Lactobacillus casei for producing gamma-glutamyl transpeptidase in high yield and application of lactobacillus casei in production of L-theanine

Abstract

The invention relates to lactobacillus casei TH139 for producing gamma-glutamyl transpeptidase in a high yield. The lactobacillus casei TH139 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.18686; the strain is used to produce the gamma-glutamyl transpeptidase through fermentation at 37 DEG C and pH of 7.0; after centrifugation is performed to collect bacterial cells, the bacterial cells are subjected to bioconversion in the presence of L-glutamine and ethylamine at 40 DEG C and pH of 9.0 to obtain L-theanine; and the reaction solution is subjected to membrane separation, ion exchange resin separation is performed, and concentration crystallization is performed to obtain a qualified L-theanine finished product. The method forproducing the L-theanine by using the lactobacillus casei developed by the invention has the advantages of food safety, environmental protection, simple operation and low costs, and has good industrial application value.

Description

technical field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a strain of Lactobacillus casei with high production of gamma-glutamyl transpeptidase and its application in the production of L-theanine. Background technique

[0002] L-theanine is a unique amino acid in tea, accounting for more than 50% of its total free amino acids. L-theanine has many beneficial physiological effects, which can promote the relaxation of the human brain, improve concentration and promote learning ability. In terms of health care, L-theanine has the functions of lowering blood pressure, preventing vascular diseases, relieving stress, protecting nerves, losing weight and reducing fat, and improving the immune system. In terms of food, L-theanine has been listed by the FDA as a safe food and drug additive, which is used to improve food quality and enhance food flavor.

[0003] The existing main preparation methods of L-theanine include extract...

Examples

Embodiment 1

[0032] Example 1: Screening of Lactobacillus casei with high yield of γ-glutamyl transpeptidase

[0033] (1) Primary screening method: Inoculate a single colony of 200 strains of Lactobacillus casei isolated from fermented dairy products in various regions of Henan Province on a plate screening medium, and culture at 37°C for 1-2 days, observe the size of the transparent circle . The plate screening medium contains L-glutamine 12 g / L, ethylamine 8 g / L, 0.1% phenolphthalein indicator 8 mL / L, and agar 1.8 g / L.

[0034] (2) Re-screening method: Select the strain with a larger transparent circle, inoculate it in LBS medium at 37°C, pH 7.0 and culture it for 12 hours, then collect the bacterial cells by centrifugation to determine the activity of γ-glutamyl transpeptidase . The amount of bacterial cells (wet weight) that catalyzes the production of 1 μmol L-theanine per minute is defined as an enzyme activity. The strain with the highest enzyme activity was selected for cryopres...

Embodiment 2

[0035] Example 2: Preparation of L-theanine by biotransformation

[0036] (1) Cell culture and collection: Take 300 μL Lactobacillus casei Lactobacillus casei TH139 bacterial liquid was inoculated into 30 mL of LBS medium, pH 7.0, 37°C, and cultured at 200 r / min for 8 h, then inoculated 30 mL of seed liquid into 3 L fermentation medium, pH 7.0, 37°C After culturing for 14 h under the condition of 200 r / min, the bacterial cells were obtained by refrigerated centrifugation.

[0037] The components of the fermentation medium are: glucose 18 g / L, peptone 8 g / L, yeast powder 5 g / L, ammonium citrate 2 g / L, sodium acetate 1 g / L, dipotassium hydrogen phosphate 2 g / L, Magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L.

[0038] (2) Preparation of L-theanine by biotransformation: Take 40 g of bacterial cells (wet weight) and put them in 1 L of Tris-HCl containing 50 g / L L-glutamine, 15 g / L ethylamine, pH 9.0 After reacting in the buffer solution for 8 h at 40 °C and 100 r / min, the su...

Embodiment 3

[0040] Example 3: Preparation of L-theanine by biotransformation

[0041] (1) Cell culture and collection: Take 300 μL Lactobacillus casei Lactobacillus casei TH139 bacterial liquid was inoculated into 30 mL of LBS medium, pH 7.0, 37°C, and cultured at 200 r / min for 9 h, then inoculated 30 mL of seed liquid into 3 L fermentation medium, pH 7.0, 37°C After culturing for 15 h under the condition of 200 r / min, the bacterial cells were obtained by refrigerated centrifugation.

[0042] The components of the fermentation medium are: glucose 19 g / L, peptone 9 g / L, yeast powder 6 g / L, ammonium citrate 3 g / L, sodium acetate 1.5 g / L, dipotassium hydrogen phosphate 2.5 g / L, Magnesium sulfate 0.8 g / L, manganese sulfate 0.3 g / L.

[0043] (2) Preparation of L-theanine by biotransformation: Take 50 g of bacterial cells (wet weight) and put them in 1 L of Tris-HCl containing 60 g / L L-glutamine, 18 g / L ethylamine, pH 9.0 After reacting in the buffer solution for 10 h at 40 °C and 100 r / min,...

technical field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a strain of Lactobacillus casei with high production of gamma-glutamyl transpeptidase and its application in the production of L-theanine. Background technique

[0002] L-theanine is a unique amino acid in tea, accounting for more than 50% of its total free amino acids. L-theanine has many beneficial physiological effects, which can promote the relaxation of the human brain, improve concentration and promote learning ability. In terms of health care, L-theanine has the functions of lowering blood pressure, preventing vascular diseases, relieving stress, protecting nerves, losing weight and reducing fat, and improving the immune system. In terms of food, L-theanine has been listed by the FDA as a safe food and drug additive, which is used to improve food quality and enhance food flavor.

[0003] The existing main preparation methods of L-theanine include extract...

Examples

Embodiment 1

[0032] Example 1: Screening of Lactobacillus casei with high yield of γ-glutamyl transpeptidase

[0033] (1) Primary screening method: Inoculate a single colony of 200 strains of Lactobacillus casei isolated from fermented dairy products in various regions of Henan Province on a plate screening medium, and culture at 37°C for 1-2 days, observe the size of the transparent circle . The plate screening medium contains L-glutamine 12 g / L, ethylamine 8 g / L, 0.1% phenolphthalein indicator 8 mL / L, and agar 1.8 g / L.

[0034] (2) Re-screening method: Select the strain with a larger transparent circle, inoculate it in LBS medium at 37°C, pH 7.0 and culture it for 12 hours, then collect the bacterial cells by centrifugation to determine the activity of γ-glutamyl transpeptidase . The amount of bacterial cells (wet weight) that catalyzes the production of 1 μmol L-theanine per minute is defined as an enzyme activity. The strain with the highest enzyme activity was selected for cryopres...

Embodiment 2

[0035] Example 2: Preparation of L-theanine by biotransformation

[0036] (1) Cell culture and collection: Take 300 μL Lactobacillus casei Lactobacillus casei TH139 bacterial liquid was inoculated into 30 mL of LBS medium, pH 7.0, 37°C, and cultured at 200 r / min for 8 h, then inoculated 30 mL of seed liquid into 3 L fermentation medium, pH 7.0, 37°C After culturing for 14 h under the condition of 200 r / min, the bacterial cells were obtained by refrigerated centrifugation.

[0037] The components of the fermentation medium are: glucose 18 g / L, peptone 8 g / L, yeast powder 5 g / L, ammonium citrate 2 g / L, sodium acetate 1 g / L, dipotassium hydrogen phosphate 2 g / L, Magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L.

[0038] (2) Preparation of L-theanine by biotransformation: Take 40 g of bacterial cells (wet weight) and put them in 1 L of Tris-HCl containing 50 g / L L-glutamine, 15 g / L ethylamine, pH 9.0 After reacting in the buffer solution for 8 h at 40 °C and 100 r / min, the su...

Embodiment 3

[0040] Example 3: Preparation of L-theanine by biotransformation

[0041] (1) Cell culture and collection: Take 300 μL Lactobacillus casei Lactobacillus casei TH139 bacterial liquid was inoculated into 30 mL of LBS medium, pH 7.0, 37°C, and cultured at 200 r / min for 9 h, then inoculated 30 mL of seed liquid into 3 L fermentation medium, pH 7.0, 37°C After culturing for 15 h under the condition of 200 r / min, the bacterial cells were obtained by refrigerated centrifugation.

[0042] The components of the fermentation medium are: glucose 19 g / L, peptone 9 g / L, yeast powder 6 g / L, ammonium citrate 3 g / L, sodium acetate 1.5 g / L, dipotassium hydrogen phosphate 2.5 g / L, Magnesium sulfate 0.8 g / L, manganese sulfate 0.3 g / L.

[0043] (2) Preparation of L-theanine by biotransformation: Take 50 g of bacterial cells (wet weight) and put them in 1 L of Tris-HCl containing 60 g / L L-glutamine, 18 g / L ethylamine, pH 9.0 After reacting in the buffer solution for 10 h at 40 °C and 100 r / min,...

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