Method for producing high-yield gamma-propalanine and application thereof

Abstract

The invention relates to a method for producing high-yield gamma-propalanine and an application thereof, belonging to the technical field of biotechnology medicine and food engineering. The method comprises the following steps: carrying out strain separated screening, acridine orange-ultraviolet ray mutagenesis and N<+> injection mutagenesis to obtain a high-yield gamma-propalanine LactobacillusbrevisTCCC (CGMCC No.3414) strain; optimizing a fermentation medium and fermentation conditions; coupling fermentation of the gonotokont of the strain with resting cell biotransformation of the strain to produce the gamma-propalanine; employing the membrane filtration technology, the adsorbent resin decoloration technology, the strong acid resin cation exchange technology, the ethanol recrystallization technology, the preparative chromatography technology and the energy-saving and cost-reducing technology to separate the gamma-propalanine from the fermentation fluid and the biotransformation fluid; and then carrying out purification to obtain the crystallized gamma-propalanine with the purity of 99%. The method has the advantages of cheap raw materials, low energy consumption for production, low production cost, good product security and easy realization of industrialized production. Applied to glutamic acid fermentation waste liquor for producing the gamma-propalanine, the strain has good potential for generating social and economic benefits.

Description

technical field

[0001] The invention belongs to the technical field of medical biology and food engineering, is an organic combination of modern biological and chemical engineering and traditional fermentation engineering technology, and is an improvement on the current production method of gamma-aminobutyric acid (GABA) by biotechnology. More specifically, Lactobacillus brevis isolated and bred from sauerkraut in northern my country Lactobacillus brevis CGMCCNO.3414, through the biotransformation of growing cells and resting cells, efficiently produces the internationally recognized safest API and health food - γ-aminobutyric acid. Background technique

[0002] γ-Aminobutyric acid (GABA), also known as aminobutyric acid, is a natural amino acid that is not a protein component. It exists widely in nature and is an important inhibitory neurotransmitter that has been studied in depth. Metabolic activity has the effects of lowering blood pressure, regulating arrhythmia, impro...

Examples

Embodiment 1

[0060] (2) Ultraviolet-acridine orange mutagenesis: Take the well-grown Lactobacillus brevis S1 seed solution, irradiate with ultraviolet light for 90s, and then transfer 600 μL of the ultraviolet-mutated bacterial suspension into a test tube containing 5 mL of mutagenic medium Incubate in a dark water bath for 12 hours at 30°C with shaking. (The mutagenesis medium contains 1 mg / mL acridine orange, the doses are 80 μL, 100 μL, 150 μL, 200 μL, 250 μL)

[0061] (3) N + Ion implantation mutagenesis: Take the well-grown Lactobacillus brevis seed liquid, dilute it to 100 times with 8% sterile glycerol solution, take 100 μL and evenly spread it on a sterile plate, dry it with sterile wind, take it out for microscopic examination Ion implantation is performed on those cells that overlap. use N + Ions were implanted, the implantation energy was 30keV, and the implantation dose was 0ions / cm 2 ~2.0×10 14 ions / cm 2 , the vacuum degree of the target chamber is 1.2×10 -3 Pa. After...

Embodiment 2

[0071] will activate the Lactobacillus brevis CGM CCN 0 The .3414 strain was inoculated in the seed medium for cultivation, and was transferred to the fermentation medium when the bacterial mass reached the fermentation conditions.

[0072] Seed medium (g / L) and cultivation method: corn saccharification solution 20, peptone 10, yeast extract powder 5, ammonium citrate 2, magnesium sulfate 0.58, manganese sulfate 0.25, sodium acetate 2, potassium dihydrogen phosphate 2. Seed culture conditions: 30 ° C incubator, static culture for 12 hours. According to the inoculation amount of 10-15%, it is inserted into a 5L fermenter.

[0073] Fermentation medium (g / L) and culture method: corn saccharification solution 20, yeast extract powder 20, corn steep liquor 10, sodium acetate 2, potassium phosphate 2, magnesium sulfate 0.4, ammonium sulfate 0.2. The culture temperature of the fermenter was controlled at 30°C and the initial pH was 6.5. Ventilate for 8 hours, the ventilation rat...

Embodiment 3

[0079] Seed medium composition (g / L): peptone 10, yeast extract 10, glucose 20, sodium acetate 5, ammonium citrate 2, MnSO 4˙ h 2 O 0.05, MgSO 4˙ 7H 2 0 0.2, Tween 80 1mL, distilled water 1000 mL, pH control 5.0-6.0. Transfer to the fermentation medium containing 8% L-sodium glutamate at an inoculum size of 10-15%.

[0080] Fermentation medium (g / L): corn steep liquor 10, glucose 22, yeast powder 15, sodium acetate 2, magnesium sulfate 0.2, (NH 4 ) 2 SO 4 0.2, pH6.0. After 16 hours of cultivation, the growth of the bacteria entered the logarithmic phase, and the pH was adjusted to 4.6 and kept at 4.6. After 60 hours of cultivation, the GABA production reached a maximum of 76.52g / L. After culturing the cells for 60 h, the cells were collected, centrifuged for 10 min, 8000 rpm, 4 °C, washed twice with 0.2 mM sodium phosphate buffer pH 7.2, resuspended in pH 4.6 phosphate buffer, and the wet cell concentration was 25 g / L.

[0081]Add 80 mM of L-sodium glutamate and 0.01 M...

technical field

[0001] The invention belongs to the technical field of medical biology and food engineering, is an organic combination of modern biological and chemical engineering and traditional fermentation engineering technology, and is an improvement on the current production method of gamma-aminobutyric acid (GABA) by biotechnology. More specifically, Lactobacillus brevis isolated and bred from sauerkraut in northern my country Lactobacillus brevis CGMCCNO.3414, through the biotransformation of growing cells and resting cells, efficiently produces the internationally recognized safest API and health food - γ-aminobutyric acid. Background technique

[0002] γ-Aminobutyric acid (GABA), also known as aminobutyric acid, is a natural amino acid that is not a protein component. It exists widely in nature and is an important inhibitory neurotransmitter that has been studied in depth. Metabolic activity has the effects of lowering blood pressure, regulating arrhythmia, impro...

Examples

Embodiment 1

[0060] (2) Ultraviolet-acridine orange mutagenesis: Take the well-grown Lactobacillus brevis S1 seed solution, irradiate with ultraviolet light for 90s, and then transfer 600 μL of the ultraviolet-mutated bacterial suspension into a test tube containing 5 mL of mutagenic medium Incubate in a dark water bath for 12 hours at 30°C with shaking. (The mutagenesis medium contains 1 mg / mL acridine orange, the doses are 80 μL, 100 μL, 150 μL, 200 μL, 250 μL)

[0061] (3) N + Ion implantation mutagenesis: Take the well-grown Lactobacillus brevis seed liquid, dilute it to 100 times with 8% sterile glycerol solution, take 100 μL and evenly spread it on a sterile plate, dry it with sterile wind, take it out for microscopic examination Ion implantation is performed on those cells that overlap. use N + Ions were implanted, the implantation energy was 30keV, and the implantation dose was 0ions / cm 2 ~2.0×10 14 ions / cm 2 , the vacuum degree of the target chamber is 1.2×10 -3 Pa. After...

Embodiment 2

[0071] will activate the Lactobacillus brevis CGM CCN 0 The .3414 strain was inoculated in the seed medium for cultivation, and was transferred to the fermentation medium when the bacterial mass reached the fermentation conditions.

[0072] Seed medium (g / L) and cultivation method: corn saccharification solution 20, peptone 10, yeast extract powder 5, ammonium citrate 2, magnesium sulfate 0.58, manganese sulfate 0.25, sodium acetate 2, potassium dihydrogen phosphate 2. Seed culture conditions: 30 ° C incubator, static culture for 12 hours. According to the inoculation amount of 10-15%, it is inserted into a 5L fermenter.

[0073] Fermentation medium (g / L) and culture method: corn saccharification solution 20, yeast extract powder 20, corn steep liquor 10, sodium acetate 2, potassium phosphate 2, magnesium sulfate 0.4, ammonium sulfate 0.2. The culture temperature of the fermenter was controlled at 30°C and the initial pH was 6.5. Ventilate for 8 hours, the ventilation rat...

Embodiment 3

[0079] Seed medium composition (g / L): peptone 10, yeast extract 10, glucose 20, sodium acetate 5, ammonium citrate 2, MnSO 4˙ h 2 O 0.05, MgSO 4˙ 7H 2 0 0.2, Tween 80 1mL, distilled water 1000 mL, pH control 5.0-6.0. Transfer to the fermentation medium containing 8% L-sodium glutamate at an inoculum size of 10-15%.

[0080] Fermentation medium (g / L): corn steep liquor 10, glucose 22, yeast powder 15, sodium acetate 2, magnesium sulfate 0.2, (NH 4 ) 2 SO 4 0.2, pH6.0. After 16 hours of cultivation, the growth of the bacteria entered the logarithmic phase, and the pH was adjusted to 4.6 and kept at 4.6. After 60 hours of cultivation, the GABA production reached a maximum of 76.52g / L. After culturing the cells for 60 h, the cells were collected, centrifuged for 10 min, 8000 rpm, 4 °C, washed twice with 0.2 mM sodium phosphate buffer pH 7.2, resuspended in pH 4.6 phosphate buffer, and the wet cell concentration was 25 g / L.

[0081]Add 80 mM of L-sodium glutamate and 0.01 M...

Images