Method for preparing rebaudioside A by fermentation and catalysis of bacillus subtilis

A Bacillus subtilis, catalyzed preparation technology, applied in the field of microbial fermentation enzyme preparations, can solve the problems of complex operation, high production cost, and inability to apply industrial production, and achieve low enzyme activity, reduce operation steps, and be conducive to industrial production. Effect

Pending Publication Date: 2020-08-28
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 that existing enzyme method and fermentation method catalyze stevioside into rebaudioside A in the production of high production cost, complicated operation and inapplicability to industrialized production, and provide a Method for preparing rebaudioside A by catalysis of bacillus fermentation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] (1) Seed preparation: link the synthesized glycosyltransferase UGT76G1 gene to the PBR322 plasmid vector, and transfer the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene into a competent Bacillus subtilis, and implant the The Bacillus subtilis with the glycosyltransferase UGT76G1 gene was inserted into the LB medium, the temperature of the LB medium was controlled at 34 °C, the rotation speed was 150 rpm, and the culture time was 22 h;

[0021] (2) Seed tank cultivation: The Bacillus subtilis obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 5% volume ratio, and the rotation speed of the seed tank was controlled at 300 rpm, and the ventilation ratio ( The aeration ratio is the volume ratio of air passing through a unit volume of culture solution per minute) is 0.5 V / V.min, cultivated at 35°C for 22 hours to obtain a seed culture solution;

[0022] (3) Fermentation tank production: put th...

Embodiment 2

[0028] (1) Seed preparation: link the synthesized glycosyltransferase UGT76G1 gene to the PBR322 plasmid vector, and transfer the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene into a competent Bacillus subtilis, and implant the The Bacillus subtilis with the glycosyltransferase UGT76G1 gene was inserted into the LB medium, the temperature of the LB medium was controlled at 30°C, the rotation speed was 200 rpm, and the culture time was 20 h;

[0029] (2) Seed tank cultivation: The Bacillus subtilis obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 5% volume ratio, and the rotation speed of the seed tank was controlled at 300 rpm, and the ventilation ratio ( The aeration ratio is the volume ratio of air passing through a unit volume of culture solution per minute) is 0.4 V / V.min, cultivated at 30°C for 20 hours to obtain a seed culture solution;

[0030] (3) Fermentation tank production: The see...

Embodiment 3

[0036] (1) Seed preparation: link the synthesized glycosyltransferase UGT76G1 gene to the PBR322 plasmid vector, and transfer the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene into a competent Bacillus subtilis, and implant the The Bacillus subtilis with the glycosyltransferase UGT76G1 gene was inserted into the LB medium, the temperature of the LB medium was controlled at 30°C, the rotation speed was 220 rpm, and the culture time was 24 h;

[0037] (2) Seed tank cultivation: The Bacillus subtilis obtained in step (1) was inserted into the seed tank containing LB medium according to the inoculation amount of 8% volume ratio, and the rotation speed of the seed tank was controlled at 400 rpm, and the ventilation ratio ( The aeration ratio is the volume ratio of air passing through a unit volume of culture solution per minute) is 0.3 V / V.min, cultivated at 30°C for 24 hours to obtain a seed culture solution;

[0038](3) Fermentation tank production: The seed...

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Abstract

The invention relates to a method for preparing rebaudioside A by fermentation and catalysis of bacillus subtilis. The method is characterized by comprising the following steps of: 1, connecting a glycosyltransferase UGT76G1 gene onto a PBR322 plasmid vector, transferring the plasmid vector to a competent state of bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, andculturing at 30-37 DEG C for 20-24 h; 2, inoculating into a seed tank according to an inoculation amount of 1-10% volume ratio, culturing, introducing air, and culturing at 30-37 DEG C for 20-24 h; 3,inoculating into a fermentation tank according to an inoculation amount of 1-10% volume ratio, culturing, introducing air, controlling the pH to be 6-8, and culturing at 30-37 DEG C for 48-72 h; 4, carrying out filter pressing to obtain bacillus subtilis thalli, re-suspending, carrying out high-pressure homogeneous crushing, and carrying out filter pressing to obtain crude enzyme solution; and 5,mixing stevioside, uridine diphosphate glucose, phosphate buffer solution and the crude enzyme solution according to a mass ratio of (5-10): (1-5): (40-60): (5-20), and carrying out a reaction at 25-40 DEG C for 12-48 h. The method in the invention has the advantages that: the yield of the rebaudioside A is increased, and can reach 85.3 g / L; and the method is few in operation step, low in production cost and easy for industrial production.

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 fermenting and catalyzing Bacillus subtilis. 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, and its energy is about 1 / 300 of that of sucrose. It is non-toxic, has no side effects, and has high stability. It is an ideal natural sweetener to replace sucrose, and it is known as the "third sugar source" internationally. Studies have reported that long-term consumption of stevia can prevent high blood pressure, diabetes, obesity, tooth decay and other diseases. Stevioside accounts for 60% to 70% of the total glycosides, and its taste is worse than that of rebaudioside A, with a certain after-bitterness. Rebaudioside A is a glycoside component in the steviol glycoside mixtur...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/54C12N9/10C12P33/20C12R1/125
CPCC12N15/75C12N9/1048C12P33/20
Inventor 夏家信祁飞陈永旭陆晓雨
Owner ANHUI JINGHE IND
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