Method for high-yield preparation of glycyrrhetinic acid monoglucuronide

A technology of aldehyde acid glycyrrhetic acid and glucose, applied in the field of biochemistry, can solve the problems of good repeatability, unfavorable conversion reaction, low production efficiency and the like, and achieve the effects of high efficiency, good repeatability and low cost

Inactive Publication Date: 2014-01-15
JIANGSU TIANSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. The biocatalyst is mainly microbial cells. Excessively high substrate concentration increases the osmotic pressure of the medium, which is not conducive to the growth of microorganisms and the accumulation of glucuronidase
[0007] 2. The conversion product monoglucuronoglycyrrhetinic acid accumulates continuously in the fermentation broth, the feedback inhibition effect on glucuronidase is enhanced, and the viscosity of the fermentation broth increases, which is not conducive to the full progress of the conversion reaction
In the invention patent of Li Chun et al., whose patent number is 201110344409.1 titled "A Method for Production of Monoglucuronyl Glycyrrhetinic Acid by Batch-Fed Fermentation", the batch operation of adding substrate glycyrrhizinic acid with batch-fed feed is used, although the improvement Substrate inhibition is achieved, but the substrate utilization rate is low, energy consumption is high, and the problem of product feedback inhibition is not solved, which makes it difficult to further increase the fermentation yield and lower production efficiency
[0009] The method for preparing monoglucuronylglycyrrhetinic acid provided by the present invention is to use a strain producing specific glucuronidase as a catalyst, and use fermentation and resin separation coupling technology to obtain high-yield monoglucuronylglycyrrhetinic acid, It has the advantages of high efficiency, low cost, good repeatability, and is suitable for industrial production. At present, there are no relevant patents and literature reports

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Preparation of seed medium: glucose 10g / L, yeast extract 2g / L, KH 2 PO 4 1g / L, MgSO 4 0.25g / L, agar 20g / L, solvent is water, pH is natural. Add 20g / L agar to the slant medium in this recipe. All the above media were sterilized at 115°C for 20 min. Pick up the ring lawn from the slant of the strain stored in the refrigerator, inoculate it into a fresh slant medium, and cultivate it at 30° C. for 48 hours to obtain an activated seed slant. Inoculate 2 rings of the activated slant pick into a 250ml Erlenmeyer flask containing 40ml of seed medium, and shake and culture at 30°C and 200rpm for 24h to obtain fermented seed liquid.

[0031] (2) Preparation of fermentation medium: glycyrrhizic acid concentration 55g / L, KH 2 PO 4 1g / L, urea 10g / L, MgSO 40.8g / L, the solvent is water; use acid and alkali to control pH6. Sterilize at 115°C for 20 minutes. Take 60ml of fermented seed liquid and inoculate it into a 5L fermenter with 3L transformation medium

[0032] (...

Embodiment 2

[0037] (1) Preparation of seed medium: glucose 20g / L, yeast extract 2g / L, KH 2 PO 4 1g / L, MgSO 4 0.8g / L, the solvent is water, and the pH is natural. Add 20g / L agar to the slant medium in this recipe. All the above media were sterilized at 115°C for 20 min. Firstly, pick out the ring lawn from the slant of the strain stored in the refrigerator, inoculate it on the fresh slant medium, and cultivate it at 28°C for 240 hours to obtain the activated seed slant. Inoculate 2 rings of the activated slant pick into a 250ml Erlenmeyer flask containing 40ml of seed medium, and shake and culture at 30°C and 200rpm for 72h to obtain first-grade seeds. Use a sterile pipette to inoculate 5ml of the primary seed solution into a 500ml shake flask containing 80ml of the seed solution, and culture at 30°C and 250rpm with shaking for 48 hours to obtain the secondary seed solution.

[0038] (2) Preparation of fermentation medium: glycyrrhizic acid concentration 27g / L, KH 2 PO 4 1g / L, am...

Embodiment 3

[0044] (1) Preparation of seed medium: glucose 10g / L, yeast extract 2g / L, KH 2 PO 4 1g / L, MgSO 4 0.25g / L, agar 20g / L, solvent is water, pH is natural. Add 20g / L agar to the slant medium in this recipe. All the above media were sterilized at 115°C for 20 min. Firstly, pick out the ring lawn from the slant of the strain stored in the refrigerator, inoculate it on the fresh slant medium, and cultivate it at 28°C for 240 hours to obtain the activated seed slant. Inoculate 2 rings of the activated slant pick into a 250ml Erlenmeyer flask containing 40ml of seed medium, and shake and culture at 28°C and 200rpm for 72h to obtain first-grade seeds. Use a sterile pipette to inoculate 5ml of the primary seed solution into a 500ml shake flask containing 80ml of the seed solution, and culture at 28°C and 250rpm for 48 hours with shaking to obtain the secondary seed solution.

[0045] (2) Preparation of fermentation medium: glycyrrhizic acid concentration 55g / L, glucose 20 / L, KH 2 P...

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Abstract

The invention provides a method for high-yield preparation of glycyrrhetinic acid monoglucuronide, and belongs to the technical field of biochemistry. The method comprises the following steps: with a bacterial strain of generating specific glucuronidase as a catalyst and glycyrrhizic acid as a substrate, removing a glycyrrhetinic acid monoglucuronide product by taking a macroporous resin as a separating medium in processes of continuous stirring and ventilating conversion reaction, releasing the substrate glycyrrhizic acid adsorbed in the resin, and discharging the glycyrrhizic acid out of a resin column together with unreacted glycyrrhizic acid in a fermentation liquor; and reflowing to a fermentation tank to continue to ferment. Simultaneous coupling of three processes of fermentation, material supplement and product separation is achieved; the problems of negative feedback inhibition and unfavourable fermentation caused by over-high viscosity of the product and a substrate fermented liquid are solved by maintaining proper substrate concentration and low product concentration in a fermentation process; the utilization efficiency of the substrate and the fermentation yield of the glycyrrhetinic acid monoglucuronide are significantly improved; meanwhile, the automatic degree is high.

Description

technical field [0001] The invention relates to a method for preparing glycyrrhetinic acid monoglucuronate with high yield, and more specifically relates to a method for preparing glycyrrhetinic acid monoglucuronate coupled with fermentation and resin separation, which belongs to the technical field of biochemistry. Background technique [0002] Glycyrrhizic acid is the main pharmacologically active ingredient in the traditional Chinese medicine licorice. Research on this traditional Chinese medicine monomer at home and abroad began in the 19th century, and it has anti-inflammatory, anti-viral, anti-tumor, and anti-allergic effects. And it is a sweetener, about 170 times sweeter than sucrose. With the deepening of research, in the 1980s, scholars found that β-glucuronidase in different organisms had the following four catalytic properties in the conversion of glycyrrhizic acid as a catalyst: (1) glycyrrhizic acid → glycyrrhetinic acid; ( 2) Glycyrrhizic acid → monoglucuron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/20C07J63/00C07H15/256C07H1/08
Inventor 魏元刚杨永安易铭钟慧金显友袁继文黄全书季浩
Owner JIANGSU TIANSHENG PHARMA
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