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Gene for encoding LsGAD (Lactobacillus senmaizukei glutamic acid decarboxylase), recombinant bacterium and Kombucha fermentation viable bacteria preparation

A technology of glutamic acid decarboxylase and recombinant engineering bacteria, which is applied in genetic engineering, fermentation, plant gene improvement, etc., can solve the problems of insufficient GABA health care efficacy and low production of GABA, and achieve good industrialization prospects and production The effect of short cycle and high content

Active Publication Date: 2020-02-14
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low level of glutamic acid decarboxylase expressed by these wild-type lactic acid bacteria in the traditional fermentation process, when the wild-type lactic acid bacteria are used for fermentation, the level of GABA production is also low, which is not enough to reflect the health benefits of GABA

Method used

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  • Gene for encoding LsGAD (Lactobacillus senmaizukei glutamic acid decarboxylase), recombinant bacterium and Kombucha fermentation viable bacteria preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Obtain the gene encoding glutamic acid decarboxylase of the present invention and construct recombinant engineering bacteria

[0021] The present invention takes a novel glutamic acid decarboxylase gene (LsGAD gene) excavated from the genome of Lactobacillus senmaizukei certified by GRAS as the target gene, and performs codon optimization with reference to the codon usage frequency table of Lactococcus lactis MG1363. After the whole gene synthesis, the engineering bacteria of Lactococcus lactis capable of high-yielding γ-aminobutyric acid were successfully constructed with the help of the food-grade lactic acid bacteria expression plasmid pNZ8149. Wherein, the nucleotide sequence of the gene encoding glutamic acid decarboxylase after codon optimization of the present invention is shown in SEQ ID NO.1; the amino acid sequence of the glutamic acid decarboxylase LsGAD is shown in SEQ ID NO.2 .

[0022] Construction of the recombinant vector: The glutamic acid de...

Embodiment 2

[0025] The preparation of embodiment 2 Kombucha

[0026] 2.1 Preparation of fermentation base liquid: Take 10g of black tea leaves, 5g of skimmed milk powder, 5g of monosodium glutamate and 100g of sucrose, mix well with 1000mL of tap water, boil for 10min, filter with 8 layers of medical gauze to obtain fermentation base liquid A, and quickly cool to room temperature, Then add nisin (nisin) mother solution to a final concentration of 1ng / mL to obtain fermentation base solution B, place it in a sterile glass bottle, wrap the mouth of the bottle with 8 layers of gauze and kraft paper to prevent bacterial contamination, and store it for future use;

[0027] 2.2 Strain pretreatment

[0028] (1) Preparation of Lactococcus lactis engineering bacteria (prepared in Example 1) seed solution

[0029] Inoculate Lactococcus lactis engineered bacteria into M17 solid medium, culture at 30°C for 48 hours to obtain a single colony, pick a single colony and inoculate it into 100mL M17 liquid...

Embodiment 3

[0058] Prepare kombucha according to the method of above-mentioned embodiment 2, and the difference with embodiment 2 is only: adopt fermentation agent 1 to carry out kombucha fermentation, wherein, the concentration of 5 kinds of strains in fermentation agent 1 is 10 6 CFU / mL; fermentation time is 8 days.

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Abstract

The invention belongs to the technical field of bioengineering, particularly relates to a gene for encoding LsGAD (Lactobacillus senmaizukei glutamic acid decarboxylase), a recombinant bacterium and aKombucha fermentation viable bacteria preparation. The gene for the encoding LsGAD has a sequence disclosed by SEQ ID NO.1. A novel glutamic acid decarboxylase gene excavated from the genome of Lactobacillus senmaizukei approved by GRAS (Generally recognized as safe) is taken as a target gene, a codon utilization frequency table of Lactococcus lactis MG1363 is taken as codon optimization, after gene synthesis is carried out, the engineering bacterium of the Lactococcus lactis capable of realizing the high yield of a Gamma-aminobutyric acid is successfully constructed in virtue of a food-gradelactobacillus expression plasmid, the research of the functional Kombucha produced by fermentation is carried out, and the content of the GABA (Gamma-aminobutyric acid) in the Kombucha can be obviously improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene encoding glutamic acid decarboxylase LsGAD, a recombinant engineering bacterium and a kombucha fermentation bacterium. Background technique [0002] γ-aminobutyric acid (Gamma-aminobutyric acid, GABA) is a natural non-protein amino acid, as an important inhibitory neurotransmitter in mammals, it mediates more than 40% of nerve inhibitory signals, and has important physiological activity. GABA has the functions of lowering blood pressure, calming the nerves, enhancing brain vitality, enhancing memory, improving sleep, and delaying aging. It has broad application prospects in medicine, food health care, and chemical industries. Glutamate decarboxylase (Glutamatedecarboxylase, GAD) plays a key role in the biosynthesis of GABA, which can specifically and irreversibly catalyze the cleavage of L-glutamic acid into GABA and carbon dioxide. Glutamic acid decar...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/74C12N1/21C12N1/16C12N1/20A23F3/16C12R1/84C12R1/25C12R1/245C12R1/02C12R1/01
CPCC12N9/88C12Y401/01015C12N15/746C12N1/16C12N1/20A23F3/166C12N2800/22
Inventor 唐存多姚伦广阚云超史红玲董自星史鸿飞焦铸锦
Owner NANYANG NORMAL UNIV
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