CN112094883A

CN112094883A - Method for preparing beta-hyodeoxycholic acid by microbial transformation

Info

Publication number: CN112094883A
Application number: CN202011039603.4A
Authority: CN
Inventors: 邓家国; 魏红霞; 李国军
Original assignee: CHANGDE YUNGANG BIOTECHNOLOGY CO LTD
Current assignee: CHANGDE YUNGANG BIOTECHNOLOGY CO LTD
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2020-12-18

Abstract

The invention discloses a method for preparing beta-hyodeoxycholic acid by microbial transformation, which converts hydroxyl of hyodeoxycholic acid at 3-position from alpha position to beta position by microbial transformation. The method comprises the following steps: preparing spores and seeds; carrying out microbial transformation on alpha-hyodeoxycholic acid; and (5) separating and purifying. The method is simple to operate, takes the alpha-hyodeoxycholic acid with rich resources and low price as a raw material, obtains the beta-hyodeoxycholic acid through biotransformation, is favorable for realizing the industrial production of the beta-hyodeoxycholic acid, and promotes the development and utilization of the beta-hyodeoxycholic acid.

Description

Method for preparing beta-hyodeoxycholic acid by microbial transformation

Technical Field

The invention belongs to the technical field of microbial transformation, and particularly relates to a method for preparing beta-hyodeoxycholic acid by microbial transformation, which is characterized in that hydroxyl at the 3-position of hyodeoxycholic acid is transformed from alpha position to beta position by using microbes.

Background

The alpha-hyodeoxycholic acid (3 alpha, 6 alpha-dihydroxycholanic acid, HDCA) is prepared by extracting and processing pig bile, and has the effects of reducing blood fat, eliminating phlegm and relieving cough. Alpha-hyodeoxycholic acid has certain inhibitory effect on Bordetella pertussis, Corynebacterium diphtheriae, Staphylococcus aureus, etc., and can be used as antiinflammatory agent for treating chronic bronchitis, infantile viral upper respiratory inflammation, etc. At present, the extraction and preparation process of alpha-hyodeoxycholic acid is mature, large-scale processing production is realized industrially, and the alpha-hyodeoxycholic acid can be used as an important raw material of an artificial bezoar formula and other traditional Chinese medicine preparation components such as a medicine qingkailing and the like based on the characteristics of the alpha-hyodeoxycholic acid.

3 beta, 6 alpha-dihydroxycholanic acid (beta-HDCA) is an isomer of alpha-hyodeoxycholic acid, only exists in trace amount in natural pig bile, and the existing preparation of beta-hyodeoxycholic acid is mainly synthesized by a chemical method, has complex process, great environmental pollution and low yield, and cannot realize large-scale production. Just because the production condition is limited, the application of the beta-hyodeoxycholic acid is also limited, and the beta-hyodeoxycholic acid is only used as a standard substance and an impurity reference substance at present and sold in a small amount on the market, and cannot meet the requirements of certain special industries.

Biotransformation is a physiological and biochemical reaction for carrying out specific structural modification on a specific part or functional group of an exogenous organic substrate by utilizing a biological system (comprising a culture system of plants, microorganisms or animal tissues) or a related enzyme preparation of the biological system so as to obtain a valuable product. The enzyme method has incomparable advantages compared with the chemical method, such as mild conditions, high chemical, regional and stereoselectivity, simple operation, environmental protection and the like, so that how to apply the biotransformation technology to the preparation of the beta-hyodeoxycholic acid is a problem which needs to be solved at present in the industry.

Disclosure of Invention

The invention aims to provide a method for preparing beta-hyodeoxycholic acid by microbial transformation, which aims to solve the problems that the existing beta-hyodeoxycholic acid preparation is mainly synthesized by a chemical method, the process is complex, the environmental pollution is large, the yield is low, and the large-scale production cannot be realized.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for preparing beta-hyodeoxycholic acid by microbial transformation is characterized by comprising the following specific preparation processes:

(1) preparation of spores and seeds

The mycelium of the Bacillus sphaericus AKU218 is picked by using an inoculating loop and streaked on a PDA plate, and the streaked mycelium is cultured in a constant-temperature incubator at the temperature of 28 ℃ for 3 to 5 days to obtain a Bacillus sphaericus AKU218 plate. The bacillus sphaericus AKU218 belongs to the existing strains, is not the protection scope of the invention, and the strain information is not described in detail.

(2) Microbial transformation of alpha-hyodeoxycholic acid

A. Under the aseptic condition, selecting a ring of mycelium of the bacillus sphaericus AKU218 prepared in the step (1) by using a sterilized inoculating ring, inoculating the mycelium into a sterilized bacterial culture medium, carrying out constant-temperature shaking culture for 48-72 hours at the temperature of 28 ℃ and the rpm of 120, centrifuging for 10 minutes at the rpm of 3000, and separating and recovering the thallus;

B. and B, dispersing the thalli obtained in the step A into an inorganic salt culture medium, adding alpha-hyodeoxycholic acid and beta-oxidation inhibitor 2, 2' -dipyridyl which are dissolved in advance into the inorganic salt culture medium, putting the inorganic salt culture medium into a closed container with a deoxidizer, and culturing for 36-72 hours at 28 ℃ under the anaerobic condition of 120 rpm.

(3) Separation and purification of the conversion product

And C, adding equal volume of ethyl acetate into the inorganic salt culture medium cultured in the step B for extraction and separation, decoloring and purifying the separated product to obtain white powdery beta-hyodeoxycholic acid, wherein the conversion rate of the alpha-hyodeoxycholic acid into the beta-hyodeoxycholic acid is 80-90%.

Further, the bacterial culture medium in the step A comprises the following components: 5g/L of tryptone, 5g/L of yeast extract, 1g/L of sugar and 1g/L of dipotassium hydrogen phosphate.

Further, the inorganic salt culture medium in the step B comprises the following components: 1g/L of dipotassium phosphate dibasic, 1g/L of monopotassium phosphate monobasic, 1g/L of ammonium sulfate, 0.2g/L of magnesium sulfate heptahydrate, 0.1g/L of sodium chloride, 0.02g/L of calcium chloride dihydrate, 0.01g/L of ferric sulfate heptahydrate and 0.25g/L of yeast extract.

Further, the alpha-hyodeoxycholic acid and the beta-oxidation inhibitor 2,2 '-Bipyridy in the step B are dissolved in ethanol in advance, wherein the concentration of the alpha-hyodeoxycholic acid is 0.1% -1.0%, and the concentration of the beta-oxidation inhibitor 2, 2' -Bipyridy is 0.01%.

Compared with the prior art, the invention has the beneficial effects that:

the invention firstly proposes that the alpha hydroxyl at the 3 rd position of hyodeoxycholic acid is converted into the beta hydroxyl by using a microbial conversion method, the beta-hyodeoxycholic acid is obtained by biotransformation by using the alpha-hyodeoxycholic acid with rich resources and low price as a raw material, the preparation process is simple, convenient and low in cost, the industrial production of the beta-hyodeoxycholic acid is favorably realized, the development and utilization of the beta-hyodeoxycholic acid are promoted, and the microbial conversion method has great social value and economic value.

The ethyl acetate extract was mixed with a 0.1% fatty acid-containing fluorescent reagent ADAM methanol solution for analysis, and then methanol was added thereto, followed by standing at room temperature for 60 minutes to form a fluorescent label. After the fluorescent labeling, the sample is analyzed by HPLC, LC-MS and GC-MS, and is analyzed by the same method as the newly purchased standard sample beta-hyodeoxycholic acid, and the result proves that the alpha-hyodeoxycholic acid can be converted into the beta-hyodeoxycholic acid after being fermented by the bacillus sphaericus.

Drawings

FIG. 1 is a diagram of HDCA transformation reaction of Bacillus sphaericus AKU 218;

FIG. 2 is HPLC of a market standard with the transformed product of Bacillus sphaericus AKU218

Comparing the graphs;

FIG. 3 is a LC-MS analysis of β -HDCA;

FIG. 4 is a GC-MS analysis chart of HDCA, β -HDCA standards and the conversion products;

FIG. 5 is a comparison of MS spectra of the standard and the transformed product.

In fig. 1: line 1, standards (from left to right CA, UDCA, HDCA, DCA, C,

CDCA, LCA); line 2, anaerobic reaction products; line 3, aerobic reaction product.

In fig. 2: line 4, standards (UDCA, HDCA, CDCA, LCA from left to right); line 5, beta-hyodeoxycholic acid standard sample; line 6, bacillus sphaericus AKU218 reaction product.

In fig. 3: line 7, TCI (-); line 8, m/z 617; line 9, m/z 619.

In fig. 4: line 10, HDCA standard; line 11, bacillus sphaericus transformation product; line 12, 3 β -HDCA standard.

In fig. 5: the upper line is a 3 beta-HDCA standard product; the lower line is the transformed product of Bacillus sphaericus.

Detailed Description

The technical scheme of the invention is clearly and completely described below by combining the embodiment,

it is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A method for preparing beta-hyodesoxycholic acid by microbial transformation comprises the following specific preparation processes:

(1) preparation of spores and seeds

Taking 200g of peeled potatoes, adding 200ml of deionized water, boiling, and maintaining for 25 min; filtering with sterilized double-layer gauze, discarding residue, and retaining filtrate; adding 20g of glucose and 20g of agar into the filtrate, heating the asbestos on a small fire, continuously stirring the mixture by using a glass rod until the mixture is completely dissolved, and adding deionized water to a constant volume of 1000 ml; subpackaging the prepared culture medium, pouring the prepared culture medium into 500mL conical flasks, plugging the bottle openings with tampons, wrapping a layer of kraft paper outside the tampons, and marking the name, the preparation date and the preparation person of the culture medium; sterilizing at 115 deg.C for 30min in a sterilizing pan, cooling to about 50 deg.C, aseptically pouring into flat plate (pouring 15ml per dish), standing at room temperature, and coagulating to obtain PDA flat plate; the mycelium of the Bacillus sphaericus AKU218 is picked by using an inoculating loop and streaked on a PDA plate, and the streaked mycelium is cultured for 3 days in a constant temperature incubator at 28 ℃ to obtain a Bacillus sphaericus AKU218 plate.

(2) The microbial transformation steps of the alpha-hyodeoxycholic acid are as follows:

pouring 4ml of sterilized bacterial culture medium into a glass test tube, wherein the formula of the bacterial culture medium is shown in table 1, burning and cooling an inoculating loop by using an alcohol lamp, selecting a loop of the mycelium of the Bacillus sphaericus AKU218 prepared in the step (1), inoculating the mycelium into the sterilized bacterial culture medium, performing shake culture at 28 ℃ and 120 rpm for 24 hours, pouring the mycelium into a 200ml conical flask containing the bacterial culture medium, and after culturing for 24 hours, centrifuging at 3000 rpm for 10 minutes to separate and recover the thallus;

the obtained thalli is dispersed in 200ml of inorganic salt culture medium, the formula of the inorganic salt culture medium is shown in table 2, 5ml of alpha-hyodeoxycholic acid ethanol solution with the concentration of 20 percent and 2ul of beta-oxidation inhibitor 2, 2' -Bipyridyl are added into the culture medium, wherein the final concentration of the alpha-hyodeoxycholic acid ethanol solution is 0.5 percent, the mixture is placed into a closed container with a deoxidizer, and the mixture is cultured for 36 hours at the temperature of 120 rpm under the anaerobic condition at the temperature of 28 ℃.

(3) Separation and extraction of the conversion product:

and (3) taking the filtered fermentation culture solution obtained in the step (2), and extracting the filtrate for 3 times by using ethyl acetate with the same volume. The organic phases are combined and concentrated to dryness under reduced pressure to give 0.67g of conversion residue. Dissolving 30mg sample in 2ml methanol, diluting the mobile phase to 20ml, detecting by high performance liquid chromatography, and detecting the reference substance by the same method. The calculated conversion was 81.5%.

The specific steps for the structural analysis of the conversion product of step (3) in example 1 are as follows:

another 2.5ul of the ethyl acetate extract was mixed with 50ul of a 0.1% aliphatic acid analysis-use fluorescent reagent ADAM methanol solution, and then 47.5ul of methanol was added thereto, followed by standing at room temperature for 60 minutes to form a fluorescent label. After the fluorescent labeling, the sample is analyzed by HPLC, the analysis conditions of the HPLC are shown in Table 3, and the newly purchased standard sample beta-hyodeoxycholic acid is analyzed by the same method. The results are shown in attached figures 1-5, and the results prove that the alpha-hyodeoxycholic acid is converted into the beta-hyodeoxycholic acid after being fermented by the bacillus sphaericus.

TABLE 1 bacterial culture Medium

Composition (I)	Concentration (g/L)
		Tryptone	5.0
Yeast cream	5.0
		Glucose	1.0
Dipotassium hydrogen phosphate	1.0

TABLE 2 inorganic salt culture Medium

Composition (I)	Concentration (g/L)
		Yeast cream	0.25
KH₂PO₄	1.0
		K₂HPO₄	1.0
(NH₄)₂SO₄	1.0
		MgSO₄·7H₂O	0.20
NaCl	0.10
		CaCl·2H₂O	0.02
FeSO₄·7H₂O	0.01

TABLE 3 HPLC analysis conditions

Item	Condition
		Chromatography column	COSMOSIL 5C18- AR-1I (φ4.6x 150 mm)
Mobile phase	Acetonitrile: 0.1% phosphoric acid solution =90: 10
		Flow rate of flow	1 mL/min
Temperature of the column	40°C
		Analysis time
	25 min
		Detection wavelength	Excitation wavelength 365 nm fluorescence wavelength 412 nm
Sample loading amount	1 μL

Example 2

(1) preparation of spores and seeds

Taking 200g of peeled potatoes, adding 200ml of deionized water, boiling, and maintaining for 25 min; filtering with sterilized double-layer gauze, discarding residue, and retaining filtrate; adding 20g of glucose and 20g of agar into the filtrate, heating the asbestos on a small fire, continuously stirring the mixture by using a glass rod until the mixture is completely dissolved, and adding deionized water to a constant volume of 1000 ml; subpackaging the prepared culture medium, pouring the prepared culture medium into 500mL conical flasks, plugging the bottle openings with tampons, wrapping a layer of kraft paper outside the tampons, and marking the name, the preparation date and the preparation person of the culture medium; sterilizing at 115 deg.C for 30min in a sterilizing pan, cooling to about 50 deg.C, aseptically pouring into flat plate (pouring 15ml per dish), standing at room temperature, and coagulating to obtain PDA flat plate; the mycelium of the Bacillus sphaericus AKU218 is picked by using an inoculating loop and streaked on a PDA plate, and the plate is cultured for 3 days in a constant temperature incubator at 28 ℃ to obtain the Bacillus sphaericus AKU218 plate.

pouring 4ml of sterilized bacterial culture medium into a glass test tube, wherein the formula of the bacterial culture medium is shown in table 1, adjusting the pH to 6.8, burning the cooled inoculating loop by using an alcohol lamp, selecting the mycelium of the Bacillus sphaericus AKU218 prepared in the step (1), inoculating the mycelium into the sterilized bacterial culture medium, performing shake culture at 28 ℃ and 120 rpm for 24 hours, pouring the mycelium into a conical flask containing 500ml of the bacterial culture medium, and after culturing for 48 hours, centrifuging at 3000 rpm for 10 minutes to separate and recover the thallus;

the obtained thalli is dispersed in 500ml of inorganic salt culture medium, the formula of the inorganic salt culture medium is shown in table 2, the PH is adjusted to 6.8, 15ml of alpha-hyodeoxycholic acid ethanol solution with the concentration of 20 percent and 5ul of beta-oxidation inhibitor 2, 2' -Bipyridyl are added into the culture medium, wherein the final concentration of the alpha-hyodeoxycholic acid ethanol solution is 0.6 percent, the mixture is placed into a closed container with a deoxidizer, and the culture is carried out for 72 hours at the temperature of 28 ℃ and the rpm of 120 under the anaerobic condition.

(3) Separation and extraction of the conversion product:

and (3) taking the filtered fermentation culture solution obtained in the step (2), and extracting the filtrate for 5 times by using ethyl acetate with the same volume. The organic phases were combined and concentrated to dryness under reduced pressure to give 2.58 g of conversion residue. Dissolving 30mg sample in 2ml methanol, diluting the mobile phase to 20ml, detecting by high performance liquid chromatography, and detecting the reference substance by the same method. The calculated conversion was 85.24%.

The procedure for the structural analysis of the conversion product in step (3) in example 2 was the same as in example 1, and the structural analysis of the conversion product was not carried out again.

Example 3

(1) preparation of spores and seeds:

(2) The specific steps of the alpha-hyodeoxycholic acid microbial transformation are as follows:

pouring 8ml of sterilized bacterial culture medium into a glass test tube, wherein the formula of the bacterial culture medium is shown in table 1, adjusting the pH to 7.0, burning the cooled inoculating loop by using an alcohol lamp, selecting the mycelium of the bacillus sphaericus AKU218 prepared in the two-loop step (1), inoculating the mycelium into the sterilized bacterial culture medium, performing shake culture at 28 ℃ and 120 rpm for 24 hours, pouring the mycelium into a conical flask containing 500ml of the bacterial culture medium, and after culturing for 48 hours, centrifuging at 3000 rpm for 10 minutes to separate and recover the thallus;

the obtained thalli is dispersed in 500ml of inorganic salt culture medium, the formula of the culture medium is shown in table 2, the PH is adjusted to be 7.0, 20ml of 20 percent alpha-hyodeoxycholic acid ethanol solution and 8ul of beta-oxidation inhibitor 2, 2' -Bipyridyl are added into the culture medium, wherein the final concentration of the alpha-hyodeoxycholic acid ethanol solution is 0.8 percent, the mixture is placed into a closed container with a deoxidizer, and the mixture is cultured for 72 hours at the temperature of 28 ℃ and the rpm of 120 under the anaerobic condition.

(3) Separation and extraction of the conversion product:

and (3) taking the filtered fermentation culture solution obtained in the step (2), and extracting the filtrate for 4 times by using ethyl acetate with the same volume. The organic phases were combined and concentrated to dryness under reduced pressure to give 3.65 g of conversion residue. Dissolving 30mg sample in 2ml methanol, diluting the mobile phase to 20ml, detecting by high performance liquid chromatography, and detecting the reference substance by the same method. The calculated conversion was 89.27%.

The procedure for the structural analysis of the conversion product in step (3) in example 3 was the same as in example 1, and the structural analysis of the conversion product was not carried out again.

Claims

1. A method for preparing beta-hyodeoxycholic acid by microbial transformation is characterized by comprising the following specific preparation processes:

(1) preparation of spores and seeds

Selecting mycelium of the bacillus sphaericus AKU218 by using an inoculating loop, scribing on a PDA (personal digital assistant) plate, and culturing in a constant-temperature incubator to obtain a bacillus sphaericus AKU218 plate;

(2) microbial transformation of alpha-hyodeoxycholic acid

Selecting a ring of mycelia of the Bacillus sphaericus AKU218 prepared in the step (1) from the Bacillus sphaericus AKU218 flat plate in the step (1), inoculating the mycelia in a sterilized bacterial culture medium, carrying out shake culture at constant temperature for 48-72 hours, and carrying out centrifugal separation to recover thalli; then inoculating the recovered thalli into an inorganic salt culture medium containing alpha-hyodeoxycholic acid and beta-oxidation inhibitor 2, 2' -Bipyridyl for fermentation;

(3) separation and purification of the conversion product

Adding equal volume of ethyl acetate into the inorganic salt culture medium cultured in the step (2) for extraction and separation, wherein the separated product is decolorized and purified to form white powdery beta-hyodeoxycholic acid, and the conversion rate of converting alpha-hyodeoxycholic acid into beta-hyodeoxycholic acid is 80-90%.

2. The method for preparing beta-hyodeoxycholic acid by microbial transformation according to claim 1, wherein the temperature in the constant temperature incubator in step (1) is 28 ℃ and the incubation time is 3-5 days.

3. The method for preparing beta-hyodeoxycholic acid by microbial transformation according to claim 1, wherein the microbial transformation of alpha-hyodeoxycholic acid in step (2) comprises the following steps:

A. selecting a ring of mycelia of the Bacillus sphaericus AKU218 prepared in the step (1) from the Bacillus sphaericus AKU218 flat plate in the step (1), inoculating the mycelia in a sterilized bacterial culture medium, performing constant-temperature shaking culture at 28 ℃ and 120 rpm for 48-72 hours, centrifuging at 3000 rpm for 10 minutes, and separating and recovering the mycelia;

4. The method for preparing beta-hyodeoxycholic acid by microbial transformation according to claim 3, wherein the bacterial culture medium in step A comprises the following components: 5g/L of tryptone, 5g/L of yeast extract, 1g/L of sugar and 1g/L of dipotassium hydrogen phosphate.

5. The method for preparing beta-hyodeoxycholic acid by microbial transformation according to claim 3 or 4, wherein the inorganic salt medium in step B comprises the following components: 1g/L of dipotassium phosphate dibasic, 1g/L of monopotassium phosphate monobasic, 1g/L of ammonium sulfate, 0.2g/L of magnesium sulfate heptahydrate, 0.1g/L of sodium chloride, 0.02g/L of calcium chloride dihydrate, 0.01g/L of ferric sulfate heptahydrate and 0.25g/L of yeast extract.

6. The method for preparing beta-hyodeoxycholic acid by microbial transformation according to claim 5, wherein the alpha-hyodeoxycholic acid and the beta-oxidation inhibitor 2,2 '-Bipyridy in step B are dissolved in ethanol in advance, wherein the concentration of the alpha-hyodeoxycholic acid is 0.1% -1.0%, and the concentration of the beta-oxidation inhibitor 2, 2' -Bipyridy is 0.01%.