A method for producing L-theanine by fermentation

Abstract

The present invention belongs to the technical field of microbial fermentation in the light industry, and discloses a method for producing L-theanine by fermentation, which mainly includes: accessing a microbial strain to a seed culture medium for cultivation, and obtaining a seed solution by controlling the culture conditions; inoculating the seed solution into a fermentation tank by a shake flask inoculation method for fermentation, and adding ethylamine in batches and adding sugar during the fermentation process to obtain a high-acid fermentation liquid. The present invention has high fermentation efficiency, low investment cost, and accurately controls the fermentation process to avoid fermentation out of control and cost increase, while reducing labor intensity, and the entire production process is simple and easy to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation in the light industry, and specifically relates to a method for producing L-theanine by fermentation. Background Art

[0002] L-Theanine, also known as glutamic acid γ-ethylamide, is a unique free amino acid in tea, accounting for more than 50% of the total free amino acids in tea. As the main component of the sweet taste of tea, theanine determines the quality of tea to a certain extent. Theanine has many beneficial effects on human health, including promoting the learning and memory function of the brain, preventing cardiovascular diseases, inhibiting fat accumulation, and acting as a tranquilizer. In the food industry, it can be used as a quality improver for tea beverages, an additive to improve food flavor, and an additive for functional foods. It can inhibit bitterness and spiciness in food and reduce astringency. Therefore, it has been widely used in the pharmaceutical and food industries and has become a highly anticipated compound in the research of tea health ingredients.

[0003] At present, theanine is mainly extracted from tea leaves or tea waste, which generally has problems such as cumbersome extraction steps, high use of organic solvents, low extraction yield and product purity, and cannot meet the needs of large-scale production. The microbial fermentation method uses cheap carbon and nitrogen sources as substrates, and synthesizes theanine directly through glucose fermentation through microbial metabolism and its own ability to produce theanine. The fermentation method has relatively low raw material source costs, mild fermentation reaction conditions, relatively stable production process, high controllability, and is easy to scale up production, making it the main method for producing theanine at present.

[0004] However, the current technical conditions for theanine production by fermentation in my country are not mature, and the acid production capacity and conversion rate of the strains are not high, resulting in high costs for theanine production by fermentation. Therefore, new technological breakthroughs and production processes are urgently needed to improve the acid production capacity and conversion rate of L-theanine strains while reducing production costs. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a method for producing L-theanine by fermentation, which has high fermentation efficiency, low investment cost, and accurately controls the fermentation process to avoid fermentation out of control and cost increase, while reducing labor intensity, and the entire production process is simple and easy to operate.

[0006] The present invention is achieved through the following technical solutions:

[0007] On the one hand, the present invention protects an Escherichia coli CTh2020-05, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with the deposit number CGMCC No.23583.

[0008] In another aspect, the present invention protects the use of Escherichia coli CTh2020-05 in the fermentation production of L-theanine and its derivatives.

[0009] In another aspect, the present invention relates to a method for producing L-theanine by fermentation, characterized in that the method comprises:

[0010] Escherichia coli CTh2020-05 was inoculated into the fermentation medium for fermentation, and the fermentation liquid was collected.

[0011] Specifically,

[0012] The fermentation medium comprises: 60 g / L carbon source, 20 g / L corn steep liquor, 1 g / L magnesium sulfate heptahydrate, 2 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 50 mg / L ferrous sulfate heptahydrate, 40 mg / L magnesium sulfate monohydrate, 10 mg / L zinc sulfate, and 5 mg / L cobalt nitrate hexahydrate.

[0013] Preferably,

[0014] The carbon source is: glucose, sucrose, lactose, glycerol or a combination of any two or more thereof.

[0015] More preferably,

[0016] The carbon source is a combination of glucose and glycerol.

[0017] Most preferably, the carbon source is a mixture of glucose and glycerol in an equal mass ratio.

[0018] Furthermore, the method comprises:

[0019] The Escherichia coli CTh2020-05 seed liquid was diluted 50 times and inoculation was started when OD=0.4. The inoculation amount was 12.5% ​​and then inoculated into a fermentation tank containing a fermentation medium for fermentation. When OD=0.3 was measured, ethylamine was added, and the initial flow rate was 1mL / h per liter of liquid, which was increased to 2mL / h after 4h. The sugar content was controlled to be not less than 1% by adding a glucose solution with a mass concentration of 70%. The stirring speed during the fermentation process was controlled to be 550rpm, pH=7.0, pressure 0.05MPa, temperature 36-40℃, dissolved oxygen control 20%, the total fermentation time was 24-36h, and the fermentation liquid was collected.

[0020] The technical solution of the present invention has the following outstanding advantages and uniqueness:

[0021] The strain of the invention has excellent performance, can produce a high amount of L-theanine, and has high industrial application value.

[0022] The present invention selects different carbon source combinations according to the physicochemical properties of the strain, selects the most suitable carbon source, improves fermentation efficiency, and reduces fermentation cost.

[0023] The present invention also discovered that ethylamine can stimulate the strain to produce high theanine by adding an auxiliary carbon source, which can be increased by 20% at most compared with not adding ethylamine. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solution in the present application, the technical solution will be fully described below in conjunction with the specific embodiments of the present application.

[0025] Example 1

[0026] Escherichia coli CTh2020-05 was deposited on October 12, 2021 in the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 23583.

[0027] The samples were collected from the L-theanine fermentation waste residue in the production workshop, treated with hot water at 48°C for 3 minutes, heated to 50°C for another 3 minutes, and then coated with LB solid culture medium. The plaques were collected at 38°C for 24 hours, and the strain XFM-7 with the highest expression of L-theanine was selected; the microbial strain identification Biology instrument was used for identification, and the identification result was Escherichia coli. The atmospheric pressure room temperature plasma technology was used for mutagenesis, and the strain with the highest expression of L-theanine was screened, which was Escherichia coli (Escherichiacoli) CTh2020-05.

[0028] Example 2

[0029] A method for producing L-theanine by fermentation, the specific operating steps are as follows:

[0030] 1) Inoculate the microbial strains into a seed culture medium containing 30 g / L glucose, 0.5 g / L magnesium sulfate heptahydrate, 6 g / L yeast powder, 2 g / L potassium dihydrogen phosphate, 2 g / L citric acid, 2 g / L peptone, 10 mg / L ferrous sulfate heptahydrate, 10 mg / L magnesium sulfate monohydrate, 6.5 mg / L zinc sulfate, and 5 mg / L cobalt nitrate hexahydrate, and sterilize the culture medium by adjusting the pH to 4.5 with ammonia water, and the temperature to 121-123°C for 25 min. After sterilization, cool the culture medium to 38°C with condensed water, control the pH to 6.8-7.2, the pressure to 0.05 MPa, and the dissolved oxygen to 30%-50%, and culture the culture medium for 8 hours to obtain the seed solution.

[0031] 2) Dilute the seed solution obtained in step 1) 50 times and start transplanting when OD=0.4, with an inoculation amount of 12.5%, and inoculate it into a fermentation tank containing fermentation medium (fermentation medium: glucose 60g / L, corn steep liquor 20g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 2g / L, potassium hydrogen phosphate 2g / L, ferrous sulfate heptahydrate 50mg / L, magnesium sulfate monohydrate 40mg / L, zinc sulfate 10mg / L, cobalt nitrate hexahydrate 5mg / L) for fermentation. When OD=0.3, ethylamine was added, and the initial flow rate was 1mL / hL, which was increased to 2mL / hL after 4h. The sugar content was controlled to be not less than 1% (mass percentage) by adding a glucose solution with a mass concentration of 70%. Control the stirring speed during the fermentation process: 550rpm, pH=7.0, pressure 0.05MPa, temperature 36-40℃, dissolved oxygen control 20%, fermentation 24h, and collect the fermentation liquid.

[0032] Example 3

[0033] Effects of different fermentation temperatures on the production of L-theanine.

[0034] The fermentation process was as in Example 2, and the yield of L-theanine in the fermentation broth is shown in Table 1:

[0035] Table 1

[0036] strain 36℃ 37℃ 38℃ 39℃ 40℃ XFM-7 31.3 35.4 35.8 33.1 27.6 CTh2020-05 46.8 48.9 53.2 54.0 47.9

[0037] As shown in Table 1 above, compared with the starting strain XFM-7, the fermentation efficiency of theanine production by CTh2020-05 was increased by about 50%, with excellent performance; and the optimal fermentation temperature was higher than that of conventional fermentation strains, which is beneficial to reduce the possibility of contamination by foreign bacteria.

[0038] Example 4

[0039] Effect of carbon source of fermentation medium on L-theanine.

[0040] Basic fermentation medium: carbon source 60g / L, corn steep liquor 20g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 2g / L, dipotassium hydrogen phosphate 2g / L, ferrous sulfate heptahydrate 50mg / L, magnesium sulfate monohydrate 40mg / L, zinc sulfate 10mg / L, cobalt nitrate hexahydrate 5mg / L.

[0041] Effects of different carbon source types on L-theanine.

[0042] Group 1: glucose; Group 2: glucose: sucrose = 1:1 mass ratio; Group 3: glucose: glycerol = 1:1 mass ratio; Group 4: glucose: lactose = 1:1 mass ratio; Group 5: sucrose; Group 6: glycerol.

[0043] The fermentation process was as in Example 2, the fermentation temperature was 39° C., and the yield of L-theanine in the fermentation broth was shown in Table 2:

[0044] Table 2

[0045] strain Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 XFM-7 33.1 31.4 27.9 29.8 27.6 32.0 CTh2020-05 54.0 52.5 57.8 53.6 45.3 51.2

[0046] As shown in Table 2 above, in Group 3, where glucose + glycerol is used as a composite carbon source, CTh2020-05 has the highest efficiency in fermenting theanine, which is about 7% higher than Group 1, where glucose is used as a carbon source. This trend is not reflected in XFM-7 and existing E. coli for L-theanine production. Glucose is the best fermentation carbon source for E. coli such as XFM-7 strains. Considering that glycerol is in large excess in the field of bioenergy and its cost is relatively low, the CTh2020-05 strain uses part of the glycerol as a carbon source, which not only improves the fermentation efficiency, but also solves the problem of excess glycerol, and has a high industrial application value.

[0047] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A type of Escherichia coli ( Escherichia coli )CTh2020-05, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.23583.

2. The Escherichia coli according to claim 1 ( Escherichia coli )Application of CTh2020-05 in fermentation production of L-theanine.

3. A method for producing L-theanine by fermentation, characterized in that: The method comprises: The Escherichia coli ( Escherichia coli ) CTh2020-05 was connected to the fermentation medium for fermentation, and the fermentation liquid was collected; The fermentation medium comprises: 60 g / L carbon source, 20 g / L corn steep liquor, 1 g / L magnesium sulfate heptahydrate, 2 g / L potassium dihydrogen phosphate, 2 g / L dipotassium hydrogen phosphate, 50 mg / L ferrous sulfate heptahydrate, 40 mg / L magnesium sulfate monohydrate, 10 mg / L zinc sulfate, and 5 mg / L cobalt nitrate hexahydrate; The carbon source is a mixture of glucose and glycerol in equal mass ratio; Escherichia coli ( Escherichia coli )CTh2020-05 seed liquid was diluted, and inoculation was started when OD=0.

4. The inoculation amount was 12.5% ​​and then accessed into a fermentation tank containing fermentation medium for fermentation. When OD=0.3 was measured, ethylamine was added, and the initial flow rate was 1mL / h per liter of liquid, which was increased to 2mL / h after 4h. The sugar content was controlled to be not less than 1% by adding 70% mass concentration of glucose solution. During the fermentation process, the stirring speed was controlled at 550rpm, pH=7.0, pressure 0.05MPa, temperature 36-40℃, dissolved oxygen was controlled at 20%, the total fermentation time was 24-36h, and the fermentation liquid was collected.