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Method for preparing rebaudioside D through fermentation catalysis of bacillus subtilis

A technology for Bacillus subtilis and catalytic preparation, applied in the field of microbial fermentation enzyme preparations, can solve the problems of complicated operation, inability to apply industrialized production, and high production cost, achieves high enzyme activity level, is beneficial to industrialized production, and reduces the effects of operation steps

Pending Publication Date: 2022-03-08
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 existing enzymatic and fermentation method to catalyze stevioside into rebaudioside D in the production of high production cost, complicated operation, and the problems that cannot be applied to industrial production, and to provide a method of using Bacillus subtilis Method for preparing rebaudioside D 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 and glycosyltransferase AtSUSY gene to the PBR322 plasmid vector, and the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene and glycosyltransferase AtSUSY gene Introduced into the competent state of Bacillus subtilis, the Bacillus subtilis implanted with the glycosyltransferase UGT76G1 gene and the glycosyltransferase AtSUSY gene was inserted into the LB medium, and the temperature of the LB medium was controlled at 35 ° C, and the rotation speed was 200 rpm. Culture time 20 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, ...

Embodiment 2

[0027] (1) Seed preparation: Link the synthesized glycosyltransferase UGT76G1 gene and glycosyltransferase AtSUSY gene to the PBR322 plasmid vector, and the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene and glycosyltransferase AtSUSY gene Introduced into the competent state of Bacillus subtilis, the Bacillus subtilis implanted with the glycosyltransferase UGT76G1 gene and the glycosyltransferase AtSUSY gene was inserted into the LB medium, and the temperature of the LB medium was controlled at 30°C, and the rotation speed was 220 rpm. Culture time 24 h;

[0028] (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 1 V / V.min, cult...

Embodiment 3

[0034] (1) Seed preparation: Link the synthesized glycosyltransferase UGT76G1 gene and glycosyltransferase AtSUSY gene to the PBR322 plasmid vector, and the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene and glycosyltransferase AtSUSY gene Introduced into the competent state of Bacillus subtilis, the Bacillus subtilis implanted with the glycosyltransferase UGT76G1 gene and the glycosyltransferase AtSUSY gene was inserted into the LB medium, and the temperature of the LB medium was controlled at 30°C, and the rotation speed was 220 rpm. Culture time 24 h;

[0035](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, cul...

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Abstract

The invention relates to a method for preparing rebaudioside D by fermentation catalysis of bacillus subtilis, which is characterized by comprising the following steps: (1) inoculating glycosyl transferase UGT76G1 and AtSUSY genes to a PBR322 plasmid vector, guiding into the bacillus subtilis, inoculating into an LB culture medium, and culturing for 20-24 hours at the temperature of 30-37 DEG C and the speed of 100-250 rpm; (2) inoculating into a seed tank according to the inoculation amount of 1%-10%, and culturing for 20-24 hours at the temperature of 30-37 DEG C at the speed of 200-400 rpm and the ventilation ratio of 0.1-1 V / V.min; (3) inoculating into a fermentation tank according to the volume ratio of 1%-10%, and culturing for 48-72 hours under the conditions that the speed is 200-1000 rpm, the ventilation ratio is 0.1-2 V / V.min, the temperature is 30-37 DEG C and the pH value is 6-8; (4) carrying out ultrafiltration on the fermentation liquor by adopting a ceramic membrane, resuspending bacteria, carrying out high-pressure homogeneous crushing, and carrying out ultrafiltration on the crushed liquor by adopting the ceramic membrane to obtain crude enzyme liquor; and (5) mixing stevioside, uridine diphosphate glucose, a Tris-HCl buffer solution and the crude enzyme solution according to a mass ratio of (5-10): (1-5): (40-60): (5-20), and reacting at 25-40 DEG C for 12-48 hours. The method has the advantages that two crude enzymes are obtained through one-time fermentation, the cost is low, and the conversion rate of rebaudioside D reaches 88-90 g / L.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation enzyme preparations, and relates to a method for preparing rebaudioside D 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 D, with a certain after-bitterness. Rebaudioside D is a glycoside component in the steviol glycoside mixtur...

Claims

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Application Information

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IPC IPC(8): C12P19/56C12P19/18C12N1/21C12N15/54C12N15/75C12R1/125
CPCC12P19/56C12P19/18C12N9/1051C12N9/1062C12N15/75C12Y204/01C12Y204/01013C12N2800/101
Inventor 杨乐祁飞张敏戴永辉
Owner ANHUI JINGHE IND
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