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Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same

A technology of mollerian inoculation mold and ginsenoside, applied in the direction of fungi, fermentation, etc., can solve the problems of high ginsenoside concentration, low yield, prolonged reaction time, etc., and achieve cost-free by-products, simple and convenient, specific effect

Inactive Publication Date: 2011-06-01
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Utilizing enzymes derived from microorganisms to convert main ginsenosides to produce ginsenoside Rd has the following common problems: the conversion product is not specific during the conversion process; the concentration of main ginsenosides used as the conversion substrate should not be too high; the substrate has an inhibitory effect on the enzyme, etc.
However, due to the inhibitory effect of ginsenosides, when the reaction substrate was replaced by the total extract of ginseng, the reaction time was longer than that with pure Rb 1 Extended 30 times; When the total ginseng extract mass concentration reached 4.2mg / mL, the productive rate of ginsenoside Rd was reduced from 80% to 12% (Biotechnology Letters such as Son J W, 2008, 30: 713-716)

Method used

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  • Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same
  • Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same
  • Ginseng endogenesis zygorhynchus moelleri mildew as well as method for preparing ginsenoside Rd by utilizing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Isolation and screening of bacterial strains

[0031]1. Isolation of endophytic bacteria from ginseng: select fresh and healthy ginseng, wash it with tap water, soak it in 0.1% mercury liter for 1-1.5 minutes, rinse it with sterile water for 4-5 times; Soak in 75% alcohol for 1 to 1.5 minutes, and rinse with sterile water for 3 to 4 times. Under sterile conditions, ginseng roots were cut into small pieces (2mm×2mm) with sterilized surgical scissors, and placed on PDA plates with a diameter of 9cm. After 5-7 days, observe whether there are colonies formed around the tissue block, and pick endophytic fungal hyphae and inoculate them on PDA slant medium for purification and preservation.

[0032] Medium: potato dextrose agar medium (PDA medium): 200 g of potatoes, 18 g of glucose, 18 g of agar, 1000 mL.

[0033] The ginseng (Panax ginseng C.A.Mey) used in this example was obtained by self-collection, and was collected by the inventor from Huanren County, Li...

Embodiment 2

[0043] Embodiment 2: conversion singleness test

[0044] Adopt the method of embodiment 1 step 2 (2), the conversion selectivity of ginsenoside substrate and product of the mollerian mold (CGMCCNo.4315) obtained by test screening, through TLC detection, the result is as attached image 3 Shown:

[0045] It can be seen that the protodiol saponin Rb 1 , Rb 2 . 1 A reaction occurs, and the conversion product is only one, and the product can be preliminarily judged to be Rd according to the Rf value. It has good substrate selectivity and product singleness.

Embodiment 3

[0046] Embodiment 3: solid conversion method prepares Rd

[0047] ① Prepare solid transformation medium (PDA): every 100mL medium contains 20g of potato, 1.8g of glucose, 1.8g of agar, and ginsenoside Rb 1 20g; prepared with deionized water, sterilized under 1.0kPa autoclave at 121°C for 30min.

[0048] ② Inoculate the activated Molderella mollerii (CGMCC No.4315) to the above-mentioned ginsenoside Rb-containing 1 cultured on PDA medium at 25°C for 5-7 days.

[0049] ③Product extraction and separation: Soak the culture in n-butanol and extract fully; the extract is centrifuged at 10000r / min for 1min, filtered through a microporous membrane with a diameter of 0.22μm, and the target product is separated by HPLC.

[0050] HPLC conditions: flow rate: 0.6mL / min; detection wavelength: 203nm; column temperature 35°C; mobile phase: A is acetonitrile, B is high-purity water;

[0051] Gradient elution mobile phase ratio:

[0052] 0min, A is 20%, B is 80%;

[0053] 18min, A is 40%, ...

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Abstract

The invention relates to ginseng endogenesis zygorhynchus moelleri mildew (CGMCC (China General Microbiological Culture Collection): No.4315). The mildew has the capability for preparing Rd by converting a substrate ginsenoside Rb1 and has higher substrate unicity and product unicity, and the preparation of the ginsenoside Rd by utilizing the mildew can adopt in-site conversion. A method for preparing the ginsenoside Rd comprising the following steps of: dibbling the mildew in a PDA (Potato Dextrose Agar) culture medium containing the ginsenoside Rb1, standing still at 25 DEG C and culturing for 5-7 days or vaccinating the mildew on an enzyme production culture medium by adopting a microorganism enzymic method and culturing at 28 DEG C for 5-7 days, collecting enzyme liquid, mixing with the ginsenoside Rb1 and reacting at 40 DEG C for 24h. The ginsenoside Rd produced by adopting the technical scheme of the invention has the advantages of strong specificity, simplicity, convenience, safety, reliability and low cost without side products, the purity of the fermentation product Rd is higher than 90 percent, and the conversion rate can be higher than 60 percent.

Description

technical field [0001] The invention relates to an endophytic fungus of ginseng, Zygorhynchus moelleri (CGMCCNo.4315), and the specific transformation substrate ginsenoside Rb by using the fungus 1 A method for ginsenoside Rd. Background technique [0002] Ginsenoside Rd is a promising candidate drug. It has the function of specifically blocking receptor-dependent calcium ion channels. It can protect kidney function, regulate immunity, inhibit HeLa cell growth, induce COX22 production, and protect against radiation. It has a unique effect that other monomeric ginsenosides do not have; in terms of analgesic and neuroprotective effects, Rd is also stronger than other monomeric ginsenosides. Therefore, ginsenoside Rd can be developed into drugs for radiation protection, treatment of cardiovascular diseases, inflammation, trauma, and internal and external hemorrhage caused by injury. However, due to the low content of ginsenoside Rd in Panax genus and its complex structure, ch...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P33/20
Inventor 吕国忠张薇孙晓东
Owner DALIAN NATIONALITIES UNIVERSITY
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