CN111073830B - Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine - Google Patents
Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine Download PDFInfo
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Abstract
The invention relates to lactobacillus casei for high yield of gamma-glutamyltranspeptidaseLactobacillus casei TH139, preserved in China general microbiological culture Collection center with preservation number CGMCC No. 18686; the strain is used for producing gamma-glutamyl transpeptidase by fermentation at 37 ℃ and pH7.0; after centrifugal collection of the somatic cells, biologically converting L-glutamine and ethylamine at 40 ℃ and pH9.0 to obtain L-theanine; and (3) carrying out membrane separation, ion exchange resin separation and concentration crystallization on the reaction solution to obtain a qualified L-theanine finished product. The method for producing L-theanine by using lactobacillus casei has the advantages of food safety, environmental protection, simple and convenient operation and low cost, and has good industrial application value.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in L-theanine production.
Background
L-theanine is a specific amino acid in tea leaves and accounts for more than 50% of total free amino acids. L-theanine has many beneficial physiological effects, and can promote brain relaxation, improve attention and promote learning ability. In the aspect of medical care, the L-theanine has the effects of reducing blood pressure, preventing vascular diseases, relieving pressure, protecting nerves, losing weight and reducing blood fat, improving the capability of an immune system and the like. In food, L-theanine has been classified as a safe food and drug additive by FDA for improving food quality and enhancing food flavor.
The existing main preparation routes of L-theanine comprise an extraction separation method, a chemical synthesis method and an enzymatic synthesis method. The extraction method has certain defects, and the steps for extracting theanine from plants are complicated, the period is long, the cost is high, the extraction amount is small, and the requirement of large-scale production cannot be met. The chemical synthesis method is simple and low in cost, is one of the main ways of industrial production at present, but the synthesized theanine is easy to exist in racemate, difficult to extract, large in pollution and energy consumption and difficult to apply in the field of food. Recently, the production of L-theanine by enzyme catalysis has attracted much attention from researchers, and there are currently mainly 4 different enzymes of bacterial origin that can serve as ideal biocatalysts and show different potentials. These 4 bacterially derived enzymes include L-glutamine synthetase, gamma-glutamyl-methacrylamide synthetase, gamma-glutamyl transpeptidase and L-glutaminase. Wherein the gamma-glutamyl transpeptidase is a catalytic enzyme capable of catalyzing the synthesis of L-theanine from a plurality of gamma-glutamyl donors without consuming ATP. However, the prior art strains for producing gamma-glutamyltranspeptidase mainly comprise bacillus subtilis and recombinant escherichia coli (ZL 200810021187.8, ZL200810021188.2, ZL201310302347.7 and ZL 200910027192.4), have the defects of low expression efficiency and low enzyme activity, and are limited in application in food.
Lactobacillus casei is one of the most deeply studied and widely used probiotics at present, and is considered as the three most classical probiotics at present together with lactobacillus acidophilus and bifidobacterium. The invention takes 200 strains of lactobacillus casei separated from fermented milk products in various regions of Henan province as research objects, screens out high-yield and high-activity gamma-glutamyl transpeptidase strains, and uses the strains in the production of food-grade L-theanine to meet the requirements of products in the fields of food and medicine.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a lactobacillus casei with high yield of gamma-glutamyltranspeptidase, and aims to provide a lactobacillus casei using the lactobacillus caseiLactobacillus casei A method for producing L-theanine by TH 139. The L-theanine is produced by the lactobacillus casei with high yield of the gamma-glutamyltranspeptidase, and has the advantages of high product yield, high purity and low costLow cost and the like.
In order to achieve the purpose, the invention adopts the specific scheme that:
lactobacillus casei with high yield of gamma-glutamyl transpeptidaseLactobacillus casei TH139, classification name of Lactobacillus casei: (Lactobacillus casei),Has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang district; the preservation date is 2019, 10 months and 15 days; the preservation number is CGMCC No. 18686.
The method for producing L-theanine by using the lactobacillus casei comprises the following steps:
(1) cell culture and collection: inoculating 300 mu L of lactobacillus casei bacterial liquid into 30 mL of LBS culture medium, culturing for 8-10 h under the conditions of pH7.0, 37 ℃ and 200 r/min, inoculating 30 mL of seed liquid into 3L of fermentation culture medium, culturing for 14-16 h under the conditions of pH7.0, 37 ℃ and 200 r/min, and freezing and centrifuging to obtain somatic cells;
(2) preparing L-theanine by biotransformation: putting 40-60 g of bacterial cells into 1L of Tris-HCl buffer solution containing 50-65 g/L L-glutamine, 15-20 g/L ethylamine and pH of 9.0, reacting for 8-12 h at 40 ℃ at 100 r/min, and centrifuging to obtain supernatant;
(3) separating and extracting L-theanine: enabling reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein; allowing the membrane filtrate to pass through cation exchange resin to adsorb L-theanine therein, and eluting with 1% ammonia water; decolorizing the eluent with medicinal active carbon to light transmittance of above 90%, concentrating and crystallizing to obtain L-theanine product.
As a further optimization of the above scheme, in the first step, the fermentation medium comprises the following components in percentage by weight: 18-20 g/L of glucose, 8-10 g/L of peptone, 5-7 g/L of yeast powder, 2-4 g/L of ammonium citrate, 1-2 g/L of sodium acetate, 2-4 g/L of dipotassium hydrogen phosphate, 0.5-1 g/L of magnesium sulfate and 0.2-0.5 g/L of manganese sulfate.
The invention also claims the application of the lactobacillus casei in the production of L-theanine.
Has the advantages that:
the invention separates and screens a lactobacillus casei strain with high yield of gamma-glutamyltranspeptidase from fermented dairy productsLactobacillus casei TH139, and is applied to the production of L-theanine. The qualified L-theanine product is obtained through the operation procedures of culture of enzyme-producing strains, cell collection, biotransformation, separation and extraction and the like.
Preservation of biological materials
Lactobacillus caseiLactobacillus casei TH139, which is classified and named as Lactobacillus casei: (Lactobacillus casei)The preservation date is No. 10 and 15 in 2019, and the preservation unit is abbreviated as: china general microbiological culture Collection center (CGMCC), the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation number is CGMCC No. 18686.
Drawings
FIG. 1 is a high performance liquid chromatogram of an L-theanine standard;
FIG. 2 is a high performance liquid chromatogram of the prepared L-theanine.
Detailed Description
Lactobacillus casei with high yield of gamma-glutamyl transpeptidaseLactobacillus casei TH139, China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 18686.
The screening method comprises the following steps:
(1) the primary screening method comprises the following steps: a single colony of 200 strains of Lactobacillus casei isolated from fermented milk products in various regions of Henan province was inoculated into a plate screening medium, subjected to static culture at 37 ℃ for 1-2 days, and the size of a transparent circle was observed. The plate screening culture medium contains 10-15 g/L of L-glutamine, 5-10 g/L of ethylamine, 1-5 mL/L of 0.1% phenolphthalein indicator and 1.5-2 g/L of agar.
(2) The secondary screening method comprises the following steps: selecting a strain with a larger transparent circle, inoculating the strain into LBS culture medium, culturing for 12 h at 37 ℃ and pH7.0, and centrifugally collecting bacterial cells to determine the activity of the gamma-glutamyl transpeptidase. And selecting the strain with the maximum enzyme activity for glycerol tube freezing preservation.
Use lactobacillus caseiLactobacillus casei The method for producing the L-theanine by the TH139 comprises the following steps:
(1) cell culture and collection: taking 300 mu L of lactobacillus caseiLactobacillus casei Inoculating TH139 bacterial liquid into 30 mL LBS culture medium, culturing for 8-10 h at pH7.0, 37 deg.C and 200 r/min, inoculating 30 mL seed liquid into 3L fermentation culture medium, culturing for 14-16 h at pH7.0, 37 deg.C and 200 r/min, and freeze centrifuging to obtain thallus cells.
The fermentation medium comprises the following components: 18-20 g/L of glucose, 8-10 g/L of peptone, 5-7 g/L of yeast powder, 2-4 g/L of ammonium citrate, 1-2 g/L of sodium acetate, 2-4 g/L of dipotassium hydrogen phosphate, 0.5-1 g/L of magnesium sulfate and 0.2-0.5 g/L of manganese sulfate.
(2) Preparing L-theanine by biotransformation: 40-60 g of bacterial cells (wet weight) are put into 1L of Tris-HCl buffer solution containing 50-65 g/L L-glutamine, 15-20 g/L ethylamine and pH9.0, and after reaction for 8-12 h under the conditions of 40 ℃ and 100 r/min, the supernatant is left after centrifugation.
(3) Separating and extracting L-theanine: enabling reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein; allowing the membrane filtrate to pass through 001 × 7 cation exchange resin to adsorb L-theanine therein, and eluting with 1% ammonia water; decolorizing the eluent with medicinal active carbon to light transmittance of above 90%, concentrating and crystallizing to obtain L-theanine product.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
Lactobacillus casei used in the inventionLactobacillus casei TH139 is a novel strain discovered by the present inventors and hasA naturally occurring fermentation strain having the ability to produce gamma-glutamyltranspeptidase.
The gamma-glutamyl transpeptidase adopted by the invention is derived from lactobacillus caseiLactobacillus casei TH139, which catalyzes glutamyl transfer reaction under alkaline conditions, for L-theanine biosynthesis.
Example 1: screening of lactobacillus casei with high yield of gamma-glutamyl transpeptidase
(1) The primary screening method comprises the following steps: a single colony of 200 strains of Lactobacillus casei isolated from fermented milk products in various regions of Henan province was inoculated into a plate screening medium, subjected to static culture at 37 ℃ for 1-2 days, and the size of a transparent circle was observed. The plate screening culture medium contains 12 g/L of L-glutamine, 8 g/L of ethylamine, 8 mL/L of 0.1% phenolphthalein indicator and 1.8 g/L of agar.
(2) The secondary screening method comprises the following steps: selecting a strain with a larger transparent circle, inoculating the strain into LBS culture medium, culturing for 12 h at 37 ℃ and pH7.0, and centrifugally collecting bacterial cells to determine the activity of the gamma-glutamyl transpeptidase. The amount of cells of the cells (wet weight) which catalyzed the production of 1. mu. mol L-theanine per minute was defined as one enzyme activity. And selecting the strain with the maximum enzyme activity for glycerol tube freezing preservation.
Example 2: biotransformation for preparing L-theanine
(1) Cell culture and collection: taking 300 mu L of lactobacillus caseiLactobacillus casei The TH139 bacterial liquid is inoculated into 30 mL LBS culture medium, cultured for 8 h under the conditions of pH7.0, 37 ℃ and 200 r/min, then 30 mL seed liquid is inoculated into 3L fermentation culture medium, cultured for 14 h under the conditions of pH7.0, 37 ℃ and 200 r/min, and frozen and centrifuged to obtain the somatic cells.
The fermentation medium comprises the following components: 18 g/L of glucose, 8 g/L of peptone, 5 g/L of yeast powder, 2 g/L of ammonium citrate, 1 g/L of sodium acetate, 2 g/L of dipotassium hydrogen phosphate, 0.5 g/L of magnesium sulfate and 0.2 g/L of manganese sulfate.
(2) Preparing L-theanine by biotransformation: 40 g of the bacterial cells (wet weight) were put in 1L of Tris-HCl buffer solution containing 50 g/L L-glutamine and 15 g/L ethylamine at pH9.0, reacted at 40 ℃ for 8 hours at 100 r/min, and then centrifuged to leave a supernatant. The concentration of L-theanine in the supernatant was 54.5 g/L, and the molar conversion of the substrate L-glutamine was 91.5%.
(3) Separating and extracting L-theanine: enabling 1L of reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein, wherein the filtration temperature is normal temperature, the filtration pressure is 0.2 MPa, and the filtration speed is 10 mL/min; allowing the membrane filtrate to pass through 001 × 7 cation exchange resin to adsorb L-theanine therein, at normal temperature and loading rate of 1 mL/min, eluting with 1% ammonia water at normal temperature and elution rate of 1.5 mL/min; decolorizing the eluate with medicinal active carbon until light transmittance is above 90%, decolorizing temperature is 60 deg.C, active carbon dosage is 5 g/L, and decolorizing time is 30 min; concentrating and crystallizing the decolorized solution to obtain a finished product of 44.8 g L-theanine. The purity of the finished product is 98.5 percent by using high performance liquid chromatography.
Example 3: biotransformation for preparing L-theanine
(1) Cell culture and collection: taking 300 mu L of lactobacillus caseiLactobacillus casei The TH139 bacterial liquid is inoculated into 30 mL LBS culture medium, cultured for 9 h under the conditions of pH7.0, 37 ℃ and 200 r/min, then 30 mL seed liquid is inoculated into 3L fermentation culture medium, cultured for 15 h under the conditions of pH7.0, 37 ℃ and 200 r/min, and frozen and centrifuged to obtain the somatic cells.
The fermentation medium comprises the following components: 19 g/L of glucose, 9 g/L of peptone, 6 g/L of yeast powder, 3 g/L of ammonium citrate, 1.5 g/L of sodium acetate, 2.5 g/L of dipotassium phosphate, 0.8 g/L of magnesium sulfate and 0.3 g/L of manganese sulfate.
(2) Preparing L-theanine by biotransformation: 50 g of the bacterial cells (wet weight) were put in 1L of Tris-HCl buffer solution containing 60 g/L L-glutamine and 18 g/L ethylamine at pH9.0, reacted at 40 ℃ for 10 hours at 100 r/min, and then centrifuged to leave a supernatant. The concentration of L-theanine in the supernatant was 64.8 g/L, and the molar conversion of the substrate L-glutamine was 90.7%.
(3) Separating and extracting L-theanine: enabling 1L of reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein, wherein the filtration temperature is normal temperature, the filtration pressure is 0.2 MPa, and the filtration speed is 10 mL/min; allowing the membrane filtrate to pass through 001 × 7 cation exchange resin to adsorb L-theanine therein, at normal temperature and loading rate of 1 mL/min, eluting with 1% ammonia water at normal temperature and elution rate of 1.5 mL/min; decolorizing the eluate with medicinal active carbon until light transmittance is above 90%, decolorizing temperature is 60 deg.C, active carbon dosage is 6 g/L, and decolorizing time is 30 min; concentrating and crystallizing the decolorized solution to obtain 54.2 g L-theanine finished product. The purity of the finished product is 98.8 percent by using high performance liquid chromatography.
Example 4: biotransformation for preparing L-theanine
(1) Cell culture and collection: taking 300 mu L of lactobacillus caseiLactobacillus casei The TH139 bacterial liquid is inoculated into 30 mL LBS culture medium, cultured for 10 h under the conditions of pH7.0, 37 ℃ and 200 r/min, then 30 mL seed liquid is inoculated into 3L fermentation culture medium, cultured for 16 h under the conditions of pH7.0, 37 ℃ and 200 r/min, and frozen and centrifuged to obtain the somatic cells.
The fermentation medium comprises the following components: 20 g/L of glucose, 10 g/L of peptone, 7 g/L of yeast powder, 4 g/L of ammonium citrate, 2 g/L of sodium acetate, 4 g/L of dipotassium hydrogen phosphate, 1 g/L of magnesium sulfate and 0.5 g/L of manganese sulfate.
(2) Preparing L-theanine by biotransformation: 60 g of the bacterial cells (wet weight) are put into 1L of Tris-HCl buffer solution containing 65 g/L L-glutamine, 20 g/L ethylamine and pH9.0, reacted for 12 h at 40 ℃ and 100 r/min, and then centrifuged to leave supernatant. The concentration of L-theanine in the supernatant was 69.3 g/L, and the molar conversion of the substrate L-glutamine was 89.5%.
(3) Separating and extracting L-theanine: enabling 1L of reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein, wherein the filtration temperature is normal temperature, the filtration pressure is 0.2 MPa, and the filtration speed is 10 mL/min; allowing the membrane filtrate to pass through 001 × 7 cation exchange resin to adsorb L-theanine therein, at normal temperature and loading rate of 1 mL/min, eluting with 1% ammonia water at normal temperature and elution rate of 1.5 mL/min; decolorizing the eluate with medicinal active carbon until light transmittance is above 90%, decolorizing temperature is 60 deg.C, active carbon dosage is 6 g/L, and decolorizing time is 30 min; concentrating and crystallizing the decolorized solution to obtain 57.4 g L-theanine finished product. The purity of the finished product is 98.7 percent by using high performance liquid chromatography.
Example 5: high performance liquid detection method of L-theanine
(1) And (3) derivatization treatment: take 200. mu.L of derivatization buffer (50 mmol/L NaHCO)3Solution) was added to 1 mL of L-theanine finished or standard solution. Collecting the supernatant 10 μ L, adding 300 μ L derivatization agent solution (1% 2, 4-dinitrofluorobenzene acetonitrile solution), mixing, placing the reaction solution in 65 deg.C water bath kettle, reacting in dark for 1 h, taking out, adding volume-constant buffer solution (50 mmol/L KH)2PO3Solution) to 1.2 mL, shaking thoroughly, passing through a 0.2 μm membrane, and performing chromatographic analysis.
(2) And (3) chromatographic detection: high performance liquid chromatography is used, a chromatographic column is Agilent ZORBAX Eclipse AAA (4.6 mm multiplied by 150 mm, 5-Micron), a mobile phase A is a 50% acetonitrile solution, a mobile phase B is 50 mmol/L sodium acetate buffer solution, the column temperature is 33 ℃, the flow rate is 1 mL/min, the detection wavelength is 360 nm, and the sample introduction amount is 10 mu L by adopting binary gradient analysis. The high performance liquid chromatogram of L-theanine standard product (figure 1) and finished product (figure 2) are shown in figure, wherein the characteristic peak of L-theanine is 11.5 min.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (4)
1. Lactobacillus casei with high yield of gamma-glutamyl transpeptidaseLactobacillus casei TH139, classification name of Lactobacillus casei: (Lactobacillus casei),Has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang district; the preservation date is 2019, 10 months and 15 days; the preservation number is CGMCC No. 18686.
2. A process for producing L-theanine by using the Lactobacillus casei as claimed in claim 1, wherein: the method comprises the following steps:
(1) cell culture and collection: inoculating 300 mu L of lactobacillus casei bacterial liquid into 30 mL of LBS culture medium, culturing for 8-10 h under the conditions of pH7.0, 37 ℃ and 200 r/min, inoculating 30 mL of seed liquid into 3L of fermentation culture medium, culturing for 14-16 h under the conditions of pH7.0, 37 ℃ and 200 r/min, and freezing and centrifuging to obtain somatic cells;
(2) preparing L-theanine by biotransformation: putting 40-60 g of bacterial cells into 1L of Tris-HCl buffer solution containing 50-65 g/L L-glutamine, 15-20 g/L ethylamine and pH of 9.0, reacting for 8-12 h at 40 ℃ at 100 r/min, and centrifuging to obtain supernatant;
(3) separating and extracting L-theanine: enabling reaction supernatant to flow through a nanofiltration membrane with the molecular weight cutoff of 300 Da to intercept soluble protein therein; allowing the membrane filtrate to pass through cation exchange resin to adsorb L-theanine therein, and eluting with 1% ammonia water; decolorizing the eluent with medicinal active carbon to light transmittance of above 90%, concentrating and crystallizing to obtain L-theanine product.
3. The method of claim 2, wherein: step one the fermentation medium comprises the following components in percentage by weight: 18-20 g/L of glucose, 8-10 g/L of peptone, 5-7 g/L of yeast powder, 2-4 g/L of ammonium citrate, 1-2 g/L of sodium acetate, 2-4 g/L of dipotassium hydrogen phosphate, 0.5-1 g/L of magnesium sulfate and 0.2-0.5 g/L of manganese sulfate.
4. Use of lactobacillus casei according to claim 1 in the production of L-theanine.
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