Breeding for efficiently converting L-glutamate into gamma-amino butyric acid lactobacillus

A technology of aminobutyric acid lactic acid bacteria and glutamic acid, which is applied in microorganism-based methods, bacteria, biochemical equipment and methods, etc., can solve the problems of high GABA, unclear physiological significance, etc. high purity effect

Active Publication Date: 2010-12-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1950, it was found that the concentration of GABA in the normal brai

Method used

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  • Breeding for efficiently converting L-glutamate into gamma-amino butyric acid lactobacillus
  • Breeding for efficiently converting L-glutamate into gamma-amino butyric acid lactobacillus
  • Breeding for efficiently converting L-glutamate into gamma-amino butyric acid lactobacillus

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Screening of strains that effectively transform L-glutamic acid into γ-aminobutyric acid

[0030] Strain screening and culture: Select soil, yogurt, homemade sauerkraut and other samples as materials, take 0.1mL of juice diluted to different concentrations, aseptically spread on the lactic acid bacteria selection medium plate, cultivate for 1-2 days at 30℃, A transparent circle is formed, and the medium around the colony becomes a single yellow colony, which is preliminarily identified as lactic acid bacteria. The plate streak method was used to repeatedly streak on the MRS plate to obtain pure strains. The separated and purified single colonies were connected to MRS solid slant medium and stored in a refrigerator at 4°C, and transferred once every 2 weeks.

[0031] Lactic acid bacteria isolation medium composition: beef extract 10g / L, yeast extract 10g / L, peptone 10g / L, glucose 5g / L, Tween 0.5g / L, tomato juice 200g / L, bromocresol green 0.1g / L, Calcium carbonate...

Embodiment 2

[0035] Example 2: Ultraviolet mutagenesis screening of high-yield γ-aminobutyric acid strains

[0036] Take the strain obtained in Example 1 as the starting strain. After the strain is activated, the bacteria are diluted with sterile normal saline (0.85% NaCl solution), and the cell density is 10 8 Pcs / mL of bacterial suspension; under 15W UV lamp, 30cm away from the irradiated bacterial suspension, by changing the irradiation time to obtain different mutagenic doses; formulate its growth curve, ultraviolet lethality curve, the bacterial suspension is induced After the transformation treatment, it was diluted and spread on a gradient plate containing the metabolite GABA, and cultured at 30°C for 1 day. By visual observation, pick out the colonies growing on the relatively thicker part of the upper layer of the gradient plate and store them on the slope.

Embodiment 3

[0037] Example 3: Study on the transformation ability of mutant strains

[0038] The strains screened in Example 2 were placed on a solid medium (casein peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 5g / L, sodium acetate 5g / L, citrate diamine 0.2 g / L, Tween 0.1g / L, dipotassium hydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, calcium carbonate 20g / L, agar 20g / L, pH 6.5) 18h, pick a single colony and inoculate it in liquid activation medium (casein peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 5g / L, sodium acetate 5g / L, citrate diamine 0.2g / L, Tween 0.1g / L, dipotassium hydrogen phosphate 0.2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, calcium carbonate 20g / L, pH 6.5), and then transferred to the seed medium (Peptone 5g / L, yeast extract 5g / L, glucose 10g / L, sodium succinate 5g / L, pH 6.5), culture for 1d; collect the bacteria by centrifugation, wash the bacteria with sterilized water, and put them in a 100...

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Abstract

The invention discloses breeding for efficiently converting L-glutamate into gamma-amino butyric acid lactobacillus strains, and belongs to the technical field of fermentation in bioengineering. The strains screened by the invention are classified and named as Lactobacillus plantarum GB 01-21 and collected in CCTCC with a collection number of CCTCCM 209102. A lactobacillus strain capable of producing gamma-amino butyric acid by using the L-glutamate is screened from homemade pickle, named as LP-GB 01 and taken as an original strain; and a positive mutant is obtained by screening after ultraviolet mutation and named as LP-GB 01-21. A preliminary conversion experiment is performed in a 100mL triangular flask; and after the conversion is stopped, the gamma-amino butyric acid in the conversion solution is measured by using an amino acid measurement instrument, wherein the concentration of the gamma-amino butyric acid is 51.9g/L and the molar conversion rate reaches 92.7 percent. When an amplified conversion experiment is performed in a 5L fermentation tank, the concentration of the finally obtained gamma-amino butyric acid is 132g/L, and the molar conversion rate is 94.3 percent. The microbial method provides basis for producing industrialized production of the gamma-amino butyric acid.

Description

Technical field [0001] The breeding of a lactic acid bacteria that efficiently transforms L-glutamic acid into γ-aminobutyric acid belongs to the field of fermentation technology in bioengineering. Background technique [0002] γ-aminobutanoic acid, also known as 4-Aminobutanoic acid (4-Aminobutanoic acid, 4-AB, GABA for short) and γ-aminobutanoic acid, has an amino group at the γ position of butyric acid and exists in an unbound state. It is easily soluble in water, insoluble in alcohol, ether and benzene. It can be supplemented by chemical methods or biosynthesis, or directly ingested from food. Existing widely in nature, it is a natural amino acid composed of non-protein. It is catalyzed by glutamic acid (Glu) by glutamate decarboxylase (EC4.1.1.15, GAD or GDC). The free form is widely present in prokaryotes and eukaryotes. It is an important inhibitory neurotransmitter in the central nervous system of mammals and has important physiological functions. The reported physiolog...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12N13/00C12Q1/68C12Q1/04C12P13/00C12R1/25
Inventor 饶志明刘婷婷杨套伟张术聪夏海锋
Owner JIANGNAN UNIV
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