A method for extracting theanine through fermentation

Abstract

The invention belongs to the field of theanine production, and discloses a method for extracting theanine by fermentation, wherein the theanine fermentation liquid is filtered by a ceramic membrane, decolorized and separated, and then concentrated and crystallized by an evaporator, and centrifuged and dried to obtain a theanine finished product. The fermentation extraction method of theanine of the invention significantly improves the yield and yield of theanine, and improves product quality.

Description

Technical Field

[0001] The invention belongs to the field of theanine production in the biological fermentation industry, and specifically provides a method for extracting theanine through fermentation. Background Art

[0002] Theanine is one of the unique chemical components of tea trees, also known as N-ethyl-Y-glutamine, a non-protein amino acid. Theanine in nature is L-type, and the pure product is white needle-shaped crystals, which are easily soluble in water, but insoluble in anhydrous ethanol and anhydrous ether. The aqueous solution is slightly acidic, stable in nature, with a caramel aroma and a fresh taste similar to MSG. It has not been found to be toxic in toxicity experiments.

[0003] Theanine affects the metabolism and release of dopamine and other substances in the brain, and related brain diseases may also be regulated or prevented. Theanine can also improve learning ability and memory through neurotransmitters. The weight loss effect of tea is the result of the combined action of multiple ingredients in tea, including theanine. Theanine is particularly effective in reducing cholesterol in the body. In recent years, with the discovery of the physiological functions and medical value of theanine, theanine has been widely used in the fields of medicine, health care, food, beverages and fine chemicals.

[0004] At present, there are four main methods for producing theanine: 1) Tea extraction method: mainly extracting, separating and purifying theanine directly from tea leaves, the extraction steps are complicated, the equipment types are many, and there is a problem of high extraction cost. 2) Chemical synthesis method: This method may leave toxic substances, and the products include D and L-type theanine isomers, which need to be split to obtain L-type products, making the separation and refining operations complicated. 3) Tissue culture method: Tea tree callus has the ability to synthesize theanine, and the accumulation of theanine in the tissue is synchronized with growth. Improve the cultivation method, such as increasing sucrose concentration, reducing light, adding ethylamine, using growth hormones, etc. to increase the accumulation of theanine. The current operating cost of this method is too high and cannot be industrialized. 4) Fermentation method: It refers to the production using microbial enzyme method, using glutamyl transpeptidase strains to ferment to produce the corresponding enzyme, and then reacting with L-glutamine and ethylamine hydrochloride to prepare theanine.

[0005] For example, Chinese patent CN102850235A discloses a purification process for L-theanine. This method uses the fermentation broth of a strain of glutamyl transpeptidase as the starting material, and obtains the theanine product through catalytic reaction, macroporous resin adsorption, ultrafiltration concentration, and freeze-drying. The purity of the product reaches 98% and the yield is more than 70%. However, the operation steps of this method are complicated and the yield is relatively low. Summary of the invention

[0006] To solve the above problems, the present invention provides a method for extracting theanine by fermentation, which has high fermentation efficiency, simple operation, energy saving and emission reduction, and high product yield and purity.

[0007] The present invention is achieved through the following technical solutions.

[0008] A method for extracting theanine by fermentation, characterized in that the method comprises the following steps:

[0009] Step 1) biological fermentation, step 2) ceramic membrane filtration, step 3) primary decolorization, step 4) decolorization by decolorization membrane, step 5) concentration and evaporation, step 6) secondary decolorization, step 7) crystallization and drying.

[0010] Furthermore, the method comprises the following steps:

[0011] Step 1) biological fermentation: inoculate the Escherichia coli CTh2020-05 seed solution at an inoculation rate of 10-15% into a fermentation tank containing a fermentation medium for fermentation for 24 hours, and collect theanine fermentation solution;

[0012] Step 2) Ceramic membrane filtration: filtering the theanine fermentation broth through a ceramic membrane, and collecting the ceramic membrane clear liquid;

[0013] Step 3) Primary decolorization: put the ceramic membrane clear liquid into the decolorization tank, add activated carbon for decolorization, the decolorization time is 50 minutes, and the decolorization temperature is 70°C;

[0014] Step 4) Decolorization with decolorization membrane: Decolorize the clear liquid obtained in step 3) in a decolorization membrane, control the membrane inlet pressure to 0.8 MPa, and the membrane outlet pressure to 0.8 MPa, to obtain a decolorized clear liquid;

[0015] Step 5) Concentration and evaporation: The decolorized clear liquid obtained in step 4) is transported to a four-effect evaporator for evaporation, concentration and crystallization, and crude crystals are collected by centrifugation;

[0016] Step 6) Secondary decolorization: The crude crystals obtained in step 5) are dissolved in water, put into a decolorization tank, and activated carbon is added for secondary decolorization. The decolorization time is 50 minutes and the decolorization temperature is 70°C.

[0017] Step 7) Crystallization and drying: The decolorized clear liquid obtained in step 6) is transported to a second-effect evaporator for evaporation and crystallization, and the crystals are collected by centrifugation and dried.

[0018] Preferably, the components of the fermentation medium are: 60 g / L glucose, 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.

[0019] Preferably, the fermentation conditions are: rotation speed 550 rpm, pH=7.0, pressure 0.05 MPa, temperature 36-40° C., and dissolved oxygen controlled at 20%.

[0020] Preferably, the drying is carried out by fluidized bed drying, and the drying temperature is controlled to be 80° C. and the drying time is 15 min.

[0021] The technical solution of the present invention has the following outstanding advantages and uniqueness.

[0022] 1. The fermented theanine liquid of the present invention is subjected to the processes of ceramic membrane, primary decolorization, decolorization membrane, evaporation concentration crystallization and secondary decolorization, fine centrifugation and the like, and the pigment removal rate reaches 89%, which greatly removes macromolecular impurities and improves the yield and purity of the product.

[0023] 2. The theanine solution extracted by the present invention is concentrated and crystallized, and the theanine pigment removal rate is 70%, and the product purity is about 98.5%.

[0024] 3. The production process of the present invention is fully automated, has low labor intensity, and can effectively save costs. DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1

[0027] Escherichia coli CTh2020-05 was deposited on October 12, 2021 in the General Microbiology Center of China Culture Collection Administration (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.

[0028] 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.

[0029] Example 2

[0030] The fermentation method of L-theanine, the specific operation steps are as follows:

[0031] 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.

[0032] 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.

[0033] Example 3

[0034] The specific steps of the extraction method of L-theanine are as follows:

[0035] 1) The theanine fermentation liquid that has been fermented in the fermentation process is received by the ceramic membrane separation process at room temperature from the fermentation process, and the feed liquid is connected to the activated ceramic membrane. The feeding frequency is controlled to be 50HZ, the production frequency is 50HZ, the slag discharge frequency is 40HZ, the filtration temperature is 60℃, the pressure is 4.2Mpa, the filtration time is 8h, and the pH value is 4.5. The feed liquid flows at a high speed in the membrane tube. Driven by pressure, the clear liquid containing small molecular components permeates outward in a direction perpendicular to it, and the concentrated liquid containing large molecular components is retained by the membrane to obtain the ceramic membrane clear liquid.

[0036] 2) The ceramic membrane clear liquid of step 1 is pumped into the decolorization tank for one decolorization. When the material is 25% filled in the decolorization tank, the stirring device is turned on to complete the self-circulation. 1% by mass of activated carbon is added to decolorize the ceramic membrane clear liquid. The decolorization time is 50 minutes and the decolorization temperature is 70°C.

[0037] 3) The clear liquid decolorized in step 2 is subjected to secondary decolorization in the decolorization membrane of the multi-stage membrane coupling system, and the membrane inlet pressure, membrane outlet pressure and feed liquid flow rate are controlled at 0.8 MPa, 0.8 MPa and 100 ³ / h, to obtain a decolorized clear liquid.

[0038] 4) The decolorized clear liquid obtained in step 3 is transported to a four-effect evaporator for evaporation concentration, crystallization, and crude product centrifugation to collect crude crystals, and the centrifugal speed is controlled at 1100 rpm and the centrifugal time is 80 min.

[0039] 5) The crude crystals obtained in step 4 are dissolved in water, put into a decolorization tank, and 1% by mass of activated carbon is added for secondary decolorization. The decolorization time is 50 minutes and the decolorization temperature is 70°C.

[0040] 6) The decolorized clear liquid obtained in step 5 is transported to a second-effect evaporator for evaporation and crystallization, and the crystals are collected by centrifugation, and the centrifugal speed is controlled at 1100 rpm and the centrifugal time is 80 min. The fluidized bed is dried, and the drying temperature is controlled at 80°C, the drying time is 15 min, and the drying moisture is 0.5%.

[0041] The theanine product obtained through the above steps has a theanine pigment removal rate of 80% and a purity of 99% after testing.

[0042] Example 4

[0043] 1. Effect of different fermentation temperatures on fermentation production of L-theanine.

[0044] The fermentation process was as in Example 2, and the yield of L-theanine in the fermentation broth is shown in Table 1:

[0045] Table 1

[0046] 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

[0047] As shown in Table 1 above, compared with the starting strain XFM-7, the fermentation efficiency of CTh2020-05 for theanine production 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.

[0048] 2. Effect of carbon source in fermentation medium on L-theanine.

[0049] 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.

[0050] Different types of carbon sources:

[0051] 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.

[0052] 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:

[0053] Table 2

[0054] 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

[0055] 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.

[0056] 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 method for extracting theanine by fermentation, characterized in that: The method comprises the following steps: Step 1) Biofermentation: Escherichia coli with a preservation number of CGMCC No. 23583 ( Escherichia coli ) CTh2020-05 seed solution was inoculated into a fermentation tank containing fermentation medium at an inoculation rate of 10-15% and fermented for 24 hours, and theanine fermentation broth was collected; The components of the fermentation medium are: 60 g / L glucose, 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; Step 2) Ceramic membrane filtration: filtering the theanine fermentation broth through a ceramic membrane, and collecting the ceramic membrane clear liquid; Step 3) Primary decolorization: put the ceramic membrane clear liquid into the decolorization tank, add activated carbon for decolorization, the decolorization time is 50 minutes, and the decolorization temperature is 70°C; Step 4) Decolorization with a decolorizing membrane: the clear liquid decolorized in step 3) is decolorized in a decolorizing membrane to obtain a decolorized clear liquid; Step 5) Concentration and evaporation: The decolorized clear liquid obtained in step 4) is transported to a four-effect evaporator for evaporation, concentration and crystallization, and crude crystals are collected by centrifugation; Step 6) Secondary decolorization: The crude crystals obtained in step 5) are dissolved in water, put into a decolorization tank, and activated carbon is added for secondary decolorization. The decolorization time is 50 minutes and the decolorization temperature is 70°C. Step 7) Crystallization and drying: The decolorized clear liquid obtained in step 6) is transported to a second-effect evaporator for evaporation and crystallization, and the crystals are collected by centrifugation and dried.

2. The method according to claim 1, characterized in that The fermentation conditions are: rotation speed 550 rpm, pH=7.0, pressure 0.05 MPa, temperature 36-40° C., and dissolved oxygen controlled at 20%.

3. The method according to claim 1, characterized in that The drying was carried out by fluidized bed drying, the drying temperature was controlled at 80° C., and the drying time was 15 min.