Probiotics with gamma-aminobutyric acid production function and application thereof

Abstract

The invention relates to a method for screening probiotics with a gamma-aminobutyric acid production function. The invention also relates to application of the probiotics screened by the method in preparation of drugs or health care products for treating or preventing inflammation, hypertension or depression, or improving brain functions or improving sleep.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically, to probiotics capable of producing gamma-aminobutyric acid, a screening method thereof, and an application thereof. Background Art

[0002] γ-Aminobutyric acid (GABA) has beneficial functions such as inhibiting inflammation, relieving hypertension, resisting depression, improving brain function or sleep, etc., and γ-aminobutyric acid in the intestine can relieve neuropsychiatric diseases such as depression and anxiety through the "brain-gut axis", which has attracted widespread attention. There are also literature reports that GABA is related to diseases related to mucosal immune disorders (including enteritis, IgA nephropathy, etc.) (https: / / www.chinagut.cn / articles / ss / 84195f646129446bb03abe1d5ed9d5f7-GABA signal-mediated intestinal mucosal immunity or affecting IgA nephropathy), so natural probiotics that can produce γ-aminobutyric acid have broad prospects for health care applications. At present, the synthesis of γ-aminobutyric acid mainly includes chemical synthesis and biosynthesis. The chemical synthesis is the reaction of potassium phthalimide and 4-chlorobutyric cyanide at 180 degrees Celsius, the product is refluxed and hydrolyzed with concentrated sulfuric acid, and then purified by crystallization; 2-pyrrolidone is hydrolyzed with calcium hydroxide and ammonium bicarbonate to obtain [1] The use of microbial GAD to catalyze L-sodium glutamate (L-MSG) to produce γ-aminobutyric acid is not limited by resources, environment and space, and is widely used. [1] .

[0003] At present, the widely accepted biosynthesis process of γ-aminobutyric acid involves two key proteins: intracellular glutamate decarboxylase (glutamate decarboxylase B, GadB), and membrane-bound antiporter (L-glutamate: 4-aminobutyrate antiporter, GadC). The former catalyzes the α-decarboxylation of L-glutamate to produce γ-aminobutyric acid, and the latter exchanges extracellular L-glutamate with intracellular γ-aminobutyric acid. Therefore, efficient co-expression of GadB and GadC is the key to increasing the production of γ-aminobutyric acid. The most commonly used strains in microbial fermentation are Lactobacillus plantarum, Corynebacterium glutamicum, and genetic engineering of Escherichia coli. The following is a brief introduction to the existing research on the production of γ-aminobutyric acid by these three bacteria.

[0004] (1) Lactobacillus plantarum (Lactiplantibacillus plantarum)

[0005] The production of γ-aminobutyric acid is mostly completed by Lactobacillus plantarum. The γ-aminobutyric acid in Lactobacillus plantarum is produced by irreversible decarboxylation of L-glutamic acid or its derivatives under the action of glutamate decarboxylase. During this process, the pH inside the lactobacillus cell maintains a steady state, allowing the bacteria to survive in an acidic environment. [2] .

[0006] (2) Escherichia coli

[0007] There are also many studies on the use of Escherichia coli for genetic engineering to produce γ-aminobutyric acid. Escherichia coli grows rapidly and is easy to ferment at high density, so it is easy to achieve high-efficiency production of γ-aminobutyric acid with the help of genetic engineering. A study constructed a dual-promoter expression system (PT7lac and PBAD promoter) with pET28a as the backbone, and inserted the GadC mutant gene (gadCΔC41) of E. coli and the GadB mutant gene (gadBΔC11) of Lactobacillus plantarum into the designed vector to increase the production of γ-aminobutyric acid. [3] Some researchers designed GAD into a highly active enzyme by increasing or decreasing the length of the C-terminal residue of the GAD enzyme structure from E. coli, which greatly increased the pH range of the enzyme and can synthesize 1M GABA within 3 hours. [4] A study obtained the GAD enzyme gene lpgad from the whole gene of Lactobacillus plantarum GB01-2 by DNPCR amplification, constructed the recombinant plasmid pET-28a-lpgad, and efficiently induced expression in E. coli BL21 (DE3). The enzyme activity was significantly improved to 8.53U / mg, and the yield after 24 hours of conversion could reach 143.5g / L. The molar conversion rate was 97.32%, which is 2.19 times that of Lactobacillus plantarum GB01-21. [5] A study has transferred glutamate decarboxylase from Lactobacillus plantarum BC114 into Escherichia coli BL21 (DE3) for heterologous expression, achieving a γ-aminobutyric acid production of 2387 mg / L. [6] The engineering of E. coli containing SsGAD (SsGAD from Streptomyces sp.) provides a promising and cost-effective bioconversion system for the industrial production of GABA. [7] .

[0008] (3) Corynebacterium glutamicum

[0009] The direct precursor of γ-aminobutyric acid is L-glutamic acid, but the cost of L-glutamic acid is relatively high in actual production. While recombinant Corynebacterium glutamicum uses cheap carbon sources to produce L-glutamic acid, its recombinant GAD system also enables L-glutamic acid to be effectively converted into γ-aminobutyric acid. [8] . There is a patent related to the use of genetic engineering technology to construct Corynebacterium glutamicum to produce γ-aminobutyric acid. This invention uses Corynebacterium glutamicum ATCC13032 as the starting strain, integrates the glutamate decarboxylase gene from Lactobacillus brevis into the Corynebacterium glutamicum genome, and constructs a γ-aminobutyric acid production strain. This strain uses glucose as a carbon source and can produce γ-aminobutyric acid by direct fermentation. The final yield of γ-aminobutyric acid obtained by batch fed fermentation is 58.3g / L [9] Another method is to express the glutamate decarboxylase gene gadB for synthesizing γ-aminobutyric acid in Lactobacillus plantarum and the glutamate dehydrogenase gene gdh for synthesizing L-glutamate in Corynebacterium glutamicum in series and introduce them into Corynebacterium glutamicum, and then ferment and produce γ-aminobutyric acid by using the Corynebacterium glutamicum after the gene tandem expression.

[10] . Summary of the invention

[0010] The present invention aims to obtain strains that can naturally produce γ-aminobutyric acid through bioinformatics methods, replacing the conventional oral γ-aminobutyric acid absorption into the blood to exert its effect, breaking through the limitation that γ-aminobutyric acid in the blood is difficult to pass through the blood-brain barrier, and allowing probiotics to directly produce γ-aminobutyric acid in the intestines, and exerting its function by affecting the central nervous system through the "brain-gut axis".

[0011] Therefore, according to one aspect of the present invention, the present invention provides a method for screening probiotics (especially probiotics with the function of producing gamma-aminobutyric acid (GABA)), the method comprising the following steps:

[0012] 1) selecting probiotics having glutamate decarboxylase (GadB) or its homologous protein from candidate probiotics;

[0013] 2) selecting a probiotic having a membrane-bound antiporter (GadC) or a homologous protein thereof from the probiotics obtained in step 1); and

[0014] 3) The probiotics obtained in step 2) are subjected to a fermentation experiment for producing γ-aminobutyric acid, and those probiotics having the function of producing γ-aminobutyric acid are selected.

[0015] In a preferred embodiment, the probiotics are intestinal probiotics, preferably edible probiotics. For example, the probiotics can be the probiotics listed in the "List of Bacteria that can be used in Food" issued by the National Health and Safety Commission.

[0016] In another preferred embodiment, the selection in step 1) and step 2) is performed by homology analysis of protein sequences; and / or in step 3) OD is selected. 600 The probiotics with the highest values ​​and / or the highest GABA concentration in the supernatant.

[0017] In another preferred embodiment, the fermentation experiment for producing γ-aminobutyric acid is carried out in an in vitro simulated intestinal fluid environment, preferably under aerobic or microaerobic conditions.

[0018] In another preferred embodiment, the probiotic is selected from the group consisting of Lactococcus lactis including Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. Cremoris, Latilactobacillus curvatus, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus fermentum, Streptococcus thermophilus, or a mixture thereof.

[0019] In another preferred embodiment, the probiotic is Lactobacillus mucilaginosus fermentans.

[0020] According to one aspect of the present invention, the present invention provides the use of probiotics in the preparation of medicines or health products for treating or preventing inflammation, hypertension or depression, or for improving brain function or improving sleep, wherein the probiotics are selected from the group consisting of: Lactococcus lactis including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris, Lactobacillus curvatus, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus mucosa fermentation, Streptococcus thermophilus or a mixture thereof, and preferably the probiotics can be obtained by screening according to the method described in the present invention.

[0021] In a preferred embodiment, the probiotic is selected from the group consisting of: Lactococcus lactis subsp. Lactis (CGMCC No. 1.1936), Lactococcus Cremoris (CICC No. 24337), Latilactobacillus curvatus (CICC No. 25172), Pediococcus acidilactici (CGMCC No. 1.4), Lactobacillus plantarum (CGMCC No. 1.12934), Lactobacillus fermentum (CGMCC No. 1.15608), Streptococcus thermophilus (CICC No. 20174), or a mixture thereof.

[0022] In a preferred embodiment, the medicine or health product is used to treat or prevent inflammation, hypertension or depression in mammals (including humans and animals), or to improve brain function or sleep in mammals (including humans and animals). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0024] Figure 1 Demonstrate the ability of each screened strain to produce GABA; and

[0025] Figure 2 Display the OD of each strain screened 600 value. DETAILED DESCRIPTION

[0026] Unless otherwise specified, the terms used herein have the general technical meanings understood by those skilled in the art.

[0027] In the present invention, the term "probiotics" refers to microorganisms that improve the microecological balance of the host and play a beneficial role, thereby improving the health level and health status of the host. The beneficial bacteria or fungi in animals mainly include: lactic acid bacteria, bifidobacteria, actinomycetes, yeasts, etc. In the present invention, probiotics are intestinal probiotics that can regulate the balance of intestinal flora, promote nutrient absorption and maintain intestinal health. All probiotics known in the prior art can be used as candidate probiotics in the screening method of the present invention.

[0028] In the present invention, the term "glutamate decarboxylase (GadB)" refers to an enzyme that catalyzes the decarboxylation of glutamate to generate γ-aminobutyric acid (GABA). In a specific embodiment, the Uniport entry of GadB is P69910.

[0029] In the present invention, the term "membrane-bound antiporter (GadC)" refers to a carrier protein that is bound to the cell membrane and can transport two solutes across the membrane in opposite directions. In a specific embodiment, the Uniport entry of GadC is P63235.

[0030] In the present invention, the term "homologous protein" refers to a protein with obvious similarity in amino acid sequence, which performs the same or similar function in different organisms or the same organism. The protein sequence for homology analysis can be newly discovered, or can be obtained through public databases such as GenBank of NCBI, UniProt, etc. Bioinformatics tools known in the art can be selected for homology search, and BLAST (Basic Local Alignment Search Tool) is one of the most widely used tools. By analyzing the alignment results, the similarity and possible evolutionary relationship between the sequences are evaluated, and based on the alignment results, the function of the target protein can be predicted and its evolutionary relationship can be inferred. Through homology analysis, the function of an unknown protein can be predicted, especially when it is similar to a protein sequence of a known function. In a specific embodiment, GadB and GadC can be used as protein sequences of known functions, and a search for homologous proteins is performed in the above public databases to obtain probiotics having the above homologous proteins. In a specific embodiment, homologous proteins with a homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% can be selected.

[0031] In the present invention, the term "probiotics having glutamate decarboxylase (GadB) or a homologous protein thereof" refers to probiotics that can express GadB protein or a homologous protein thereof, or that contain a gene encoding GadB protein or a homologous protein thereof in their genome.

[0032] In the present invention, the term "probiotics having a membrane-bound antiporter (GadC) or a homologous protein thereof" refers to probiotics that can express GadC protein or a homologous protein thereof, or that contain a gene encoding GadC protein or a homologous protein thereof in their genome.

[0033] In the present invention, the term "simulated intestinal fluid" refers to artificial intestinal fluid, also known as synthetic intestinal fluid, which is a solution of components and pH of simulated intestinal fluid. Generally, the simulated intestinal fluid is mainly composed of phosphate, trypsin, etc., and the pH value is about 6.8. Those skilled in the art can prepare the simulated intestinal fluid according to the provisions of the Chinese Pharmacopoeia, for example. Alternatively, the simulated intestinal fluid can also be purchased from commercial channels.

[0034] In a preferred embodiment of the method for screening probiotics having the function of producing γ-aminobutyric acid (GABA) according to the present invention, the conserved domains of GadB and GadC protein sequences and the list of edible probiotics are used as input and comparison databases for homology analysis, respectively, and the experimental strains are selected from the homology analysis results based on the annotation content and the degree of similarity.

[0035] Specifically, the present invention analyzes the key enzymes and transporters in the γ-aminobutyric acid synthesis pathway. By means of bioinformatics, the key proteins and transporters are screened within the scope of the summarized probiotic list, and the EC number of the key step is determined to be 4.1.1.15. The synthesis pathway is that L-glutamate (L-glutamate) is decarboxylated to generate γ-aminobutyric acid (4-aminobutanoate) and carbon dioxide under the catalysis of glutamic acid decarboxylase (glutamatedecarboxylase, GAD).

[0036] In a specific embodiment, the γ-aminobutyric acid synthesis pathway is retrieved using the MetaCyc database (https: / / metacyc.org / ), and the keyword GABA is entered on the homepage to search for its synthesis pathway and EC number. The EC number in the synthesis pathway obtained by searching the MetaCyc database is entered on the homepage of the KEGG database to obtain the key enzyme for synthesizing γ-aminobutyric acid. Through literature research on the synthesis of γ-aminobutyric acid by microorganisms, transport proteins that play a key role in the pathway are sought. After screening, they were finally determined to be: intracellular glutamate decarboxylase (GadB, encoded by gadB), and membrane-bound antiporter (GadC, encoded by gadC). A representative Uniport entry for GadB is P69910; a representative Uniport entry for GadC is P63235.

[0037] The databases used for strain search include eggNOG, Gotenzymes, NCBI, etc., which are used to search for probiotics with the function of synthesizing γ-aminobutyric acid. Enter the keywords GadB and GadC on the eggNOG main page, search for strains containing GadB and GadC in the database respectively, and take the intersection for analysis, and select representative probiotics from the results for experimental verification.

[0038] Probiotics with γ-aminobutyric acid synthesis pathway were obtained by searching key proteins through BLAST and Gotenzymes pathways. Specifically, 7 species were selected: Lactiplantibacillus plantarum, Streptococcus thermophilus, Propionibacteriumfreudenreichii subsp. Shermanii, Latilactobacillus curvatus, Pediococcus acidilactici, Lactobacillus reuteri, Lactococcus lactis subsp. Lactis and Limosilactobacillusfermentum. Among them, one: Streptococcus thermophilus belongs to the "List of Probiotics that Can Be Used in Health Foods" issued by the National Health and Safety Commission, and six: Lactiplantibacillus plantarum, Propionibacteriumfreudenreichii subsp. Shermanii, Latilactobacillus curvatus, Pediococcus acidilactici, Lactococcus lactissubsp.Lactis and Limosilactobacillus fermentum belong to the "List of Bacteria that Can Be Used in Food" issued by the National Health and Safety Commission. These 7 strains were cultured in vitro and their ability to synthesize γ-aminobutyric acid was tested. The 7 strains are Lactococcus lactissubsp.Lactis (CGMCC No. 1.1936), Lactococcus Cremoris (CICC No. 24337), Latilactobacillus curvatus (CICC No. 25172), Pediococcus acidilactici acidilactici (CGMCC No. 1.4), Lactobacillus plantarum (CGMCC No. 1.12934), Lactobacillus fermentum (CGMCC No. 1.15608) and Streptococcus thermophilus (CICC No. 20174). The results of the wet test are shown in the figure. Figure 1 and Figure 2 As shown, the results showed that all seven strains had a certain ability to produce GABA, among which Lactobacillus fermentum had the strongest ability to produce GABA.

[0039] The present invention completes the process of metabolic pathway investigation and bioinformation mining, so that the efficiency of selecting the required probiotics from the database is improved, the cost is reduced, and the time is shortened. Natural probiotics have the advantages of high safety performance and easy conversion, and are the best choice for screening functional probiotics. However, conventional screening methods rely on high-throughput screening, and all strains need to be inoculated and grown on a selective culture medium, and then the required strains are selected. The experimental cycle is long, the efficiency is low, and it is difficult to obtain the required results. The present invention uses core characteristic gene homology analysis, compared to cultivating and screening out the expected strains from natural probiotics, and has the advantage of purposefully analyzing and finding targets, and can quickly find the required strains through homology analysis of the core gene sequence.

[0040] The present invention is further described in the following examples. These examples are only for illustration and are not intended to limit the scope of the present invention. The chemical substances used in the following reactions are all commercially available products unless otherwise indicated.

[0041] Example 1

[0042] Search for key genes in synthetic pathways

[0043] The γ-aminobutyric acid synthesis pathway was searched using the MetaCyc database (https: / / metacyc.org / ). The keyword GABA was entered on the homepage to search for its synthesis pathway and EC number. The EC number in the synthesis pathway obtained by searching the MetaCyc database was entered on the homepage of the KEGG database to obtain the key enzymes for synthesizing γ-aminobutyric acid. Through literature research on the synthesis of γ-aminobutyric acid by microorganisms, transport proteins that play a key role in the pathway were found. After screening, they were finally determined to be: intracellular glutamate decarboxylase (GadB, encoded by gadB), and membrane-bound antiporter (GadC, encoded by gadC). A representative Uniport entry for GadB is P69910; a representative Uniport entry for GadC is P63235.

[0044] Example 2

[0045] Strain screening

[0046] The databases used for strain search include eggNOG, Gotenzymes, NCBI, etc., which are used to search for probiotics with the function of synthesizing γ-aminobutyric acid. Enter the keywords GadB and GadC on the eggNOG main page, search for strains containing GadB and GadC in the database respectively, and take the intersection for analysis. According to the characteristics of the strains, representative probiotics are selected from the results for experimental verification.

[0047] Example 3

[0048] Wet test verification

[0049] The selected experimental strains were purchased from China General Microorganism Culture Collection Center (CGMCC) and China Industrial Microorganism Culture Collection Center (CICC): Lactococcus lactis subsp. Lactis (CGMCC No. 1.1936), Lactococcus Cremoris (CICC No. 24337), Latilactobacillus curvatus (CICC No. 25172), Pediococcus acidilactici (CGMCC No. 1.4), Lactobacillus plantarum (CGMCC No. 1.12934), Lactobacillus fermentum (CGMCC No. 1.15608) and Streptococcus thermophilus (CICC No. 20174). The glycerol bacteria were inoculated into De MRS broth medium (Thermo Fisher, catalog number: CM0359B) for subculture. After three inoculations, the probiotic strains were inoculated again into 7 mL of fresh MRS liquid medium at a 1% inoculation amount and cultured aerobically or anaerobically in a 37°C constant temperature incubator for 20 h. Each strain had one tube of bacterial liquid.

[0050] Then, 1.5 ml of each bacterial suspension was taken out and the OD of each strain was measured using a spectrophotometer. 600 , the results are shown in Figure 2 OD of Latilactobacillus curvatus (CICC No. 25172) 600 The OD of Pediococcus acidilactici (CGMCC No. 1.4) was 1.432. 600The OD of Lactobacillus fermentum (CGMCC No. 1.15608) was 1.444. 600 The OD of Lactococcus lactis subsp. Lactis (CGMCC No. 1.1936) was 1.507. 600 The OD of Streptococcus thermophilus (CICC No. 20174) was 1.176. 600 The OD of Lactobacillus plantarum (CGMCC No. 1.12934) was 0.496. 600 The OD of Lactococcus Cremoris (CICC No. 24337) was 1.618. 600 The yield of γ-aminobutyric acid was 1.355. Each tube of bacterial solution was then divided into 3 tubes to determine the yield of γ-aminobutyric acid. First, the bacterial solution was centrifuged at 2000rcf for 4 minutes, the precipitate was discarded, and the supernatant was placed in a centrifuge tube. The yield of γ-aminobutyric acid was then determined using a γ-aminobutyric acid kit (ELISA, catalog number: m1092747). The principle is that phenol and sodium hypochlorite react with γ-aminobutyric acid to produce a blue-green product with a maximum absorbance at 640nm.

[0051] Determination steps: After completing the operation according to the kit instructions, take 200 μL from each centrifuge tube and place it in a 96-well plate to measure the absorbance value A at 640 nm. 测定 With A 空白 , ΔA=A 测定 -A 空白 Record the data and use the calculation formula of absorbance and GABA concentration in the kit instructions (the regression equation under standard conditions is y=0.0265x-0.0163, R 2 =0.9953; x is the concentration of the standard (umol / mL), y is the absorbance value), and the GABA production of each strain was calculated. The specific results are shown in Figure 1The plotting method is the mean value plus or minus the standard deviation. Among them, the average GABA production of Latilactobacillus curvatus (CICC No. 25172) is 6.1497mM (mmol / L), the average GABA production of Pediococcus acidilactici (CGMCC No. 1.4) is 5.8604mM, the average GABA production of Lactobacillus fermentum (CGMCC No. 1.15608) is 12.4264mM, the GABA production of Lactococcus lactis subsp. Lactis (CGMCC No. 1.1936) is 9.6214mM, the GABA production of Streptococcus thermophilus (CICC No. 20174) is 8.6151mM, and the GABA production of Lactobacillus plantarum (CGMCC No. 1.15608) is 12.4264mM. plantarum) (CGMCC No. 1.12934) had an average GABA production of 6.4138 mM, and Lactococcus Cremoris (CICC No. 24337) had a GABA production of 7.4578 mM. All seven target strains had the ability to produce GABA, among which Lactobacillus fermentum had the highest production, verifying the reliability of the strain mining and selection process.

[0052] Those skilled in the art should understand that, although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, without departing from the spirit of the present invention, those skilled in the art can make appropriate modifications or improvements, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

[0053] References

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[0055] [2]Tajabadi N, Baradaran A, Ebrahimpour A, Rahim RA, Bakar FA, Manap MY, Mohammed AS, Saari N. Overexpression and optimization of glutamatedecarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production. Microb Biotechnol. 2015Jul; 8(4):623-32.

[0056] [3] Lili Yao, Changjiang Lyu, Yuting Wang, Sheng Hu, Weirui Zhao, HongweiCao, Jun Huang, Lehe Mei, High-level production of γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli, Engineering Microbiology, Volume 3, Issue 2, 2023, 100077, ISSN2667-3703.

[0057] [4]KANG TJ, HO NA, HACK SP. Buffer-free production of gamma-aminobutyricacid using an engineered glutamate decarboxylase from Escherichia coli. Enzymeand Microbial Technology, 2013, 53(3):200-205.

[0058] [5] Tian Lingzhi, Xu Meijuan, Rao Zhiming. Construction of a recombinant Escherichia coli / pET-28a-lpgad and optimization of its conversion conditions for efficient production of γ-aminobutyric acid [J]. Chinese Journal of Biotechnology, 2012, 028(001): 65-75.

[0059] [6] Tan Xiao, Sun Qing, Zeng Lin, et al. Cloning and expression of glutamate decarboxylase gene from a γ-aminobutyric acid-producing Lactobacillus plantarum strain [J]. Food Science, 2018, 39(18): 159-165.

[0060] [7] Yuan H, Wang H, Fidan O, Qin Y, Xiao G, Zhan J. Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli. J Biol Eng. 2019 Mar 21; 13:24.

[0061] [8] Zhang Heng, Liu Huiyan, Pan Lin, Wang Hongyan, Li Xiaofang, Wang Tong, Fang Haitian. Research strategies for biosynthesis of γ-aminobutyric acid. Chinese Journal of Biotechnology, 2021, 41(8): 110-119.

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Claims

1. A method for screening probiotics having the function of producing gamma-aminobutyric acid (GABA), the method comprising the following steps: 1) selecting probiotics having glutamate decarboxylase (GadB) or its homologous protein from candidate probiotics; 2) selecting a probiotic having a membrane-bound antiporter (GadC) or a homologous protein thereof from the probiotics obtained in step 1); and 3) The probiotics obtained in step 2) are subjected to a fermentation experiment for producing γ-aminobutyric acid, and those probiotics having the function of producing γ-aminobutyric acid are selected.

2. The method according to claim 1, wherein the probiotics are intestinal probiotics, preferably edible probiotics.

3. The method according to claim 1 or 2, wherein: The selection in step 1) and step 2) is performed by homology analysis of protein sequences; and / or in step 3) OD is selected 600 The probiotics with the highest values ​​and / or the highest GABA concentration in the supernatant.

4. The method according to any one of claims 1 to 3, wherein the fermentation production of γ-aminobutyric acid is carried out in an in vitro simulated intestinal fluid environment, preferably under aerobic or microaerobic conditions.

5. The method according to any one of claims 1 to 4, wherein the probiotic is selected from the group consisting of Lactococcus lactis including Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. Cremoris, Latilactobacillus curvatus, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus fermentum, Streptococcus thermophilus, or a mixture thereof. The method according to claim 5 , wherein the probiotic is Lactobacillus mucilaginosus ferment.

7. Use of probiotics in the preparation of medicines or health products for treating or preventing inflammation, hypertension or depression, or for improving brain function or sleep, wherein the probiotics are selected from the group consisting of: Lactococcus lactis including Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris, Lactobacillus curvatus, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus mucosa fermentation, Streptococcus thermophilus, or a mixture thereof, and preferably the probiotics can be obtained by screening according to the method according to any one of claims 1 to 6.

8. The use according to claim 7, wherein the probiotic is selected from the group consisting of: Lactococcus lactis subsp. lactis (CGMCC No. 1.1936), Lactococcus lactis subsp. cremoris (CICC No. 24337), Lactobacillus curvatum (CICC No. 25172), Pediococcus acidilactici (CGMCC No. 1.4), Lactobacillus plantarum (CGMCC No. 1.12934), Lactobacillus fermentans (CGMCC No. 1.15608), Streptococcus thermophilus (CICC No. 20174), or a mixture thereof.

9. The use according to claim 7 or 8, wherein the medicine or health product is used to treat or prevent inflammation, hypertension or depression in mammals (including humans and animals), or to improve brain function or sleep in mammals (including humans and animals).

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