阅读说明：本技术 一种利用废弃肠道内容物进行生物转化获得熊去氧胆酸的方法 (Method for obtaining ursodeoxycholic acid by biotransformation of waste intestinal contents ) 是由 吴庆喜 刘彩霞 王栋 吴志 于 2021-07-28 设计创作，主要内容包括：本发明公开了一种利用废弃肠道内容物进行生物转化获得熊去氧胆酸的方法,是以鹅去氧胆酸为底物,利用废弃的动物肠道内容物进行生物转化,从而获得熊去氧胆酸。本发明利用废弃肠道内容物成功催化转化得到了熊去氧胆酸,使废弃肠道内容物得到有效利用,为熊去氧胆酸的生物转化提供了一条新的思路。(The invention discloses a method for obtaining ursodeoxycholic acid by utilizing waste intestinal contents to carry out biotransformation, which takes chenodeoxycholic acid as a substrate and utilizes the waste animal intestinal contents to carry out biotransformation, thereby obtaining the ursodeoxycholic acid. The invention utilizes the waste intestinal contents to successfully catalyze and convert to obtain the ursodeoxycholic acid, effectively utilizes the waste intestinal contents, and provides a new idea for biotransformation of the ursodeoxycholic acid.)

1. A method for obtaining ursodeoxycholic acid by carrying out biotransformation on waste intestinal contents is characterized by comprising the following steps: and (3) performing biotransformation by using the waste animal intestinal contents by using chenodeoxycholic acid CDCA as a substrate to obtain ursodeoxycholic acid UDCA.

2. The method of claim 1, comprising the steps of:

(1) collecting the waste animal intestinal contents at a low temperature of 0-4 ℃;

(2) dissolving CDCA in phosphate buffer;

(3) placing the intestinal contents collected in the step (1) in an Eppendorf tube with a cover, adding a proper amount of phosphate buffer solution dissolved with CDCA, and fully and uniformly mixing to obtain a biotransformation system;

(4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 160-;

(5) and after the conversion is finished, centrifuging the biotransformation system, taking supernatant, and then filtering to obtain a crude product water solution containing the UDCA.

3. The method according to claim 1 or 2, characterized in that: the animal intestinal tract content is the content in rectum, cecum or colon of chicken, duck, goose, mouse or rat.

4. The method of claim 2, wherein: in the step (2), the pH value of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.1 mol/L.

5. The method of claim 2, wherein: in the step (2), the concentration of CDCA dissolved in phosphate buffer solution is 0.5-2.0 mg/mL.

6. The method of claim 2, wherein: in the step (3), the volume ratio of the intestinal contents to the phosphate buffer solution dissolved with the CDCA is 1: 2-6.

7. The method of claim 2, wherein: in the step (5), the rotation speed of the centrifugation is 8000-12000rpm, and the time is 20-30 min.

8. The method of claim 2, wherein: in the step (5), the filtration is performed by using a 0.45 μm microporous membrane.

Technical Field

The invention relates to a method for biotransformation and reutilization of waste intestinal contents, and belongs to the field of biocatalytic conversion.

Background

In recent years, with the improvement of living standard of people and the progress of science and technology, livestock and poultry breeding in China develops rapidly, and livestock and poultry can generate a large amount of waste intestinal contents in the slaughtering process, thereby bringing about serious environmental load. In addition, according to statistics, the number of mice and rats used for scientific experiments in China is as large as hundreds of millions, and waste after slaughtering causes a series of environmental pollution problems. In order to maximize resource utilization and practice a green sustainable development concept, it is important to find a technology for recycling waste intestinal contents.

Bile acid plays an important role in the metabolic cycle of organisms, primary bile acid is synthesized by cholesterol in the liver, and a small part of the primary bile acid enters the intestinal tract and is converted by intestinal microbial flora to form various secondary bile acids. Wherein, ursodeoxycholic acid (UDCA) is a secondary bile acid, is the main component in bear gall, has the functions of reducing blood pressure, benefiting gall bladder, dissolving gallstone and the like, is clinically applied to treating diseases in the aspect of liver and gall, has good curative effect and has strong market demand. However, the bear is a national primary protection animal, and the bear can not kill the bear to obtain the UDCA, so that the search for a new UDCA source is significant.

In order to solve the problem of limited source of UDCA, cholic acid can be converted into UDCA through a certain way, the conventional method for obtaining UDCA mainly comprises a chemical conversion method and a biological conversion method, and the chemical method has serious pollution and large environmental load in the conversion process, so that a novel biological conversion way is developed, and the method accords with the green development concept. The biological conversion to obtain UDCA is mainly catalytic conversion by using enzyme produced by microorganism. Among them, the oxyhydrogen-eration of primary bile acids is a common way of converting to secondary bile acids by intestinal microorganisms. In the prior art, pure strains are mostly used for converting Cholic Acid (CA) and chenodeoxycholic acid (CDCA) into UDCA in vitro. However, this method requires isolation, identification, and the like of the bacterial species, and is complicated.

Disclosure of Invention

Aiming at the problems existing in the current situation, the invention provides a method for obtaining ursodeoxycholic acid by carrying out biotransformation on waste intestinal contents, and aims to simplify the technological process for obtaining UDCA by the existing biotransformation method.

The invention adopts the following technical scheme for realizing the purpose:

a method for obtaining ursodeoxycholic acid by carrying out biotransformation on waste intestinal contents is characterized by comprising the following steps: and (3) performing biotransformation by using the waste animal intestinal contents by using chenodeoxycholic acid CDCA as a substrate to obtain ursodeoxycholic acid UDCA. The method specifically comprises the following steps:

(1) collecting the waste animal intestinal contents at a low temperature of 0-4 ℃;

(2) dissolving CDCA in phosphate buffer;

(3) placing the intestinal contents collected in the step (1) in an Eppendorf tube with a cover, adding a proper amount of phosphate buffer solution dissolved with CDCA, and fully and uniformly mixing to obtain a biotransformation system;

(4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 160-;

(5) and after the conversion is finished, centrifuging the biotransformation system, taking supernatant, and then filtering to obtain a crude product water solution containing the UDCA.

Further, the intestinal contents of the animals are the contents in the rectum, caecum or colon of chickens, ducks, geese, mice or rats.

Further, in the step (2), the pH value of the phosphate buffer solution is 8.0-10.0, and the concentration is 0.1 mol/L.

Further, in step (2), CDCA is dissolved in phosphate buffer solution at a concentration of 0.5-2.0 mg/mL.

Further, in the step (3), the volume ratio of the intestinal content to the phosphate buffer solution dissolved with the CDCA is 1: 2-6.

Further, in step (5), the rotation speed of the centrifugation is 8000-12000rpm, and the time is 20-30 min.

Further, in the step (5), the filtration is performed with a 0.45 μm microporous membrane.

The invention has the beneficial effects that:

1. the method utilizes the waste intestinal contents, generates an intermediate product 7-ketolithocholic acid by biotransformation by using microbial flora in the intestinal contents and 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase generated by the microbial flora and using CDCA as a substrate, and further converts the intermediate product into a final product UDCA, wherein the yield of the final product UDCA can reach 19.3% by high performance liquid chromatography determination.

2. The invention effectively utilizes the waste animal intestinal contents, has low cost, meets the requirement of green development, obtains the UDCA with high added value, and provides a new way for the bioconversion of the UDCA.

3. The conversion method of the invention has simple operation and mild reaction conditions.

Drawings

FIG. 1 is a high performance liquid chromatogram of three standards of chenodeoxycholic acid, 7-ketolithocholic acid and ursodeoxycholic acid, which shows that: the peak time of the ursodeoxycholic acid standard product is 12.8min, the peak time of the 7-ketolithocholic acid standard product is 18.0min, and the peak time of the chenodeoxycholic acid standard product is 29.1 min.

FIG. 2 is a high performance liquid chromatogram of the product of biotransformation with waste intestinal contents in example 4, from which it can be seen that the transformation product shows peaks at retention times of 12.5min and 18.9min, and it can be determined that the transformation product is ursodeoxycholic acid and the intermediate product 7-ketolithocholic acid.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The following disclosure is merely exemplary and illustrative of the inventive concept, and those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Example 1

In this example, the method for generating UDCA by converting CDCA with the content of the discarded chicken rectum includes the following specific steps:

(1) collecting the slaughtered and discarded chicken rectum contents at the low temperature of 0 ℃.

(2) Phosphate buffer solution with a pH of 8.0 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 0.8 mg/mL).

(3) And (2) placing 10mL of the content of the chicken rectum collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 20mL of phosphate buffer solution dissolved with CDCA, and fully mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 170r/min, the temperature is 28 ℃, and the conversion time is 72 h.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 10000rpm, the time is 30min), taking the supernatant, and then filtering with a 0.45 mu m microporous filter membrane to obtain a crude product water solution containing UDCA.

The concentration of the crude aqueous product was determined by High Performance Liquid Chromatography (HPLC): the mobile phase is as follows: acetonitrile/0.05% aqueous phosphate (V/V) ═ 50/50, detection wavelength 192nm, flow rate 0.5-1.0 mL/min.

The yield of the final product UDCA was calculated to be 13.8% and the conversion of CDCA was calculated to be 83.4%.

Example 2

In this embodiment, the method for converting CDCA into UDCA by using waste duck colon contents comprises the following specific steps:

(1) collecting the waste duck colon contents after slaughtering at the low temperature of 0 ℃.

(2) Phosphate buffer solution with a pH of 8.5 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 1 mg/mL).

(3) And (2) placing 6mL of contents of the duck colon collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 30mL of phosphate buffer solution dissolved with CDCA, and fully mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 200r/min, the temperature is 30 ℃, and the conversion time is 72 hours.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 10000rpm, the time is 30min), taking the supernatant, and then filtering with a 0.45 mu m microporous filter membrane to obtain a crude product water solution containing UDCA.

The final product, UDCA, was obtained in this example in 14.7% yield and CDCA conversion of 89.5% by HPLC analysis in the same manner as in example 1.

Example 3

In this embodiment, the waste goose cecum content is converted into CDCA to generate UDCA, and the specific steps are as follows:

(1) collecting the cecal content of the killed and abandoned goose at the low temperature of 0 ℃.

(2) Phosphate buffer solution with a pH of 9.0 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 1.5 mg/mL).

(3) And (3) placing 5mL of the goose cecum content collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 20mL of phosphate buffer solution dissolved with CDCA, and fully and uniformly mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 180r/min, the temperature is 35 ℃, and the conversion time is 96 h.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 12000rpm, the time is 20min), taking the supernatant, and then filtering with a 0.45 mu m microporous filter membrane to obtain a crude product water solution containing UDCA.

The final product, UDCA, was obtained in this example in 16.2% yield and CDCA conversion of 90.8% by HPLC analysis in the same manner as in example 1.

Example 4

In this example, the cecal content of the discarded mice is transformed into CDCA to generate UDCA, and the specific steps are as follows:

(1) the cecal contents of the mice discarded after slaughter were collected at a low temperature of 4 ℃.

(2) Phosphate buffer solution with a pH of 8.0 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 1.0 mg/mL).

(3) And (3) placing 5mL of the mouse cecal content collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 25mL of phosphate buffer solution dissolved with CDCA, and fully mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 240r/min, the temperature is 35 ℃, and the conversion time is 180 hours.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 9000rpm, and the time is 30min), taking supernatant, and then filtering with a 0.45-micron microporous filter membrane to obtain a crude product water solution containing UDCA.

According to the same procedure as in example 1, the final product UDCA yield of this example was 18.5% and CDCA conversion was 93.6%.

Example 5

In this example, the contents of the waste mouse colon are used to transform CDCA to UDCA, and the specific steps are as follows:

(1) the contents of the colons of the slaughtered mice were collected at a low temperature of 0 ℃.

(2) Phosphate buffer solution with a pH of 10.0 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 1.7 mg/mL).

(3) Placing 8mL of the colon contents of the mice collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 30mL of phosphate buffer solution dissolved with CDCA, and fully mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 160r/min, the temperature is 28 ℃, and the conversion time is 96 h.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 10000rpm, the time is 30min), taking the supernatant, and then filtering with a 0.45 mu m microporous filter membrane to obtain a crude product water solution containing UDCA.

The final product of this example, UDCA, was obtained in 14.1% yield and CDCA conversion was 82.8% by HPLC as in example 1.

Example 6

In this example, the cecal content of the discarded rat is converted into CDCA to generate UDCA, and the specific steps are as follows:

(1) the cecal contents of the sacrificed rats were collected at a low temperature of 0 ℃.

(2) Phosphate buffer solution with a pH of 10.0 and a concentration of 0.1mol/L was prepared, and CDCA was dissolved in the phosphate buffer solution (final concentration of 1.5 mg/mL).

(3) And (3) placing 10mL of the rat cecal content collected in the step (1) into a 50mL Eppendorf tube with a cover, adding 30mL of phosphate buffer solution dissolved with CDCA, and fully mixing to obtain a biotransformation system.

(4) And (4) putting the biological conversion system obtained in the step (3) into a shaking table for biological conversion, wherein the rotation speed of the shaking table is 180r/min, the temperature is 35 ℃, and the conversion time is 120 h.

(5) After the conversion is finished, centrifuging the biotransformation system (the centrifugal speed is 12000rpm, the time is 20min), taking the supernatant, and then filtering with a 0.45 mu m microporous filter membrane to obtain a crude product water solution containing UDCA.

According to the same procedure as in example 1, the yield of UDCA and the conversion of CDCA in this example were 19.3% and 95.5%, respectively.

The present invention is not limited to the above exemplary embodiments, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.