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Method for producing rebaudioside A through double-enzyme fermentation catalysis

A technology of bacterial fermentation and fermenter, which is applied in the field of microbial fermentation enzyme preparations, can solve the problems of low enzyme activity and high production cost of rebaudioside A, and achieve high enzyme activity, important market value and application value, and low production cost Effect

Pending Publication Date: 2020-09-15
ANHUI JINGHE IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of high production cost and low enzyme activity of existing rebaudioside A, and to provide a method for the catalytic production of rebaudioside A by dual bacterial fermentation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Seed preparation: the glycosyltransferase UGT76G1 gene and the sucrose synthase AtSUSY gene were connected to the pUC18 plasmid vector, introduced into DH5α Escherichia coli competent cells, inserted into LB medium, and controlled the temperature of LB medium to 33°C , rotation speed 180rpm, culture time 18h;

[0025] (2) Seed tank culture: The E. coli obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 2% volume ratio, and the rotation speed of the seed tank was controlled at 180 rpm, and the ventilation ratio (per minute Expressed by the air volume ratio per unit volume of culture solution, V / V.min) is 0.5V / V.min, cultured at 33°C for 9 hours to obtain seed culture solution;

[0026] (3) Fermentation tank production: put the seed culture solution into a fermenter equipped with fermentation medium according to the inoculum amount of 2% volume ratio for cultivation, control the rotation speed of the fermen...

Embodiment 2

[0032] (1) Seed preparation: the glycosyltransferase UGT76G1 gene and the sucrose synthase AtSUSY gene were connected to the pUC18 plasmid vector, introduced into DH5α Escherichia coli competent cells, inserted into LB medium, and the temperature of LB medium was controlled at 30°C. The rotation speed is 220rpm, and the incubation time is 16h;

[0033] (2) Seed tank cultivation: Inoculate the Escherichia coli obtained in step (1) with an inoculum volume of 3% by volume into a seed tank containing LB medium for cultivation, control the rotation speed of the seed tank to 220 rpm, and the ventilation ratio to 0.6V / V.min, cultivated at 30°C for 10 h to obtain the seed culture solution;

[0034] (3) Fermentation tank production: put the seed culture solution into a fermenter equipped with fermentation medium according to the inoculum amount of 3% volume ratio for cultivation, control the rotation speed of the fermenter to 800 rpm, and the aeration ratio to 1.5 V / V.min, At a tempe...

Embodiment 3

[0040] (1) Seed preparation Glycosyltransferase UGT76G1 gene and sucrose synthase AtSUSY gene were connected to pUC18 plasmid vector, introduced into DH5α Escherichia coli competent cells, inserted into LB medium, controlled the temperature of LB medium at 25°C, and the rotation speed 250rpm, culture time 18h;

[0041] (2) Seed tank culture: The E. coli obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 4% volume ratio, and the rotation speed of the seed tank was controlled to 250 rpm, and the ventilation ratio was 0.8V / V.min, cultivated at 25°C for 12 h to obtain the seed culture solution;

[0042](3) Fermentation tank production: put the seed culture solution into a fermenter equipped with fermentation medium according to the inoculum amount of 4% volume ratio for cultivation, control the rotation speed of the fermenter to 1000 rpm, and the aeration ratio to 2 V / V.min, At a temperature of 37°C, control the pH to...

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Abstract

The invention relates to a method for producing rebaudioside A through double-bacterium fermentation catalysis. The method is characterized by comprising the following steps: (1) connecting a glycosyltransferase UGT76G1 gene and a sucrose synthase AtSUSY gene to a pUC18 plasmid vector, transferring the pUC18 plasmid vector to DH5alpha escherichia coli competent cells, inoculating an LB culture medium, and culturing at 25-37 DEG C and 200-250 rpm for 10-18 h; (2) inoculating to a seed tank according to the inoculum size of 0.5%-15%, and culturing for 5-16 h at the speed of 150-400rpm and the ventilation ratio of 0.1-1.5 V / V.min at the temperature of 25-37 DEG C; (3) inoculating into a fermentation tank according to the inoculum size of 1%-10%, and culturing for 20-40 h at the speed of 100-1,000 rpm, the ventilation ratio of 0.2-2 V / V.min and the pH of 6.6-8.5 at the temperature of 25-37 DEG C; (4) adding an inducer when the OD600 value reaches 20-100, wherein the concentration of the inducer is 0.1-1.5 mmol / L; (5) carrying out filter pressing, resuspending, crushing and filter pressing on the fermentation liquid to obtain a crude enzyme liquid; and (6) mixing stevioside, uridine diphosphate, a phosphate buffer solution and the crude enzyme solution according to a mass ratio of (40-100):(1-4):(400-600):(50-100), and performing reaction at 25-40 DEG C for 24-48 h. The method has the advantages that two crude enzyme solutions are obtained through one-time fermentation, and operation steps are few; the enzyme activity is high and the production cost is low.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation enzyme preparations, and relates to a method for catalytically producing rebaudioside A by double-bacteria fermentation. Background technique [0002] Stevioside (steviol glycoside) is a natural sweetener extracted from the dried leaves of Stevia rebaudiana. Its sweetness is 200-300 times that of sucrose. very good. The stevioside component in stevia rebaudiana accounts for 60% to 70% of the total glycosides, and is the main source of bitterness; rebaudioside A accounts for about 15% to 20% of the total glycosides, and the taste is better than stevioside, closer to Sucrose, while having higher stability and safety. [0003] At present, there are few studies on the microbial fermentation of stevioside, the cost of plant extraction and separation of stevioside and rebaudioside A is relatively high, and the development of stevioside industry is relatively slow. With the improvement ...

Claims

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Application Information

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IPC IPC(8): C12P19/56C12N1/21C12R1/19
CPCC12P19/56C12N9/1048C12N9/1062C12Y204/01013
Inventor 孙多龙祁飞孙彩军陆晓雨
Owner ANHUI JINGHE IND
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