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Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide

A technology of glycyrrhizin and endophytic fungi, which is applied in the field of biotransformation, can solve the problems of poor catalytic orientation, pollute the environment, low conversion rate and the like, and achieve the effects of less by-products, high reaction efficiency and simple steps.

Active Publication Date: 2014-08-20
JIANGXI NORMAL UNIV
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Problems solved by technology

[0007] The object of the present invention is to provide a Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 to convert glycyrrhizic acid into glycyrrhetin, It overcomes the disadvantages of poor catalytic orientation, environmental pollution, high energy consumption and low conversion rate in the prior art

Method used

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  • Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide
  • Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide
  • Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: Dongxiang wild rice endophytic fungus of the present invention Aspergillus flavus Isolation of DX-SEL2

[0028] (1) Collect complete Dongxiang wild rice and process it immediately after returning to the laboratory.

[0029] (2) Roots, stems and leaves are preliminarily sterilized by soaking in 75% ethanol for 5 minutes, then soaking in 0.1% mercuric acid for 8 minutes, and rinsing with sterile water to sterilize the surface of the tissues.

[0030] (3) Cut off the two ends of the above-mentioned treated stem with sterilized scissors, then cut the stem with two ends longitudinally, divide it into two, and cut it into small pieces of 0.1×0.5 cm. The edge of the leaf is cut off with sterilized scissors, and the edge-cut blade is also cut into small pieces of about 0.2×0.5 cm. They were respectively placed on PDA medium supplemented with 60 μg / L streptomycin and 0.5 g / L potassium dichromate, and cultured in an incubator at 28°C. In order to check the surf...

Embodiment 2

[0033] Example 2: The present invention screens out the Dongxiang wild rice endophytic fungus that directional transforms glycyrrhizic acid into glycyrrhetin Aspergillus flavus DX-SEL2.

[0034] (1) Plate primary screening: the isolated endophytic fungi of O. sativa Dongxiang were activated and inoculated in a solid screening medium with glycyrrhizic acid as the sole carbon source. In the control group, glucose was used instead of glycyrrhizic acid as the sole carbon source. At 28°C, cultivate for 7 days, and screen out the strains that can grow well.

[0035] The screening medium is; every liter of medium contains 3.0 g of glycyrrhizic acid, KH 2 PO 4 2.2 g, NH 4 NO 3 3.0 g, NaCl 0.5 g, yeast powder 0.05 g, MgSO 4 0.12g, CaCl 2 0.014g, FeSO 4 0.014g, ZnSO 4 ·7H 2 O 2.9 mg, MnCl 2 4H 2 O 2.0mg, CuSO 4 ·5H 2 O 0.25mg, CoCl 2 ·6H 2 O 0.24 mg, Na 2 MoO 4 2H 2 O 0.24mg, H 3 BO 3 0.03mg, 20g agar powder (not added to liquid medium), the rest is pure wate...

Embodiment 3

[0038] Example 3: Endophytic fungi Aspergillus flavus Conversion of Glycyrrhizic Acid to Glycyrrhetin by DX-SEL2

[0039] Dongxiang wild rice endophytic fungus Aspergillus flavus DX-SEL2 was inserted into the seed medium for 3 days at 28°C and 250 rpm, and then transferred to the transformation medium at 30% inoculum size, and cultured on a shaker at 35°C and 250 rpm for 7 days. The bacteria were removed by centrifugation, the fermentation broth was collected, and the transformation product was detected by HPLC.

[0040] The composition of the seed medium is as follows: every liter of medium contains 25g of glucose, 0.2g of glycyrrhizic acid (or ammonium glycyrrhizinate), KH 2 PO 4 2.0g, NH 4 NO 3 3g, NaCl 0.5g, yeast powder 0.1g, MgSO 4 0.12g, CaCl 2 0.2g, FeSO 4 0.014g, ZnSO 4 ·7H 2 O 2.9 mg, MnCl 2 4H 2 O2.0mg, CuSO 4 ·5H 2 O 0.25mg, CoCl 2 ·6H 2 O 0.24mg, Na 2 MoO 4 2H 2 O 0.24mg, H 3 BO 3 0.03mg, the rest is pure water, adjust the pH to 6.0....

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Abstract

The invention provides a Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide. The strain is identified and named as Aspergillus flavus DX-SEL2 and preserved in China Center for Type Culture Collection, with an accession number of CCTCC NO: M2013686. The strain can induce generation of beta-glucuronidase in a transformation medium with a glycyrrhizic acid as an inductive agent, then hydrolysis is carried out so as to remove a glucuronyl group at the terminal of a glycyrrhizic acid molecule, so glycyrrhetinic acid monoglucuronide is produced (secreted in fermentation broth). Organic solvent extraction, normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography and the like are used for further separation and purification so as to obtain glycyrrhetinic acid monoglucuronide. According to the invention, microbial catalytic conversion is employed, and the advantages of greenness, environmental protection, no pollution, simple transformation steps, a single product, a high transformation rate, etc. are obtained.

Description

technical field [0001] The invention relates to the technical field of biotransformation, in particular to an endophytic fungus of Dongxiang wild rice that transforms glycyrrhizic acid into glycyrrhetin. Background technique [0002] Glycyrrhizic acid (GL) is one of the main active ingredients in licorice. The structure of glycyrrhizic acid is as image 3 , is composed of pentacyclic triterpene saponins linking two glucuronic acids through glycosidic bonds. Glycyrrhizic acid is hydrolyzed by β-glucuronidase to remove a glucuronic acid group at its terminal to generate glycyrrhizin (such as image 3 ). [0003] Glycyrrhetin is also known as monoglucuronyl glycyrrhetinic acid (GAMG). The application value of glycyrrhizin (GAMG) is far greater than that of glycyrrhizic acid. The sweetness of glycyrrhizin is 5 times that of glycyrrhizic acid and 941 times that of sucrose. It is a new type of sweetener with high sweetness and low calories, which can effectively reduce obesity ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P33/20C12R1/67
Inventor 朱笃李平张志斌高波良
Owner JIANGXI NORMAL UNIV
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