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Process for preparing artificial bear gall powder through large-scale fermentation culture of engineered strain

A technology of fermentation culture and bear bile powder, applied in the biological field, can solve the problems of high cost, cumbersome TUDCA steps, large environmental pollution and the like

Active Publication Date: 2018-09-28
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of TUDCA by chemical synthesis has not been widely used due to cumbersome steps, high cost and high environmental pollution.

Method used

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  • Process for preparing artificial bear gall powder through large-scale fermentation culture of engineered strain
  • Process for preparing artificial bear gall powder through large-scale fermentation culture of engineered strain
  • Process for preparing artificial bear gall powder through large-scale fermentation culture of engineered strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] 1 The influence of secondary seed medium on growth curve

[0075] 1) Coat the engineered bacteria stored in the refrigerator at -80°C into the LB solid plate medium containing 100mg / L ampicillin by streaking, and activate the culture at 37°C for 12 hours;

[0076] The composition of the LB solid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, and agarose 15.0g / L.

[0077] 2) Pick a single colony from the LB solid plate medium, inoculate it in the LB liquid medium supplemented with 100mg / L ampicillin, and cultivate it with shaking at 225rpm and 37°C for 9 hours to obtain first-class seeds;

[0078] The composition of the LB liquid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L;

[0079] 3) The above-mentioned cultures were respectively inoculated into 2-YT liquid medium and the same LB liquid medium as described in 2) at a ratio of 1:20, cultured with shaking at 225 rpm / min and 37°C, and measured both Growth curve;...

Embodiment 2

[0085] Optimization experiment of M9-GY optimized medium

[0086] 1) Using the Box-Behnken Design design method in Design-Expert.V8.0.6.1 software, based on the composition of M9 basic medium, design a four-factor test with glucose, glycerol, yeast extract and tryptone, Glucose, yeast extract, and tryptone are selected at the three levels of 0, 10, and 20g / L, and glycerol at the three levels of 0, 15, and 30ml / L. With OD600 and conversion rate as the inspection indicators, 29 sets of programs and The results are as follows:

[0087]

[0088]

[0089] 2) The specific fermentation process is as follows: the first-level seeds are obtained according to the operation in Example 1, the first-level seeds are inoculated into liquid 2-YT medium at a ratio of 1:20, and cultured at 225 rpm and 37°C for 4 hours to obtain two Grade seeds; inoculate the secondary seeds in the above-mentioned various liquid media at a ratio of 1:20. The liquid media are all based on the M9 basic medium formula a...

Embodiment 3

[0093] Optimization of the induction culture phase

[0094] 1 The influence of IPTG concentration on conversion rate

[0095] 1) Obtain first-level seeds according to the operation in Example 1, inoculate the first-level seeds into liquid 2-YT medium at a ratio of 1:20, and shake culture at 225rpm and 37°C for 4 hours to obtain second-level seeds;

[0096] 2) Inoculate the secondary seeds into the M9-GY optimized medium liquid medium at a ratio of 1:20, in triplicate, and shake culture at 225rpm and 37℃ for 5 hours;

[0097] 3) Add IPTG to the final concentration of 0.1, 0.3, 0.6, 0.8, 1.0 mM, and shake culture at 225 rpm and 30°C for 3 hours; add refined chicken gall powder as a substrate at a final concentration of 5 g / L, at 225 rpm, Incubate with shaking for 6 hours at 25℃ for fermentation; figure 1 a shows:

[0098] 2 Combination optimization of induction conditions

[0099] Select the early logarithmic phase (4 hours), mid-logarithmic phase (5.5 hours), and late logarithmic phase ...

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Abstract

The invention provides a process for preparing artificial bear gall powder through large-scale fermentation culture of an engineering strain BL21<*>-alpha 1 beta 2, belonging to the field of biotechnology. The preparation process is simple and controllable, the prepared finished product is qualified in quality, the raw materials are cheap and easily available, and the process belongs to the breakthrough for preparing the artificial bear gall powder through the biotechnology means.

Description

Technical field [0001] The invention relates to a process for preparing artificial bear bile powder by large-scale fermentation culture of recombinant engineering bacteria. Specifically, it is a process of using recombinant engineering bacteria to carry out large-scale fermentation culture in a fermentor and purify and prepare artificial bear bile powder, and perform quality evaluation on the artificial bear bile powder produced by the process, which belongs to the field of biotechnology. Background technique [0002] Bear bile powder is a powder product of the gallbladder of the bear family animal black bear or brown bear. It has the effects of clearing heat, calming liver, improving eyesight, promoting choleretics, antispasmodic, and dissolving stones. Clinically, it is often used in children with fever, convulsion, epilepsy, convulsions, and jaundice; external use to treat carbuncle, hemorrhoids, red eyes and clouding. Bear bile powder mainly contains bile acid, and its main ...

Claims

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Application Information

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IPC IPC(8): C12P33/00C12R1/19
CPCC12P33/00
Inventor 赵淑娟王峥涛许盈芃杨莉周吉燕胡之璧张金家
Owner SHANGHAI UNIV OF T C M
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