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Method for preparing rebaudioside A by liquid fermentation

A technology of liquid fermentation and fermenter, which is applied in the directions of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of high production cost, complicated operation and low enzyme activity of rebaudioside A , to achieve the effect of low fermentation cost, easy control of fermentation process and high enzyme activity

Pending Publication Date: 2020-09-04
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 provide a method for preparing rebaudioside A by liquid fermentation in order to solve the problems of high production cost, low enzyme activity and complicated operation of existing rebaudioside A

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Seed preparation: The glycosyltransferase UGT76G1 gene was connected to the pUC18 plasmid vector, and the plasmid vector was transferred to DH5α E. coli competent cells, and the E. coli was inserted into LB medium, and the temperature of LB medium was controlled at 30° C, rotating speed 200 rpm, incubation time 15h;

[0025] (2) Seed tank culture: The Escherichia coli obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 1% volume ratio, and the rotation speed of the seed tank was controlled at 250 rpm, and the ventilation ratio (per minute Expressed by the air volume ratio per unit volume of culture solution, V / V.min) is 0.2 V / V.min, cultured at 30°C for 10 h 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 1% volume ratio for cultivation, control the rotation speed...

Embodiment 2

[0032] (1) Seed preparation: The glycosyltransferase UGT76G1 gene was connected to the pUC18 plasmid vector, and the plasmid vector was transferred to DH5α E. coli competent cells, and the E. coli was inserted into LB medium, and the temperature of LB medium was controlled at 33° C, the rotation speed is 220 rpm, and the incubation time is 12 hours;

[0033] (2) Seed tank culture: The Escherichia coli obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculum amount of 2.5% volume ratio, and the rotation speed of the seed tank was controlled at 200 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 8 hours to obtain seed culture solution;

[0034] (3) Fermentation tank production: The seed culture solution was inserted into a fermenter equipped with a fermentation medium according to the inoculum amount of 5% volume ratio for cult...

Embodiment 3

[0040] (1) Seed preparation: The glycosyltransferase UGT76G1 gene was connected to the pUC18 plasmid vector, and the plasmid vector was transferred to DH5α E. coli competent cells, and the E. coli was inserted into LB medium, and the temperature of LB medium was controlled at 36° C, the rotating speed is 250 rpm, and the incubation time is 18h;

[0041] (2) Seed tank cultivation: The Escherichia coli obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 3% volume ratio, and the rotation speed of the seed tank was controlled at 250 rpm, and the ventilation ratio (per minute Expressed by the air volume ratio per unit volume of culture solution, V / V.min) is 0.75V / V.min, cultured at 30°C for 12 hours to obtain seed culture solution;

[0042] (3) Fermentation tank production: put the seed culture solution into a fermenter with a fermentation medium at an inoculum volume of 7.5% by volume, and control the rotation speed of the...

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Abstract

The invention relates to a method for preparing rebaudioside A by liquid fermentation. The method includes: (1) transferring a glycosyltransferase UGT76G1 gene to a pUC18 plasmid vector, transferringto DH5alpha escherichia coli competent cells, inoculating into an LB medium, and culturing for 10-18 hours at 30-37 DEG C and 200-250 rpm; (2) inoculating into a seed tank according to an inoculationamount of 0.1-10% by volume, and culturing for 5-12 hours at 100-400 rpm, a ventilation ratio of 0.2-1 V / V. min and 30-37 DEG C; (3) inoculating into a fermentation tank according to an inoculation amount of 1-15% by volume, and culturing for 20-30 hours at 100-800 rpm, a ventilation ratio of 0.5-2 V / V. min and 30-37 DEG C; (4) adding an inducer when cell concentration OD600 reaches 20-60, whereinthe concentration is 0.05-1 mmol / L; (5) carrying out pressure filtration, resuspension, high-pressure homogenization and crushing, and pressure filtration to obtain a crude enzyme liquid; and (6) mixing stevioside, uridine diphosphate glucose, a phosphate buffer solution and the crude enzyme liquid, and reacting at 25-40 DEG C for 12-48h. The method high enzyme activity, high catalytic substrateconcentration, high conversion rate, easy control of the fermentation process, simple steps and low fermentation cost.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation enzyme preparations, and relates to a method for preparing rebaudioside A by liquid state fermentation. Background technique [0002] Stevioside (steviol glycoside) is a natural sweetener extracted from the dried leaves of stevia rebaudiana. The stevioside component in stevia rebaudiana accounts for 60% to 70% of the total glycosides, but stevioside has a certain degree of menthol flavor, and The post-bitter taste is more obvious, and the overall sweet taste is not very good. Rebaudioside A accounts for about 15% to 20% of the total glycosides, and the taste is closer to sucrose, with higher stability and safety. Stevia glycosyltransferase UGT76G1 can convert stevioside into rebaudioside A, improve the taste of stevioside products, and increase the market value of products. [0003] In recent years, as food and beverage companies have increased their requirements for the taste of s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/70C12N9/10C12P19/56C12R1/19
CPCC12N9/1048C12N15/70C12N1/20C12P19/56
Inventor 夏家信杨志健祁飞王余杰
Owner ANHUI JINGHE IND
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