Method for preparing diosgenin through biological method conversion

A technology of diosgenin and biological method, which is applied in the field of biological conversion and preparation of diosgenin, can solve the problems of low saponin yield, low conversion rate, poor hydrolysis efficiency, etc. Beneficial for separation and purification

Pending Publication Date: 2021-03-30
BEIJING UNIV OF CHEM TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In China's published patent applications (Chinese patent application publication numbers: 107177662A, 101012474B, 103146795A), direct microbial fermentation is used to prepare saponin, but the yield of saponin is generally low, and the direct microbial fermentation period is long and the conversion rate is not high , the steps are cumbersome, and the separation and purification in the later stage are also difficult
In China's published patent applications (Chinese patent application publication numbers: 105925559A, 101857855B), enzyme catalysis is used to produce saponin, but the enzymes involved are many and have poor specificity, resulting in poor hydrolysis efficiency and low substrate conversion rate, which limits the production of saponin. Industrial production of saponin

Method used

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  • Method for preparing diosgenin through biological method conversion
  • Method for preparing diosgenin through biological method conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Take by weighing 500g turmeric tubers ( figure 2 c), it is dried and pulverized and then passed through a 100-mesh sieve ( figure 2d); Take 100g of turmeric powder and 500mL of ethanol solution with a concentration of 80% by volume and add them to a 1L round bottom flask, and reflux extraction in a water bath at 70°C for 2h; after the reflux extraction, the extract is filtered, and the The filtrate was concentrated by rotary evaporation and dried to obtain the crude extract of dioscin, and finally the crude extract was ground into a powder of 100-200 mesh ( figure 2 b).

[0032] Get the lyophilized powder of Myrothecium verrucaria CGMCC NO.3.0781 and place it on the slant of the test tube containing PDA medium to carry out activation culture, then transfer the bacterial classification to the PDA flat medium for cultivation and recovery for 3 days ( figure 2 a).

[0033] Put the revived Mycobacterium verrucosus into PDA liquid medium, ferment and cultivate it for ...

Embodiment 2

[0035] Example 2 Fermentation of Mycobacterium verrucae to produce enzyme

[0036] Prepare microbial 2L fermentation medium, the formula is: glucose concentration 3g / L, yeast powder concentration 8g / L, turmeric powder concentration 3g / L, ammonium sulfate concentration 1g / L, KH 2 PO4 concentration is 3g / L, MgSO 4 ·7H 2 The concentration of O is 2g / L, the volume percentage of Tween 80 is 0.8‰, the solvent is water, and the pH is adjusted to 5.5.

[0037] Add 10mL of Myrocarina verrucosa seed solution to 1L of sterilized fermentation medium, place it in a constant temperature shaking incubator for fermentation, the shaker speed is 160r / min, the temperature is 28°C, and the fermentation time is 3 days.

[0038] After the fermentation and cultivation, the fermentation broth was directly filtered to remove the mycelia to obtain 900mL rhamnosidase crude enzyme solution, in which the enzyme activity of the crude enzyme solution was 0.15U / mL; enzyme activity definition (U): hydrolysi...

Embodiment 3

[0039] Example 3 Two-step enzyme-catalyzed conversion of diosgenin to prepare saponin

[0040] The first step of catalysis: prepare 200mL of phosphate buffer solution with a pH of 5 (200mM), add 100mg of diosgenin crude extract, and then add 20mL of rhamnose crude enzyme solution to it, at 50℃, 200r / min Under full reaction for 24h, at this time the S in the substrate 1 , S 2 , S 3 Components are completely converted to S 4 , S 5 , S 6 , the hydrolytic cleavage is the rhamnoside bond ( image 3 and Figure 4 ).

[0041] The second step of catalysis: after the first step of catalysis, add 200uL cellulase to the system to catalyze the S in the substrate 4 , S 5 , S 6 The glucosidic bond of the component ( image 3 and Figure 4 ), the reaction temperature is 50°C, the shaking table speed is 200r / min, and the reaction time is 24h, finally 18.4mg of diosgenin is obtained, and the conversion rate reaches 99%.

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Abstract

The invention discloses a method for preparing diosgenin through biological method conversion, and belongs to the technical field of biological conversion and production of natural products. The method comprises the following steps: by taking a dioscin crude extract as a substrate, performing catalytic hydrolysis on rhamnosidase crude enzyme liquid prepared by fermenting Myrothecium verrucaria CGMCC NO.3.0781 to break rhamnoside bonds on the outer side of a dioscin C3 position in the substrate, and hydrolyzing residual glucoside bonds on the dioscin C3 position by using cellulase to obtain a final product diosgenin. According to the invention, a two-step enzyme catalysis method is used for converting the dioscin crude extract to prepare diosgenin, and the method is characterized in that the conversion rate of dioscin in a substrate is up to 99%, no by-product is produced, and the method is simple and easy to implement, mild in condition, low in equipment requirement, free of pollutionto the environment and suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biotransformation of natural products, and in particular relates to a method for preparing diosgenin through biological transformation. Background technique [0002] Turmeric is currently the plant species with the highest diosgenin content in the world. Its scientific name is Dioscores zingiberensis C.H.Wright (abbreviated as DZW). It is mainly distributed in Shaanxi, Gansu, and Lianghu in my country. The rhizome fruit of turmeric can be directly used as medicine, which can be used to dispel dampness, clear away heat and detoxify. Diosgenin, commonly known as saponin, exists in various forms of diosgenin in natural plants, such as diosgenin, diosgenin, and diosgenin A. The structures of related substances are as follows: figure 1 shown. [0003] As the main precursor for the synthesis of steroid hormones, diosgenin can be used to synthesize as many as 300 compounds with medicinal value, including Dunye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/20C12N9/24C12N9/42
CPCC12P33/20C12N9/2402C12N9/2437
Inventor 袁其朋王钰涵程磊雨
Owner BEIJING UNIV OF CHEM TECH
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