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Method for preparing glycyrrhetinic acid monoglucuronide

A technology of glycyrrhetinic acid and glucose, applied in the direction of microbial-based methods, biochemical equipment and methods, bulk chemical production, etc., can solve the problems that GAMG cannot be realized

Active Publication Date: 2014-04-16
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the content of glycyrrhizic acid is the highest in licorice, it is not possible to directly use the total extract of licorice or the crude triterpenes of licorice as a carbon source to produce GAMG through biotransformation.

Method used

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  • Method for preparing glycyrrhetinic acid monoglucuronide
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  • Method for preparing glycyrrhetinic acid monoglucuronide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, only using glycyrrhizic acid monoammonium salt as inducer to produce enzyme implementation method:

[0044] Inoculate the Penicillium purpurea Li-3 strain on the slant medium, and cultivate it at a constant temperature of 30°C for 3 days. The composition of the slant medium is: glucose 0.5g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, distilled water 100mL, agar 1.5g, adjust the pH to 5.5, sterilize at 121°C for 20min, and cool to room temperature.

[0045] Take the spores on the slant medium, inoculate them into the seed medium, and culture them on a shaker at 30°C at 170r / min for 72h, then transfer 5% of the inoculum into the secondary seed medium at 30°C on a shaker at 170r / min Cultivate for 24 hours to obtain secondary seed liquid, the composition of the seed medium is: glucose 0.5g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, 100mL of distilled water, adju...

Embodiment 2

[0047] Example 2, the implementation method of adding exogenous accelerator to produce enzymes of monoammonium glycyrrhizinate:

[0048] Slope culture and seed culture are the same as in Example 1.

[0049] The secondary seed solution was inserted into the enzyme-producing medium according to the inoculation amount of 10% by volume, and the fermented liquid reached the maximum specific enzyme activity of 82.3U / ml (whole cell enzyme activity) for 72 hours at 30°C and 170r / min shaker culture , wherein the conversion rate of GL reached 93.6%, and the yield of GAMG reached 92.7%. The composition of the enzyme production medium: monoammonium glycyrrhizinate 0.6g, licorice total extract 0.4g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO4 ·7H 2 O0.001g, 100mL of distilled water, adjust the pH to 5.5, sterilize at 121°C for 20min, and cool to room temperature.

Embodiment 3

[0051] Slope culture and seed culture are the same as in Example 1.

[0052] The secondary seed liquid was inserted into the enzyme-producing medium according to the inoculum amount of 10% by volume, and cultivated on a shaker at 30°C and 170r / min for 72 hours. Enzyme solution 100mL, specific enzyme activity is 141477U / ml (enzyme activity of broken cell crude enzyme solution). Composition of enzyme production medium: 1.2g monoammonium glycyrrhizinate, 0.8g total extract of licorice, NH 4 NO 3 0.6g, KH 2 PO 4 0.2g, KCl0.1g, MgSO 4 ·7H 2 O0.1g, FeSO 4 ·7H 2 O0.002g, distilled water 200mL, pH4.8, after sterilizing at 121°C for 20min, cool to room temperature.

[0053] Take a 1000mL Erlenmeyer flask with 400mL of substrate solution inside, add 100mL of crude enzyme solution of broken cells to the transformation system, culture on a shaker at 30°C for 6 hours, centrifuge, and take the supernatant for detection by high performance liquid chromatography , the conversion rate...

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Abstract

The invention relates to a method for preparing glycyrrhetinic acid monoglucuronide (GAMG), and belongs to the field of food additives. The method comprises the steps that accelerants of a liquorice total extract, or liquorice total polysaccharide and / or liquorice total flavone are added to a culture medium containing glycyrrhizic acid or salt of glycyrrhizic acid; Penicillium purpurogenum Li-3 is induced to generate beta-glucuronidase; the preservation number of Penicillium purpurogenum Li-3 is CGMCC No. 5446 (China General Microbiological Culture Collection Center Number 5446); enzyme production is 5-48h ahead compared with the condition that the accelerants are not added; the enzyme activity is increased by 0.5-5 times; a beta-glucuronidase crude enzyme preparation is prepared by thalli after the enzyme production or fermentation liquor containing the thalli; GAMG is generated by converting the liquorice total extract or glycyrrhizic acid and an analogue of glycyrrhizic acid in liquorice total triterpene; and GAMG in the fermentation liquor is separated and purified. The method improves the enzyme activity, avoids a complicated extraction and separation course by taking glycyrrhizic acid (or salt of glycyrrhizic acid) as carbon source glycyrrhizic acid (or salt of carbon source glycyrrhizic acid), and lowers the production cost.

Description

technical field [0001] The invention relates to a method for preparing monoglucuronyl glycyrrhetinic acid, belonging to the field of food additives (sweeteners). Background technique [0002] Licorice is a shrub-like perennial herb of the leguminous Glycyrrhizae genus, which is an important food additive and traditional Chinese medicine. Glycyrrhizic acid (GL) is a pentacyclic triterpenoid compound with the highest content (3-11%) in licorice. It is one of the main active ingredients in licorice. Potassium excretion increases, resulting in certain side effects. GL hydrolyzes a glucuronic acid group to produce a product - monoglucuronic acid glycyrrhetinic acid (GAMG), see the structure figure 1 . The sweetness of GAMG is 5 times that of glycyrrhizic acid and nearly 1000 times that of sucrose. It is a new type of sweetener with high sweetness and low calorie. Animal experiments show that the LD50 of GAMG is 5000mg / kg, far greater than the LD50805mg / kg of glycyrrhizic acid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/56C12N9/24C12R1/80
CPCY02P20/54
Inventor 刘桂艳李春王栋汪竹高自强刘冬羽
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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