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Method for producing glycyrrhetinic acid monoglucuronide through intermittent feed supplement fermentation

A technology of glucuronic acid-based and glycyrrhetinic acid, which is applied in the field of intermittent fed-batch fermentation to produce mono-glucuronic acid-based glycyrrhetinic acid, which can solve the problems of limiting the large-scale application of GAMG, unfavorable industrial production, and high extraction costs, and achieve product orientation Good performance, improved substrate utilization, and low yield of by-product GA

Active Publication Date: 2014-04-09
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The β-glucuronidases that have been found to convert glycyrrhizic acid into GAMG exist in intestinal bacteria, fungi and animal tissues, etc., but these enzymes generally have the disadvantage of low selectivity and produce a large amount of by-product glycyrrhetinic acid (GA ); In addition, the enzymes derived from animal tissues, the extraction cost is too high, which is not conducive to industrial production
[0006] At present, the methods of biotransforming glycyrrhizic acid into GAMG generally have the disadvantages of poor orientation, low yield and high cost, which limit the large-scale application of GAMG.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Inoculate the Penicillium purpurea strain into the slant of the culture medium, culture at a constant temperature of 25°C for 9 days, add 0.75% normal saline to wash the mycelium, and obtain a spore content of 4.1×10 6 spore suspension. The composition of the slant medium is: glucose 6g, NaNO 3 3g, K 2 HPO 4 1g, KCl 0.5g, MgSO 4 ·7H 2 O 0.5g, FeSO 4 ·7H 2 O 0.01g, distilled water 1000mL, agar 20g, adjust the pH to 5.0, sterilize at 118°C for 15min, and cool to room temperature.

[0026] Inoculate the spore suspension of Penicillium purpurea into the seed medium with an inoculum of 5% by volume, cultivate at 30°C at 170r / min for 84h, then transfer it to the secondary seed medium with an inoculum of 1%, at 30°C , 170r / min cultured for 24h, to obtain secondary seed liquid. The composition of the seed medium is: glucose 6g, NaNO 3 3g, K 2 HPO 4 1g, KCl 0.5g, MgSO 4 ·7H 2 O 0.5g, FeSO 4 ·7H 2 O 0.01g, distilled water 1000mL, adjust the pH to 5.0, sterilize...

Embodiment 2

[0031] Inoculate the Penicillium purpurea strain into the slant of the culture medium, culture at 32°C for 7 days, add 0.75% saline to wash the mycelium, and obtain a spore content of 5.8×10 6 spore suspension. The composition of the slant medium is: 200 grams of potatoes, 20 grams of glucose, 18 grams of agar, 1000 mL of distilled water, natural pH, sterilized at 118 ° C for 15 minutes, and cooled to room temperature.

[0032]Inoculate the spore suspension of Penicillium purpurea into the seed medium with an inoculum of 5% by volume, cultivate at 32°C at 170r / min for 72h, then transfer it to the secondary seed medium with an inoculum of 1%, at 32°C , 170r / min cultured for 24h, to obtain secondary seed liquid. The composition of seed culture medium is with embodiment 1.

[0033] Put the secondary seed liquid into the 2.5L fermenter according to the inoculum volume ratio of 10%. It is 4.5~5.0, and the fermentation time is 54h. The composition of the fermentation medium is t...

Embodiment 3

[0037] Inoculate the Penicillium purpurea strain on the slant of the culture medium, culture at 30°C for 7 days, add 0.75% saline to wash the mycelium, and obtain a spore content of 6.4×10 6 spore suspension. The composition of the slant medium is the same as in Example 1.

[0038] Inoculate the spore suspension of Penicillium purpurea into the seed medium with an inoculum of 5% by volume, cultivate at 30°C at 170r / min for 72h, then transfer it to the secondary seed medium with a 3% inoculum, at 30°C , 170r / min cultured for 24h, to obtain secondary seed liquid. The composition of seed culture medium is with embodiment 1.

[0039] Put the secondary seed liquid into the 2.5L fermenter according to the inoculum volume ratio of 10%. It is 4.5~5.0, and the fermentation time is 48h. The composition of the fermentation medium is the same as in Example 1.

[0040] After 48 hours of fermentation, add the aseptic substrate glycyrrhizic acid at a final concentration of 6g / L every 18...

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Abstract

The invention relates to a method for producing glycyrrhetinic acid monoglucuronide (GAMG) through intermittent feed supplement fermentation, belonging to the technical field of biological transformation. The method specifically comprises the steps of: firstly, cultivating and inducing microorganisms capable of inducing to generate beta-glucuronidase in a seed culture medium and a fermentation culture medium containing glycyrrhizic acid to obtain corresponding beta-glucuronidase; and secondly, specifically splitting a terminal glucosidic bond in a glycyrrhizic acid molecular structure by using the beta-glucuronidase generated by the microorganisms through induction, generating the GAMG, and secreting the GAMG to a fermentation solution and intermittently replenishing glycyrrhizic acid and inorganic salts as a substrate for multiple times. Through replenishing the glycyrrhizic acid and the inorganic salts as the substrate, nutrient substances are replenished, thallus can grow in a proper environment and promote the synthesis of a target product; and after the fermentation is ended, the yield of the GAMG reaches 13-24g / L. According to the method disclosed by the invention, the fermentation yield of the GAMG can be effectively increased, the transformation period is prolonged, the equipment utilization rate is increased, and the product production cost is reduced.

Description

technical field [0001] The invention relates to a method for producing monoglucuronyl glycyrrhetinic acid (GAMG) by intermittent fed-batch fermentation, belonging to the technical field of biotransformation. Background technique [0002] In recent years, due to the sharp reduction of resources, the output of traditional Chinese medicinal materials has decreased year by year, and the supply is tight; while the active ingredients of natural medicinal materials are often low in content, complex in structure, and difficult to synthesize. In real life, many drugs are transformed by various enzymes in the stomach and intestines to exert their medicinal effects. The transformation is very limited, and a large number of drugs are directly excreted out of the body without being absorbed, and cannot play a role. If the medicine is directly converted into medicinal components in vitro, the utilization rate of Chinese herbal medicines can be greatly improved. Modern biotransformation c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/56C12R1/80
Inventor 李春杨晓刚周娟娟黄申
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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