of life style and the popularity of western high-fat and high-sugar diets, the prevalence rate of common metabolic chronic diseases such as hyperglycemia, hyperlipidemia and non-alcoholic fatty liver disease is rapidly increasing, and the disease is a non-negligible national health threat.

Because most metabolic diseases belong to chronic diseases and have the characteristic of long medicine taking period and the like, the current means for reducing blood sugar and blood fat which are commonly used clinically mainly comprises oral medicines, the commonly used blood sugar reducing medicines comprise sulfonylureas and biguanides oral medicines, and the main medicine for reducing blood fat is statins. Most of the structures of these drugs are artificially synthesized and not naturally occurring in the host; the long-term taking of the medicaments can cause side effects with different degrees, for example, biguanide medicaments can cause abnormal reaction of the digestive tract or lactic acidosis; prolonged statin administration can lead to hyperglycemia, cognitive impairment, and liver and muscle damage. Therefore, it has been paid attention to the long-term drug development of chronic metabolic diseases such as hyperglycemia and hyperlipidemia by searching for safe and effective natural products.

Disclosure of Invention

A first object of the present invention is to provide cholic acid derivatives having the following chemical formula:

wherein R is

The invention also provides a preparation method of the cholic acid derivative, which comprises the following steps: culturing SJ-2 strain in improved GAM liquid culture medium for 24-48 hr to obtain culture solution; inoculating the culture solution into an improved GAM liquid culture medium for culturing for 24-48 hours in an inoculation amount of 1-5% by volume, centrifuging under anaerobic conditions, and collecting the strain to obtain a collecting solution; resuspending the harvest with fermentation medium and adjusting the biomass of SJ-2 bacteria in the fermentation medium to OD _600nm About 1 to obtain a resuspension; culturing the re-suspension at 37 deg.c for 12-24 hr under anaerobic condition, centrifuging and collecting supernatant to obtain cholic acid derivative.

In a specific embodiment of the present invention, the improved GAM broth is prepared by: casein peptone 10 g, soybean peptone 3 g, tryptone 15g, digestive serum 13.5 g, yeast extract 5g, beef powder 2 g, beef liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, naCl3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46g, hemin 0.01g, resazurin 0.001g, clarified rumen fluid 10% (v/v), distilled water to 1L, pH 7.2 + -0.1, and autoclaving at 115 ℃ for 25 minutes.

In a specific embodiment of the invention, the fermentation medium is prepared by adding a mixture of 1g/L cholate solution and 10mM small-molecule acid to the modified GAM broth.

In a specific embodiment of the present invention, the preparation method of the small molecule acid mixed solution comprises: equal molar ratios of acetic acid, propionic acid, butyric acid and valeric acid were mixed and the pH was adjusted to 7.0.

The cholic acid derivative provided by the invention has better technical effects in preparing antagonists for inhibiting FXR activation, hypoglycemic drugs, hypolipidemic drugs and fatty liver treatment drugs.

Description of the drawings:

FIG. 1.SJ-2 colony morphology.

FIG. 2 is a morphological diagram of SJ-2 cells.

FIG. 3 is a LC-MS detection result chart of ACA compounds produced by SJ-2 biotransformation.

FIG. 4 is a liquid chromatography-LC-MS detection result chart of pure ACA compound.

FIG. 5 ace-CA ¹ H NMR chart.

FIG. 6.Ace-CA ¹³ C NMR chart.

FIG. 7 Pro-CA ¹ H NMR chart.

FIG. 8 Pro-CA ¹³ C NMR chart.

FIG. 9.But-CA ¹ H NMR chart.

FIG. 10.But-CA ¹³ C NMR chart.

FIG. 11.Val-CA ¹ H NMR chart.

FIG. 12.Val-CA ¹³ C NMR chart.

FIG. 13 is a sectional view of mouse liver tissue.

The specific implementation mode is as follows:

examples

1. Obtaining, culturing and identifying production strain for producing ACA compound by fermentation

Obtaining, separating and identifying a production strain:

the strain was isolated anaerobically by the patent inventors on 6.17.2019 from fresh feces provided by the feces donors after signing informed consent.

Colony morphology: the culture medium is cultured in improved GAM broth under anaerobic condition at 37 deg.C for 2-3 days, and the colony diameter is 1-2mm, white, smooth, and free of water soluble pigment (FIG. 1). Under transmission electron microscope, the cells were observed to be short rods or spindle-shaped, about 1.2-1.6 microns long and 0.6-0.8 microns wide (FIG. 2).

The modified GAM broth contains per liter: 10 g of casein peptone, 3 g of soybean peptone, 15g of tryptone, 13.5 g of digested serum, 5g of yeast extract, 2 g of beef powder, 1.2 g of beef liver extract, 3 g of glucose, 0.3 g of soluble starch, 0.5 g of L-cysteine hydrochloride, 0.5 g of L-arginine, 0.3 g of L-tryptophan, 2 g of sodium bicarbonate, 2.5 g of potassium dihydrogen phosphate, 3 g of NaCl, 0.15 g of sodium thioglycolate, 2.46g of sodium acetate, 0.01g of hemin, 0.001g of Resazurin, 10% (v/v) of clarified rumen fluid, 1L of distilled water, pH 7.2 +/-0.1, and sterilization at 115 ℃ for 25 minutes and autoclaving. An additional 15g of agar was added to the solid medium.

The molecular identification method comprises the following steps:

the full sequence analysis of 16S rRNA, the identification and classification are named as Christensella minuta, the corresponding Chinese is named as Klisteinella minutissima, the strain is named as SJ-2, the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2021, 4 and 2 days, and the preservation number is: CGMCC No.22122, the preservation unit address is: xilu No.1 Hospital No. 3, beijing, chaoyang, north. Namely the patent strain deposit number is: CGMCC No.22122; the 16S rRAN sequence homology marked with the Klebsiella parvum is 99 percent, and the sequence is shown as SEQ ID NO.1 in the sequence table.

The culture and preservation method of the strain comprises the following steps:

klisteinella minutissima SJ-2 was activated 1 time in modified GAM broth supplemented with 10% clarified rumen fluid. Inoculating into improved GAM liquid culture medium with the same components at a volume ratio of 0.1% -10%, and anaerobically culturing at 37 deg.C for 1-7 days.

The long-term preservation method of the bacteria comprises the following steps:

centrifuging the bacterial liquid at 4 ℃,10000rpm for 2 minutes to collect precipitated cells, washing the cells by PBS buffer solution, resuspending the cells in 5:1 containing 15% -25% skimmed milk powder solution, freeze-drying the cells, and then storing the cells in a sealed manner, or freezing the cells in a preservation solution containing 15% glycerol and 85% serum by liquid nitrogen and then storing the cells at-80 ℃.

2. Method for generating ACA (acyl-amino acid A) by converting cholic acid with SJ-2 bacteria

Culturing SJ-2 strain in improved GAM liquid culture medium for 24-48 hr to obtain culture solution;

the preparation method of the improved GAM liquid culture medium (g/L) comprises the following steps: casein peptone 10 g, soybean peptone 3 g, tryptone 15g, digestive serum 13.5 g, yeast extract 5g, beef powder 2 g, beef liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, naCl3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46g, hemin 0.01g, resazurin 0.001g, clarified rumen fluid 10% (v/v), distilled water to 1L, pH 7.2 + -0.1, and autoclaving at 115 ℃ for 25 minutes. An additional 15g of agar was added to the solid medium.

After the growth of SJ-2 bacteria enters a post-logarithmic phase, inoculating the culture solution into an improved GAM liquid culture medium by an inoculation amount of 1-5% in volume ratio for culturing for 24-48 hours, and centrifuging and collecting the SJ-2 bacteria entering the post-logarithmic phase under an anaerobic condition to obtain a collection solution;

resuspending the harvest with fermentation medium and adjusting the biomass of SJ-2 bacteria in the fermentation medium to OD _600nm After about 1, a resuspension is obtained;

the fermentation culture medium is prepared by adding a cholate solution with a final concentration of 1g/L and a single small molecular acid salt or a mixed solution of the small molecular acid salt with a final concentration of 10mM into an improved GAM liquid culture medium; the single small molecular acid salt is a salt of acetic acid, propionic acid, butyric acid or valeric acid with the pH of 7.0; the mixed solution of the small molecular acid salt is a mixture of salts of acetic acid, propionic acid, butyric acid and valeric acid with equal molar ratio and pH of 7.0.

Under anaerobic condition, culturing the re-suspension at 37 deg.c for 12-24 hr, centrifuging and collecting supernatant to obtain ACA for ACA conversion detection, purification and other experiments.

LCMS (liquid Crystal display System) detection method of ACA (Aminoacetic acid) compound

Preparation of a detection sample: 1ml of fermentation supernatant is taken and filtered by a 0.22 micron filter membrane, and then is directly loaded after being centrifuged for 30 minutes by a high-speed centrifuge for 1.5 ten thousand revolutions.

The conditions of the liquid chromatography are as follows: a C8 chromatographic column with the column length of 100mm, the diameter of 2.1mm, the particle size of 1.7 mu m, the flow rate of 0.2mL/min, the column temperature of 35 ℃ and the sample injection volume of 5 mu L (considering the volume effect can be properly adjusted); the liquid chromatography mobile phase A is 10mM NH ₄ HCO ₃ Aqueous solution, B phase pure acetonitrile, using gradient elution, initially 25% B, after 0.5min, followed by a linear increase to 40% B within 12 min. Continuously raise to 90% B within 1min, maintain for 3 min, restore to 25% B in 0.5min, equilibrate for 2.5 min.

The mass spectrum detection conditions are as follows: the ESI source negative ion mode is adopted, the spraying voltage is 3kV, and the temperature of the capillary is set to be 300 ℃. The sheath gas and the auxiliary gas are both nitrogen, the flow rates are 45and 10 (orbit units), and the heating temperature of the auxiliary gas is 350 ℃. The method uses a full scan mode with resolution set to 120000 and a scan range set to m/z 73.4 to 1100.

4, calculating efficiency of generating ACA by SJ-2 strain conversion:

it is known that cholic acid added to a medium is all converted into an ACA compound of C2-C5 acyl group under the conversion by SJ-2 bacteria, and thus the total conversion efficiency of ACA is calculated as a reduction ratio of a peak area in an LCMS detection peak pattern after 24 hours of conversion catalysis of the added cholic acid at 0 time: (cholic acid peak area at time 0-cholic acid peak area at 24 hours)/cholic acid peak area at time 0 × (100%).

5. Separation and purification method of bile acid acylation derivative ACA

Adding equal volume of ethyl acetate into the fermentation broth supernatant after 24 hours of conversion, performing ultrasonic extraction for 3 times with the ultrasonic time of 3 multiplied by 0.5 hours, collecting ethyl acetate extract by a separating funnel, and concentrating by a vacuum rotary evaporator to obtain a crude extract. The crude extract was dissolved in 10mL of methanol and separated using a C18 preparative column.

The liquid phase conditions are as follows: c18 preparing a column, wherein the length of the column is 250mm, the diameter of the column is 20mm, the flow rate is 10ml/min, and the sample injection volume is 100 mu L; liquid chromatography mobile phase A is 0.01% aqueous solution of trifluoroacetic acid, phase B is pure methanol, the equal degree of A is eluted by 70% B +30%, the corresponding component of the target peak appearing under A205nm is taken, the separated component is detected by LCMS method, and then is weighed for standby after being dried by nitrogen.

6. Results of the experiment

LCMS detection result and SJ-2 conversion rate of acylcholic acids with different carbon chain lengths

SJ-2 cells were transformed into cholic acid supernatants at 0 and 24 hours, and each sample was assayed by LCMS. As shown in FIG. 3, the results of the samples at time 0 are shown in FIGS. 3a and b (CA + SA-0 h), and the results after 24 hours of transformation are shown in FIGS. 3c-h (CA + SA-24 h). The result shows that only characteristic peak of cholic acid (figure 3 b) with mass-to-charge ratio (m/z) of 407.3 is detected at 0 moment, peak area after 407.3m/z characteristic peak is extracted is 93413948.47, 4 new peaks can be obviously seen after 24-hour conversion, m/z of mass spectrogram is 449.3,463.3,477.3 and 491.3 respectively, corresponding to acetylcholic acid, propionylcholic acid, butyrylcholic acid and valerylcholic acid, peak area after peak extraction is 18071866.68,20406632.73,22769156.54 and 8969173.10 respectively, peak area of cholic acid (407.3) after reaction is 45837961.92, and conversion rate of cholic acid into ACA series compounds by SJ-2 bacteria is 50.9% by calculation.

Isolation and purification of microbially converted ACA products

The separation and purification experiment of ACA conversion product by liquid chromatography using C18 semi-preparative column, LCMS detection result of collected characteristic peak component after methanol is evaporated to dryness and redissolved is shown in FIG. 4, and four ACA compounds can be effectively separated and purified by liquid chromatography. Four pure unknown cholic acid derivatives with molecular weights of 450.3, 464.3, 478.3 and 492.3 of the isolated compounds respectively are detected by nuclear magnetic resonance as follows: the method comprises the following steps of one-dimensional 1H spectrum experiment, one-dimensional 13C spectrum experiment, two-dimensional 1H-1H correlation spectrum (adjacent hydrocarbon coupling relation), two-dimensional 1H-13C correlation spectrum (directly-bonded hydrocarbon coupling relation), two-dimensional NOESY correlation spectrum (distance of proton in a molecular three-dimensional space structure), and two-dimensional 1H-13C remote correlation experiment (remote hydrocarbon relation, definitely primary, secondary and tertiary carbon).

TABLE 1 ¹³ C NMR Data(δC)for Compounds 1-4in CDCl3

TABLE 2 ¹ H NMR Data[δH,mult(J in Hz)]for Compounds 1-4in CDCl3

Finally determining the chemical structure, wherein R groups are acetyl, propionyl, butyryl and valeryl respectively.

The structural formula of the compound is as follows:

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wherein R is

7. The use and effect of the compound and the preparation thereof are as follows:

luciferase reporter gene detection cell assay:

farnesoid X Receptor (FXR) is a bile acid receptor, plays an important role in bile acid metabolism and cholesterol metabolism, and is expected to become a therapeutic target for reducing cholesterol and treating certain cardiovascular diseases and liver diseases.

24 hours after transfection of the luciferase reporter system of FXR, cells were exposed to treatment groups of 20mmol/L of FXR agonist chenodeoxycholic acid CDCA (positive control), or 20mmol/L of CDCA and 50mmol/L of acetylcholic acid (Ace-CA), propionocholic acid (Pro-CA), butyrocholic acid (But-CA) and valerocholic acid (Val-CA), respectively; cells were not treated as a negative control.

Luciferase assays were performed using a commercial dual luciferase assay system (Promega).

Firefly and Renilla luciferase activities were measured using a Veritas microplate luminometer (Turner Biosystems).

To quantify the dose response inhibition of FXR by different ACA compounds, the fluorescence was measured after 8 hours of exposure of the cells to Ace-CA, pro-CA, but-CA and Val-CA concentrations ranging from 5mmol/L to 200mmol/L (upper solubility limit) and the half inhibitory concentrations IC of the compounds were calculated using GraphPad Prism 6 in the "log (inhibitor) and normalized response-variable slope" modes ₅₀ 。

Luciferase reporter gene detection cell experiment results show that the ACA series compounds can inhibit FXR activation induced by CDCA and play a role of antagonist.

The inhibitory effect of ACA compounds on FXR is measured in doses, with Ace-CA, pro-CA, but-CA and Val-CA, and their IC 50's of 35.52, 29.61,17.87 and 11.18. Mu.M, respectively.

TABLE 3 relative fluorescence intensity values of the FXR inhibition effect of the four acylcholic acids on the Bifluorescein fluorescence System

TABLE 4 relative fluorescence intensity values for the FXR inhibition effect of different concentrations of acetylcholinergic acid on the bifluoride fluorescent System

TABLE 5 relative fluorescence intensity values of different concentrations of butyrylcholic acid for the FXR inhibition effect of the bifluorescent System

TABLE 6 relative fluorescence intensity values of different concentrations of propionocholic acid for FXR inhibition effect of bifluorescent systems

TABLE 7 relative fluorescence intensity values for FXR inhibition of the Bifluorescein Fluorescence System with different concentrations of valerylcholic acid

Animal experiment design:

experiments were performed using high fat diet induced obese C57 mice (DIO mice), male, 15 weeks old. The frequency of intragastric administration is once a day, and the ACA group (3 ACA groups) intragastric administration is 50mg/kg body weight of Ace-CA, pro-CA, but-CA and Val-CA pure normal saline solution every day; the preparation group (3) comprises 20mg/kg body weight ACA mixture (mixed by ethyl, propyl, butyl, valeryl cholic acid, etc.) in improved GAM culture medium as effective component for intragastric administration every day; the control group (3) was filled with the same amount of physiological saline daily. The total experimental period was 8 weeks.

The improved GAM culture medium is (g/L): casein peptone 10 g, soybean peptone 3 g, tryptone 15g, digestive serum 13.5 g, yeast extract 5g, beef powder 2 g, beef liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, naCl3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46g, hemin 0.01g, resazurin 0.001g, clarified rumen fluid 10% (v/v), distilled water to 1L, pH 7.2 + -0.1, sterilized at 115 ℃ for 25 minutes and used after autoclaving)

End point sampling: a. plasma samples (supernatant collected by anticoagulation centrifugation), parenchymal organs such as B and liver (liver photographing and weighing, fixing paraformaldehyde on the left and right of a middle leaf, then performing paraffin embedding and frozen section, performing red oil staining and HE staining after section, and accurately weighing 100mg of liquid nitrogen quick-frozen 3 tubes for other index detection, C, intestinal tract and content in the rest liver.

Detection indexes of the tissue sample are as follows:

a) Blood glucose related index: free blood glucose, fasting blood glucose, glucose tolerance (OGTT), insulin resistance (ITT) tests of venous blood;

b) Blood lipid related index: total cholesterol and total triglyceride content in plasma;

c) Liver function is related: h & E staining and oil red staining, and expression levels of cholesterol 7 alpha-hydroxylase (Cyp 7a 1) and gluconeogenesis rate-limiting enzyme glucose-6-phosphatase (G6 pase), which are bile acid synthesis rate-limiting enzymes, in liver tissues.

The related indexes of blood sugar are detected by a glucometer, and other physiological and biochemical indexes except blood sugar are detected by a commercial ELISA kit after protein crude extraction is carried out on blood or homogenized liver tissues.

And (3) statistical test:

whether the data of each treatment group is statistically different from the data of the control group is tested for significance by a two-sided new double-pole difference method (Dunnett-t test), and the difference is considered significant when the P value is less than 0.05 by using the control group as a control class.

The experimental results are as follows:

ACA compounds and related preparations reduce free blood glucose in model mice:

the data results in table 8 show that ACA compounds and related preparations have significant effects of reducing free blood glucose and alleviating symptoms of hyperglycemia.

TABLE 8 free blood glucose concentration (mmol/L) of venous blood 8 weeks after model mouse intervention

ACA compounds and related formulations reduce fasting blood glucose in model mice:

the data results in table 9 show that the ACA compounds and related preparations have significant effects of reducing fasting blood glucose in model mice and relieving hyperglycemia symptoms.

TABLE 9 fasting blood glucose concentration (mmol/L) after 8 weeks of model mouse intervention

ACA compounds and related preparations decrease glucose tolerance in model mice:

the results in table 10 show that the ACA compound and the related preparations can significantly increase the glucose tolerance of the model mice and alleviate the impaired glucose tolerance.

TABLE 10 area under the curve of glucose tolerance after 7 weeks of model mouse intervention (AUC)

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ACA compounds and related formulations reduce insulin resistance in model mice:

the results in table 11 show that ACA compounds and related formulations can significantly reduce insulin resistance symptoms in model mice.

TABLE 11 area under the curve of insulin resistance (AUC) after 8 weeks of model mouse intervention

The ACA compound and related preparations reduce the total triglyceride content in blood of model mice:

the results in table 12 show that ACA compounds and related preparations can significantly reduce the total triglyceride content in blood of model mice and alleviate the symptoms of hyperlipidemia.

TABLE 12 endpoint blood Total triglyceride concentration (mmol/L) after 8 weeks of model mouse intervention

The ACA compound and the related preparation reduce the total cholesterol content in blood of the model mice:

the results in table 13 show that ACA compounds and related preparations can significantly reduce the total cholesterol level in the blood of model mice and alleviate the symptoms of high cholesterol.

TABLE 13 endpoint blood Total Cholesterol concentration (mmol/L) 8 weeks after model mouse intervention

The ACA compound and related preparations reduce pathological indexes such as liver fat accumulation and liver cell injury:

as can be seen from FIG. 13, the fat accumulation of the liver of the mice treated with ACA and the preparation is obviously reduced, fat vacuoles are rarely seen in the tissue section, and the vesicular fat is easy to basically disappear. After the model mouse is cured, the hepatic lobular structural disorder and the clear boundary are corrected, the hepatic cell reticular structure is reduced or basically disappears, the shape is normal, and the nucleus is round and centered. The above phenotype indicates that the symptoms of nonalcoholic fatty liver and liver injury in disease model mice are greatly alleviated.

ACA compound and related preparation for up-regulating Cyp7a1 expression level and down-regulating G6pase expression level in liver tissue

By detecting the expression levels (table 15) of cholesterol 7 alpha-hydroxylase (Cyp 7a 1) (table 14) and gluconeogenesis rate-limiting enzyme glucose-6-phosphatase (G6 pase) which are bile acid synthesis rate-limiting enzymes in liver tissues of mice of two treatment groups, the results show that the ACA compound and related preparations can accelerate the conversion of cholesterol into bile acid to be discharged into intestinal tracts by up-regulating the expression of Cyp7a1 in the liver, thereby playing the role of reducing cholesterol; meanwhile, the G6pase is regulated down, so that gluconeogenesis in the liver is inhibited, the rise of blood sugar is slowed down, and the effect of regulating the blood sugar is exerted.

TABLE 14 Cyp7a1 protein content (pg/mg) in liver milling samples 8 weeks after model mouse intervention

TABLE 15G 6pase protein content (pg/mg) in liver milling samples 8 weeks after model mouse intervention

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Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> cholic acid derivatives and process for producing the same

<141> 2022-02-22

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1391

<212> DNA

<213> Microcressessensella (Christensella minuta)

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agaaagtcgc cttcgccact ggtgttcctc ctaatatcta cgcatttcac cgctacacta 780

ggaattccac ttccctctcc tgtactcaag tcacacagtt tcaaatgcaa ccccggggtt 840

aagccccggt ctttcacatc tgacttacat gaccacctac gcacccttta cgcccagtaa 900

ttccggacaa cgcttgctcc ctacgtatta ccgcggctgc tggcacgtag ttagccggag 960

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actgctgcct cccgtaggag tctggaccgt gtctcagttc cagtgtggcc gatcaccctc 1140

tcaggtcggc tacccatcgt tgacttggtg ggccgttacc tcaccaacta tctaatggga 1200

cgcgagccca tcctgcatcg aataaatcct tttacctcaa aaccatgcgg tttcgtggtc 1260

tcatgcggta ttagcagtcg tttccaactg ttgtcccccg ttgcagggca ggttgctcac 1320

gcgttactca cccgtccgcc actcggtata cccacagttc ctcccgaagg attcacaaag 1380

ggcaacctcg t 1391

Claims

1. A process for the preparation of a cholic acid derivative having the following chemical formula:

；

wherein R is

、/>

、 />

Or>

；

The method is characterized by comprising the following steps:

inoculating the culture solution into an improved GAM liquid culture medium for culturing for 24-48 hours in an inoculation amount of 1-5% by volume, centrifuging under anaerobic conditions, and collecting the strain to obtain a collecting solution;

resuspending the collected liquid with fermentation medium and adjusting the biomass of SJ-2 strain in the fermentation medium to OD _600nm Obtaining resuspension after 1;

under anaerobic condition, culturing the re-suspension at 37 deg.c for 12-24 hr, centrifuging and collecting supernatant to obtain cholic acid derivative;

the preparation method of the improved GAM liquid culture medium comprises the following steps: casein peptone 10 g, soybean peptone 3 g, tryptone 15g, digested serum 13.5 g, yeast extract 5g, beef powder 2 g, beef liver extract 1.2 g, glucose 3 g, soluble starch 0.3 g, L-cysteine hydrochloride 0.5 g, L-arginine 0.5 g, L-tryptophan 0.3 g, sodium bicarbonate 2 g, potassium dihydrogen phosphate 2.5 g, naCl3 g, sodium thioglycolate 0.15 g, sodium acetate 2.46g, hemin 0.01g, resazurin 0.001g, clarified rumen fluid volume ratio 10%, distilled water to 1L, pH 7.2 + -0.1, autoclaving at 115 ℃ for 25 minutes;

the preparation method of the fermentation medium culture comprises the steps of adding a cholate solution with the final concentration of 1g/L and a single small molecular acid salt or a mixed solution of the small molecular acid salt with the final concentration of 10mM into an improved GAM liquid culture medium;

the single small molecular acid salt is as follows: a salt of acetic acid, propionic acid, butyric acid or valeric acid at a pH of 7.0.

2. The method for preparing a cholic acid derivative according to claim 1, wherein the small molecule acid salt mixture is: any molar ratio of salts of acetic acid, propionic acid, butyric acid and valeric acid at a pH of 7.0.