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Culturing method of Lb.plantarum capable of highly producing gamma-aminobutyric acid (GABA) and application

A technology of plantaractobacillus and cultivation method, which is applied in the field of cultivation of high-yielding γ-aminobutyric acid plantaractobacillus, can solve the problems of limited metabolic capacity of strains and unreasonable cultivation methods, and achieve short cultivation time and high production of γ-aminobutyric acid. The effect of high yield and low inoculation amount

Inactive Publication Date: 2015-10-28
黑龙江省乳品工业技术开发中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The reasons for the above problems are, on the one hand, the limited metabolic capacity of the strain itself, and on the other hand, the cultivation method is not reasonable enough.

Method used

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  • Culturing method of Lb.plantarum capable of highly producing gamma-aminobutyric acid (GABA) and application
  • Culturing method of Lb.plantarum capable of highly producing gamma-aminobutyric acid (GABA) and application
  • Culturing method of Lb.plantarum capable of highly producing gamma-aminobutyric acid (GABA) and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Identification, preservation and activation of the bacterial classification of embodiment 1

[0037] 1. Identification and preservation of strains

[0038] In order to determine the purity and species of the purchased strains, add an appropriate amount of liquid MRS to the purchased freeze-dried bacterial powder for culture, mix evenly, spread the appropriately diluted bacterial liquid on the solid MRS medium, and cultivate at 30°C until a single colony is formed . Pick 5 single colonies at random, inoculate them in 4 mL of liquid MRS medium, and culture them purely at 30°C for 12 hours until they reach the logarithmic growth phase, and store them at 4°C for later use.

[0039] Gram-stained five single-colony liquids were magnified at 1000 times, and examined under a microscope. At the same time, re-inoculate the pure culture bacteria solution into the corresponding MRS medium according to the ratio of 2%, culture at constant temperature to reach the logarithmic growt...

Embodiment 2

[0045] The optimization of embodiment 2 culture conditions

[0046] 1. Optimization of initial pH of fermentation

[0047] The specific operation is as follows:

[0048] 1) Prepare MRS medium with pH values ​​of 4.0, 4.2, 4.5, 5.0, 5.8, 6.0, and 6.8, respectively, pack them in 200mL Erlenmeyer flasks, sterilize under high temperature and high pressure, and store them at 4°C for later use. Three parallels were set for each pH condition;

[0049] 2) After activating the strain according to the method in 2.3.1, inoculate 200mL of PLP containing 100mM L-MSG and 20μM according to 3%, and let it stand for anaerobic fermentation at 37°C for 24h;

[0050] 3) The fermentation broth was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was taken and kept at 4°C for later use, and the content of γ-aminobutyric acid in it was detected by high performance liquid chromatography.

[0051] The measured results are as figure 1 shown. from figure 1 It can be seen that the ini...

Embodiment 3

[0078] The determination of embodiment 3 fermentation time

[0079] The frozen strains were streaked in three areas on the MRS solid medium plate to obtain a single colony, and the single colony was picked and cultured in 4 mL of MRS liquid medium at 30°C for 12 hours, and the seeds obtained after continuous expansion for two generations were used for fermentation. The fermentation medium was MRS medium, the fermentation system was 2L, the initial pH value of the fermentation was 5.5, the fermentation temperature was 37°C, and the fermentation time was 72h. In the induction group, 100 mM L-MSG was additionally added, and in the control group, except that L-MSG was not added, other fermentation conditions were the same as those of the induction group. Experimental results such as Image 6 shown.

[0080] from Image 6 It can be seen that in the initial stage of fermentation, the difference in γ-aminobutyric acid content in the fermentation broth between the experimental grou...

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Abstract

The invention discloses a culturing method of Lb.plantarum capable of highly producing gamma-aminobutyric acid (GABA) and application, and belongs to the technical field of microorganism fermentation. The method disclosed by the invention includes the steps that: Lb.plantarum ATCC14917 is activated, and the activated Lb.plantarum is cultured in a fermentation system according to the inoculum size of 3%, wherein the culture medium is MRS culture medium, the initial pH value for fermentation is 4.0-6.8, the concentration of L-sodium glutamate is 0-1 mol / L, the addition amount of pyridoxal phosphate is 0-0.001 mol / L, the fermentation temperature is 25-42 DEG C, and the fermentation time is 8-72 h. The method provided by the invention has the characteristics of short culture time, less inoculum size, and high yield of GABA, and has the advantages that the inoculum size is only 3%, the culture time is only 36 h, while the yield of GABA is 360.67 mM / L of fermentation broth, being 7.77 times higher than the control group.

Description

technical field [0001] The invention relates to a cultivation method and application of high-yielding γ-aminobutyric acid plantarum lactobacillus, belonging to the technical field of microbial fermentation. Background technique [0002] γ-Aminobutyric acid (GABA) is a non-protein amino acid widely present in eukaryotes and prokaryotes. It was first discovered in the late nineteenth century and successfully synthesized. In addition to being found in the brain and spinal cord, GABA has been found in nearly 30 peripheral tissues of various mammals, but its concentration is low, only 1% in brain tissue. The chemical molecular formula of GABA is C4H9NO2 and the molecular weight is 103.12. GABA is in the state of white flaky or needle-like crystals at room temperature, with a decomposition point of 202°C, no optical activity, slightly odorous, deliquescent, easily soluble in water, soluble in hot ethanol, insoluble in cold ethanol, ether and phenol. [0003] At present, GABA is...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/00C12R1/25
CPCC12P13/005
Inventor 刘鹏迟涛方景泉
Owner 黑龙江省乳品工业技术开发中心
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