阅读说明：本技术 一种双酶发酵催化生产莱鲍迪苷a的方法 (Method for producing rebaudioside-A through double-enzyme fermentation catalysis ) 是由 孙多龙 祁飞 孙彩军 陆晓雨 于 2020-05-25 设计创作，主要内容包括：本发明涉及一种双菌发酵催化生产莱鲍迪苷A的方法,其特征在于：(1)糖基转移酶UGT76G1基因和蔗糖合酶AtSUSY基因连到pUC18质粒载体上,导转到DH5α大肠杆菌感受态细胞中,接入LB培养基,25～37℃、200～250 rpm培养10～18h；(2)按0.5%～15%接种量接到种子罐,150～400rpm,通气比0.1～1.5V/V.min,25～37℃培养5～16 h；(3)按1%～10%接种量接入发酵罐,100～1000 rpm,通气比0.2～2 V/V.min,pH 6.6～8.5,25～37℃培养20～40h；(4)OD<Sub>600</Sub>值达20～100时加诱导剂,浓度0.1～1.5 mmol/L；(5)发酵液压滤、重悬、破碎、压滤得粗酶液；(6)甜菊糖苷、尿苷二磷酸、磷酸缓冲液、粗酶液按质量比40-100：1-4：400-600：50-100混合,25-40℃反应24-48 h即可。本发明优点：一次发酵获得两种粗酶液,操作步骤少；酶活性高,生产成本低。(The invention relates to a method for producing rebaudioside A through double-bacterium fermentation catalysis, which is characterized by comprising the steps of (1) connecting a glycosyltransferase UGT76G1 gene and a sucrose synthase AtSUSY gene to a pUC18 plasmid vector, transferring the genes to a DH5 α escherichia coli competent cell, inoculating an LB culture medium, culturing for 10-18 h at 25-37 ℃ and 200-250 rpm, (2) inoculating the genes to a seeding tank according to 0.5-15% of inoculum size, culturing for 5-16 h at 25-37 ℃, inoculating the genes to a fermentation tank according to 1-10% of inoculum size, culturing for 100-1000 rpm and 0.2-2V/V.min at 25-37 ℃, and culturing for 20-40 h at pH 6.6-8.5 and 25-37 ℃, (4) inoculating OD (OD) 600 Adding an inducer when the value reaches 20-100, wherein the concentration is 0.1-1.5 mmol/L; (5) filter pressing, resuspending, crushing and filter pressing the fermentation liquor to obtain a crude enzyme solution; (6) stevioside, uridine diphosphate, phosphate buffer solution and crude enzyme solution by massThe ratio of 40-100: 1-4: 400-600: mixing 50-100 parts of the raw materials, and reacting at 25-40 ℃ for 24-48 h. The invention has the advantages that: two crude enzyme liquids are obtained by one-time fermentation, and the operation steps are few; high enzyme activity and low production cost.)

1. The method for producing rebaudioside A through double-bacterium fermentation catalysis is characterized by comprising the following steps:

(1) seed preparation: the glycosyltransferase UGT76G1 gene and the sucrose synthase AtSUSY gene are connected to a pUC18 plasmid vector, transferred to DH5 alpha escherichia coli competent cells, inoculated to an LB culture medium, and cultured for 10-18 h at 25-37 ℃ and 200-250 rpm;

(2) seed tank culture: inoculating the escherichia coli obtained in the step (1) into a seeding tank filled with an LB (lysogeny broth) culture medium according to an inoculation amount of 0.5-15% by volume, controlling the rotation speed of the seeding tank to be 150-400 rpm and the aeration ratio to be 0.1-1.5V/V.min, and culturing at the temperature of 25-37 ℃ for 5-16 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 100-1000 rpm, the aeration ratio to be 0.2-2V/V.min, controlling the pH to be 6.6-8.5 at the temperature of 25-37 ℃, culturing for 20-40 hours, and obtaining fermentation liquor after the fermentation is finished;

(4) and (3) inducing thalli: when the thallus concentration OD is in the production process of the fermentation tank₆₀₀When the value reaches 20-100, adding an inducer isopropyl- β -D-thiogalactoside, and controlling the concentration of the inducer to be 0.1-1.5 mmol/L;

(5) carrying out filter pressing on the fermentation liquor obtained in the step (4) to obtain escherichia coli thalli, carrying out heavy suspension for 10-20 min, carrying out high-pressure homogenization crushing at 0.1-0.5 Mp to obtain an escherichia coli crushing liquid, and then carrying out filter pressing on the escherichia coli crushing liquid to obtain a crude enzyme liquid;

(6) the catalytic system is as follows: mixing stevioside, uridine diphosphate, a phosphate buffer solution and a crude enzyme solution according to a mass ratio of 40-100: 1-4: 400-600: 50-100, and then reacting for 24-48 h at 25-40 ℃ to obtain the rebaudioside A.

2. The method for catalytic production of rebaudioside a by two-strain fermentation according to claim 1, wherein: the LB culture medium in the step (1) is preferably at the temperature of 30-36 ℃, the rotating speed of 220-235 rpm and the culture time of 13-15 h.

3. The method for catalytic production of rebaudioside a by two-strain fermentation according to claim 1, wherein: in the step (2), the seeding tank is preferably set to rotate at a speed of 220-350 rpm and a ventilation ratio of 0.5-1V/V.min, and is cultured for 8-10 h at a temperature of 30-36 ℃.

4. The method for catalytic production of rebaudioside a by two-strain fermentation according to claim 1, wherein: in the step (3), the rotation speed of the fermentation tank is preferably 450-780 rpm, the aeration ratio is 0.5-1.5V/V.min, the pH is controlled to be 7-8 at the temperature of 30-36 ℃, and the fermentation is cultured for 25-35 h.

5. The method for catalytic production of rebaudioside a by two-strain fermentation according to claim 1, wherein: the concentration of the isopropyl-beta-D-thiogalactoside in the step (4) is preferably 0.5-1 mmol/L.

6. The method for catalytic production of rebaudioside A by two-strain fermentation according to any one of claims 1-5, wherein: 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 2-10 g of monopotassium phosphate, 1-10 g of diammonium phosphate, 0.5-9 g of citric acid, 2-20 g of yeast powder, 5-50 g of glucose and 1-20 g of microelement mother liquor, adjusting the pH value to 6.6-8.5 by using a sodium hydroxide solution, and fixing the volume to 1000 ml by using deionized water.

7. The method for catalytic production of rebaudioside A by two-strain fermentation according to claim 6, wherein: the preparation method of the microelement mother liquor comprises the following steps: according to the proportion of 0.84g of EDTA-2Na, 0.25g of cobalt chloride hexahydrate, 1.5 g of manganese chloride tetrahydrate, 0.22 g of copper sulfate pentahydrate, 0.3 g of boric acid, 0.182 g of ammonium molybdate, 1.7 g of zinc sulfate, 13.75 g of ferric ammonium citrate and 5 drops of concentrated sulfuric acid, the mixture is prepared, and deionized water is used for keeping the volume to 1000 ml.

Technical Field

The invention belongs to the technical field of microbial fermentation enzyme preparations, and relates to a method for producing rebaudioside A through double-bacterium fermentation catalysis.

Background

Stevioside (stevioside) is a natural sweetener extracted from dried leaves of stevia rebaudiana, the sweetness of the stevioside is 200-300 times that of cane sugar, menthol taste is provided to a certain degree, the afterbitterness is obvious, and the overall sweet taste is not good. The stevioside component in the stevia accounts for 60 to 70 percent of the total amount of the glucoside, and is the main source of bitter taste; rebaudioside A accounts for about 15% -20% of the total glycoside, tastes better than stevioside, is closer to sucrose, and has higher stability and safety.

At present, the research on a stevioside microbial fermentation method is less, the cost for extracting and separating stevioside and rebaudioside A from plants is higher, and the development of the stevioside industry is slower. With the improvement of the requirements on the taste of the sweetener, the demand on high-end products such as rebaudioside A and the like is more and more increased. Stevia rebaudiana glycosyltransferase can convert stevioside into rebaudioside A, uridine diphosphate glucose needs to be added as a raw material in the process of catalyzing a substrate by the existing enzyme catalysis technology, the uridine diphosphate glucose is high in market price, the cost is high, and the industrial production is limited to a certain extent.

Disclosure of Invention

The invention aims to solve the problems of high production cost and low enzyme activity of the existing rebaudioside-A, and provides a method for producing rebaudioside-A through double-bacterium fermentation catalysis.

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

the method for producing rebaudioside A through double-bacterium fermentation catalysis is characterized by comprising the following steps:

(1) seed preparation: the glycosyltransferase UGT76G1 gene and the sucrose synthase AtSUSY gene are connected to a pUC18 plasmid vector, transferred to DH5 alpha escherichia coli competent cells, inoculated to an LB culture medium, and cultured for 10-18 h at 25-37 ℃ and 200-250 rpm;

(2) seed tank culture: inoculating the escherichia coli obtained in the step (1) into a seeding tank filled with an LB (lysogeny broth) culture medium according to an inoculation amount of 0.5-15% by volume, controlling the rotation speed of the seeding tank to be 150-400 rpm and the aeration ratio to be 0.1-1.5V/V.min, and culturing at the temperature of 25-37 ℃ for 5-16 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 100-1000 rpm, the aeration ratio to be 0.2-2V/V.min, controlling the pH to be 6.6-8.5 at the temperature of 25-37 ℃, culturing for 20-40 hours, and obtaining fermentation liquor after the fermentation is finished;

(4) and (3) inducing thalli: when the thallus concentration OD is in the production process of the fermentation tank₆₀₀When the value reaches 20-100, adding an inducer isopropyl- β -D-thiogalactoside, and controlling the concentration of the inducer to be 0.1-1.5 mmol/L;

(5) carrying out plate-and-frame filter pressing on the fermentation liquor obtained in the step (4) to obtain escherichia coli thalli, then carrying out heavy suspension on the escherichia coli for 10-20 min, carrying out high-pressure homogenization crushing at 0.1-0.5 Mp to obtain an escherichia coli crushing liquid, and then carrying out plate-and-frame filter pressing on the escherichia coli crushing liquid to obtain a crude enzyme liquid;

(6) the catalytic system is as follows: mixing stevioside, uridine diphosphate, a phosphate buffer solution and a crude enzyme solution according to a mass ratio of 40-100: 1-4: 400-600: 50-100, and then reacting for 24-48 h at 25-40 ℃ to obtain the rebaudioside A.

Further, the LB culture medium in the step (1) is preferably at the temperature of 30-36 ℃, the rotating speed of 220-235 rpm, and the culture time of 13-15 h.

Further, in the step (2), the seeding tank is preferably cultured for 8-10 hours at the temperature of 30-36 ℃ at the rotating speed of 220-350 rpm and the aeration ratio of 0.5-1V/V.min.

Further, in the step (3), the rotation speed of the fermentation tank is preferably 450-780 rpm, the aeration ratio is 0.5-1.5V/V.min, the pH is controlled to be 7-8 at the temperature of 30-36 ℃, and the fermentation is cultured for 25-35 hours.

Further, the concentration of isopropyl-beta-D-thiogalactoside in the step (4) is preferably 0.5-1 mmol/L.

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 2-10 g of monopotassium phosphate, 1-10 g of diammonium phosphate, 0.5-9 g of citric acid, 2-20 g of yeast powder, 5-50 g of glucose and 1-20 g of microelement mother liquor, adjusting the pH value to 6.6-8.5 by using a sodium hydroxide solution, and fixing the volume to 1000 ml by using deionized water.

Further, the preparation method of the microelement mother liquor comprises the following steps: according to the proportion of 0.84g of EDTA-2Na, 0.25g of cobalt chloride hexahydrate, 1.5 g of manganese chloride tetrahydrate, 0.22 g of copper sulfate pentahydrate, 0.3 g of boric acid, 0.182 g of ammonium molybdate, 1.7 g of zinc sulfate, 13.75 g of ferric ammonium citrate and 5 drops of concentrated sulfuric acid, the mixture is prepared, and deionized water is used for keeping the volume to 1000 ml.

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

1. the preparation method of the invention obtains two crude enzyme solutions through one-time fermentation, thereby reducing the operation steps;

2. the crude enzyme solution is obtained by a double-bacterium fermentation method, the activity of the enzyme is high and can reach 32932.8-35178.2U/ml, the production cost is low, and the method has important market value and application value.

Detailed Description

A method for producing rebaudioside A through double-bacterium fermentation catalysis comprises the following specific implementation steps: