阅读说明：本技术 一种利用枯草芽孢杆菌发酵催化制备莱鲍迪苷a的方法 (Method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis ) 是由 夏家信 祁飞 陈永旭 陆晓雨 于 2020-05-25 设计创作，主要内容包括：本发明涉及一种利用枯草芽孢杆菌发酵催化制备莱鲍迪苷A的方法,其特征在于：1.糖基转移酶UGT76G1基因连接到PBR322质粒载体上,将质粒载体导转到枯草芽孢杆菌的感受态中,将枯草芽孢杆菌接入LB培养基,30～37℃培养20～24 h；2.按1%～10%体积比的接种量接入种子罐中培养,通气,30～37℃培养20～24 h；3.按1%～10%体积比的接种量接入发酵罐中培养,通气,控制pH为6～8,30～37℃培养48～72h；4.压滤得到枯草芽孢杆菌菌体,重悬,高压均质破碎,压滤得粗酶液；5.将甜菊糖苷、尿苷二磷酸葡萄糖、磷酸缓冲液、粗酶液按质量比5-10：1-5：40-60：5-20混合,在25-40℃反应12-48 h即可。本发明优点：提高了莱鲍迪苷A的产量,可达85.3 g/L；操作步骤少,生产成低本,易于工业化生产。(The invention relates to a method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis, which is characterized by comprising the following steps: 1. the glycosyltransferase UGT76G1 gene is connected to a PBR322 plasmid vector, the plasmid vector is transferred to the competence of the bacillus subtilis, the bacillus subtilis is inoculated into an LB culture medium and cultured for 20-24 h at the temperature of 30-37 ℃; 2. inoculating the mixture into a seeding tank according to the inoculation amount of 1-10% of the volume ratio for culture, ventilating, and culturing for 20-24 h at 30-37 ℃; 3. inoculating the mixture into a fermentation tank according to the inoculation amount of 1-10% of the volume ratio for culture, ventilating, controlling the pH to be 6-8, and culturing for 48-72 h at 30-37 ℃; 4. filter-pressing to obtain bacillus subtilis thallus, resuspending, homogenizing and crushing under high pressure, and filter-pressing to obtain crude enzyme solution; 5. mixing stevioside, uridine diphosphate glucose, a phosphate buffer solution and a crude enzyme solution according to a mass ratio of 5-10: 1-5: 40-60: 5-20, and reacting at 25-40 ℃ for 12-48 h. The invention has the advantages that: the yield of rebaudioside-A is improved and can reach 85.3 g/L; the operation steps are few, the production cost is low, and the industrial production is easy to realize.)

1. A method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis is characterized by comprising the following steps:

seed preparation: connecting the synthesized glycosyltransferase UGT76G1 gene to a PBR322 plasmid vector, transferring the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene into the competence of the bacillus subtilis, inoculating the bacillus subtilis implanted with the glycosyltransferase UGT76G1 gene into an LB culture medium, controlling the temperature of the LB culture medium to be 30-37 ℃, the rotation speed to be 100-250rpm, and the culture time to be 20-24 h;

(2) seed tank culture: inoculating the bacillus subtilis obtained in the step (1) into a seeding tank filled with an LB (lysogeny broth) culture medium according to the inoculation amount of 1-10% by volume, controlling the rotating speed of the seeding tank to be 200-400 rpm and the aeration ratio to be 0.1-1V/V.min, and culturing at the temperature of 30-37 ℃ for 20-24 hours to obtain a seed culture solution;

(3) fermentation tank production: inoculating the seed culture solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 1-10% of the volume ratio for culturing, controlling the rotation speed of the fermentation tank to be 200-1000 rpm and the aeration ratio to be 0.1-2V/V.min, controlling the pH to be 6-8 at the temperature of 30-37 ℃, culturing for 48-72 hours, and obtaining fermentation liquor after the fermentation is finished;

(4) filter-pressing the fermentation liquor obtained in the step (3) to obtain bacillus subtilis, then carrying out heavy suspension on the bacillus subtilis, carrying out high-pressure homogenizing and crushing to obtain a bacillus subtilis thallus crushing liquid, and then carrying out filter pressing on the bacillus subtilis thallus crushing liquid to obtain a crude enzyme liquid;

(5) the catalytic system is as follows: mixing stevioside, uridine diphosphate glucose, a phosphate buffer solution and a crude enzyme solution according to a mass ratio of 5-10: 1-5: 40-60: 5-20, and then reacting for 12-48 h at 25-40 ℃ to obtain the rebaudioside A.

2. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to claim 1, wherein the method comprises the following steps: the temperature of the LB medium in the step (1) is 33-35 ℃, the rotation speed is 150-.

3. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to claim 1, wherein the method comprises the following steps: in the step (2), the rotation speed of the seeding tank is 280-330 rpm, the aeration ratio is 0.3-0.7V/V.min, the culture temperature is 33-35 ℃, and the culture time is 20-23 h.

4. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to claim 1, wherein the method comprises the following steps: in the step (3), the rotating speed of the fermentation tank is 400-700 rpm, the aeration ratio is 0.5-1.8V/V.min, the culture temperature is 30-37 ℃, the pH value is 6.5-7.5, and the culture time is 55-65 h.

5. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to claim 1, wherein the method comprises the following steps: the preparation method of the fermentation medium in the step (3) comprises the following steps: preparing a fermentation culture medium according to the proportion of 10-40 g of bean cake powder, 20-50 g of bran, 30-60 g of corn flour, 1-10 g of diammonium hydrogen phosphate, 1-10 g of disodium hydrogen phosphate and 0.1-5g of calcium chloride, adjusting the pH to 6-8 by using a sodium hydroxide solution, and fixing the volume to 1000 ml by using deionized water.

6. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to claim 1, wherein the method comprises the following steps: in the step (4), the resuspension time is 10-20 min, and the pressure of high-pressure homogenizing and crushing is 0.1-0.5 Mp.

7. The method for preparing rebaudioside A through fermentation catalysis of bacillus subtilis according to any one of claims 1-6, wherein the method comprises the following steps: and (3) adopting a plate-and-frame filter pressing method for pressure filtration in the steps (3) and (4).

Technical Field

The invention belongs to the technical field of microbial fermentation enzyme preparations, and relates to a method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis.

Background

Stevioside (stevioside) is a natural sweetener extracted from dry leaves of stevia rebaudiana, the sweetness of the stevioside is 200-300 times that of cane sugar, the energy of the stevioside is 1/300 of the cane sugar, the stevioside is non-toxic and free of side effects, has higher stability and safety, is an ideal natural sweetener for replacing cane sugar, and is internationally known as a third sugar source. Research reports that long-term consumption of stevioside can prevent diseases such as hypertension, diabetes, obesity, decayed teeth and the like. Stevioside accounts for 60-70% of the total glycoside, has a taste inferior to rebaudioside A, and has a certain aftertaste. Rebaudioside A is a glycoside component in the stevioside mixture, has the highest sweetness in the stevioside component, has the closest mouthfeel to sucrose, and has higher stability.

At present, enzyme method and fermentation method are reported to modify stevioside, and a method for synthesizing rebaudioside A by high-density fermentation method is reported in patent publication No. CN108486160A, but the components in the fermentation liquor are complex, the yield of stevioside is low, and the separation cost is high; the whole cell catalysis efficiency is low, a large amount of cell thalli are needed for catalysis, the cost is high, and the industrial production is difficult.

Disclosure of Invention

The invention aims to solve the problems of high production cost, complex operation and incapability of being applied to industrial production in the production of catalyzing stevioside into rebaudioside A by the existing enzyme method and fermentation method, and provides a method for preparing rebaudioside A by utilizing bacillus subtilis to ferment and catalyze.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis is characterized by comprising the following steps:

(1) seed preparation: connecting the synthesized glycosyltransferase UGT76G1 gene to a PBR322 plasmid vector, transferring the PBR322 plasmid vector carrying the glycosyltransferase UGT76G1 gene into the competence of the bacillus subtilis, inoculating the bacillus subtilis implanted with the glycosyltransferase UGT76G1 gene into an LB culture medium, controlling the temperature of the LB culture medium to be 30-37 ℃, the rotation speed to be 100-250rpm, and the culture time to be 20-24 h;

(2) seed tank culture: inoculating the bacillus subtilis obtained in the step (1) into a seeding tank filled with an LB (lysogeny broth) culture medium according to the inoculation amount of 1-10% by volume, controlling the rotating speed of the seeding tank to be 200-400 rpm and the aeration ratio to be 0.1-1V/V.min, and culturing at the temperature of 30-37 ℃ for 20-24 hours to obtain a seed culture solution;

(3) fermentation tank production: inoculating the seed culture solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 1-10% of the volume ratio for culturing, controlling the rotation speed of the fermentation tank to be 200-1000 rpm and the aeration ratio to be 0.1-2V/V.min, controlling the pH to be 6-8 at the temperature of 30-37 ℃, culturing for 48-72 hours, and obtaining fermentation liquor after the fermentation is finished;

(4) performing plate-and-frame filter pressing on the fermentation liquor obtained in the step (3) to obtain bacillus subtilis thallus, then performing heavy suspension on the bacillus subtilis thallus for 10-20 min, performing high-pressure homogenization and crushing at 0.1-0.5 Mp to obtain bacillus subtilis thallus crushing liquid, and then performing plate-and-frame filter pressing on the bacillus subtilis thallus crushing liquid to obtain crude enzyme liquid;

(5) the catalytic system is as follows: mixing stevioside, uridine diphosphate glucose, a phosphate buffer solution and a crude enzyme solution according to a mass ratio of 5-10: 1-5: 40-60: 5-20, and then reacting for 12-48 h at 25-40 ℃ to obtain the rebaudioside A.

Further, the preparation method of the fermentation medium in the step (3) is as follows: preparing a fermentation culture medium according to the proportion of 10-40 g of bean cake powder, 20-50 g of bran, 30-60 g of corn flour, 1-10 g of diammonium hydrogen phosphate, 1-10 g of disodium hydrogen phosphate and 0.1-5g of calcium chloride, adjusting the pH to 6-8 by using a sodium hydroxide solution, and fixing the volume to 1000 ml by using deionized water.

Compared with the prior art, the invention has the following advantages:

1. according to the method for preparing rebaudioside A, the crude enzyme liquid is obtained through a fermentation method, so that the yield of rebaudioside A is improved, the operation steps are reduced, the production cost is reduced, the industrial production is facilitated, and the problems of low enzyme activity, complex operation and high production cost of the existing fermentation method are solved;

2. the host bacteria in the invention are bacillus subtilis, so that the food safety performance is high;

3. the bean cake powder, the bran, the corn flour and the like used in the fermentation medium are cheap and widely available raw materials, so that the production cost is greatly reduced;

4. according to the method for catalyzing rebaudioside A, the yield of the rebaudioside A product can reach 85.3 g/L, which is much higher than the yield reported in the prior art, so that the production cost is reduced, and the industrial production is facilitated.

Detailed Description

A method for preparing rebaudioside A by utilizing fermentation catalysis of bacillus subtilis comprises the following specific implementation steps: