Lactiplantibacillus plantarum strain JN9, preparation, probiotic composition and use thereof

Abstract

Disclosed in the present invention are a Lactiplantibacillus plantarum strain JN9, a preparation, a probiotic composition and the use thereof. The present application finds a novel Lactiplantibacillus plantarum strain JN9 by means of screening, and the accession number thereof is: CCTCCNO: M20241647c. By means of analysis of basic characteristics of the strain (growth curve, acid tolerance, bile salt tolerance and gastrointestinal fluid tolerance), safety evaluation (antibiotics sensitivity, hemolysis capacity, toxin production capacity, cytotoxicity and bioamine production capacity), adhesion evaluation, GABA production evaluation, alcohol tolerance evaluation and acetaldehyde dehydrogenase production capability evaluation, said strain is found to be a safe strain, and exhibits GABA production performance, alcohol tolerance and acetaldehyde dehydrogenase activity

Description

Lactobacillus plantarum JN9, preparations, probiotic compositions and their uses

[0001] This application claims priority to Chinese Patent Application No. 202411199513.X, filed on August 29, 2024, entitled "Lactobacillus plantarum JN9, Preparations, Probiotic Compositions and Uses Thereof", the contents of which shall be construed as incorporated herein by reference. Technical Field

[0002] This article generally relates to the field of bioapplication technology, especially to a plant lactobacillus JN9, its preparations, probiotic compositions and their uses. Background Technology

[0003] Gamma-aminobutyric acid (GABA) is a non-protein, naturally occurring amino acid widely found in animals, plants, and microorganisms. GABA is an important inhibitory neurotransmitter in the central nervous system, regulating disorders associated with depression and other mental health problems. Studies have shown that adequate GABA intake can improve sleep, lower blood pressure, treat epilepsy, and regulate mood. Animal and clinical trials have also confirmed GABA's efficacy in improving depressive-like behavior in mice and alleviating depression in humans. With further research, GABA has been developed into a novel functional factor and is widely used in the pharmaceutical, food, and agricultural industries.

[0004] GABA can be prepared by chemical synthesis or biosynthesis. Biosynthesis of GABA may be more promising than chemical synthesis due to its simple reaction steps, high catalytic efficiency, mild reaction conditions, and good environmental compatibility.

[0005] Probiotics are live microorganisms that colonize the human body and alter the composition of the gut microbiota in a specific area, thus providing beneficial effects to the host. Probiotics promote nutrient absorption and maintain gut health by regulating the host's mucosal and systemic immune functions or by modulating the balance of gut microbiota, thereby producing single microorganisms or well-defined mixtures of microorganisms that contribute to health. Finding a highly safe probiotic strain that can produce GABA is of great significance for improving mood, combating depression, and improving sleep quality. Summary of the Invention

[0006] This application provides a Lactiplantibacillus plantarum JN9, wherein the classification name of Lactiplantibacillus plantarum is Lactiplantibacillus plantarum, and the accession number is CCTCC NO: M 20241647.

[0007] On the other hand, this application also provides a formulation prepared from Lactobacillus plantarum JN9 as described herein.

[0008] In another aspect, this application also provides a probiotic composition comprising Lactobacillus plantarum JN9 as described herein or a formulation as described herein.

[0009] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of medicaments for reducing stress, improving mood, treating depression, improving sleep quality, and / or lowering blood pressure.

[0010] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of medicaments for liver protection and / or prevention of alcohol hangovers.

[0011] On the other hand, this application also provides the use of the Lactobacillus plantarum JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of food or health products.

[0012] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9 described herein in the production of GABA.

[0013] This application provides a strain of *Lactobacillus plantarum* capable of producing GABA, tolerant to alcohol, and exhibiting good safety, acid, bile salt, and gastrointestinal tolerance, as well as antibiotic sensitivity. This strain is named *Lactobacillus plantarum* JN9. It is deposited at the China Center for Type Culture Collection (CCTCC), School of Life Sciences, Wuhan University, Wuhan, Hubei Province, China, on July 22, 2024. The accession number is CCTCC NO: M 20241647.

[0014] Analysis of the strain's basic characteristics (growth curve, acid resistance, bile salt resistance, and gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolytic ability, toxin production ability, cytotoxicity, and ability to produce biogenic amines), adhesion evaluation, GABA production evaluation, alcohol tolerance evaluation, and acetaldehyde dehydrogenase production ability revealed that *Lactobacillus plantarum* JN9 is a safe strain with excellent GABA production performance, high alcohol tolerance, and high acetaldehyde dehydrogenase activity. Based on the efficacy of the produced GABA, strain JN9 can be used in products that improve mood (such as reducing stress, anti-depression, improving sleep quality), lower blood pressure, and have anti-diabetic, anti-cancer, antioxidant, anti-inflammatory, antimicrobial, and anti-allergic effects. Based on strain JN9's strong alcohol tolerance and high acetaldehyde dehydrogenase activity, it can be used in products that enhance alcohol tolerance during drinking and alleviate post-drinking discomfort.

[0015] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and the accompanying drawings. (Summary of Drawings)

[0016] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0017] Figure 1 is a pie chart of the *Lactobacillus plantarum* JN9 genome in the embodiments of this application. The pie chart shows seven types of information from the outside in: the first circle is the genomic location information, the second circle is the GC content information, the third circle is the coding gene on the positive strand (marked in red), the fourth circle is the coding gene on the negative strand (marked in green), the fifth circle is the ncRNA on the positive strand (marked in blue), the sixth circle is the ncRNA information on the negative strand (marked in purple), and the seventh circle is the long repetitive sequence information on the genome (marked in orange).

[0018] Figure 2 shows the colony morphology and Gram staining results of *Lactobacillus plantarum* JN9 in the embodiments of this application. (a) shows the colony morphology of *Lactobacillus plantarum* JN9, and (b) shows the cell morphology of *Lactobacillus plantarum* JN9.

[0019] Figure 3 shows the results of the API 50CH sugar fermentation test of Lactobacillus plantarum JN9 in the embodiments of this application.

[0020] Figure 4 shows the growth curve of Lactobacillus plantarum JN9 in the embodiments of this application within 48 hours.

[0021] Figure 5 shows the tolerance assessment of *Lactobacillus plantarum* JN9 in the embodiments of this application. (a) shows the survival rate of *Lactobacillus plantarum* JN9 after 3 hours in MRS at pH 3.0 or containing 0.3% bile salts; (b) shows the survival rate of *Lactobacillus plantarum* JN9 after 30 minutes and 1 hour in a sterile electrolyte solution containing 100 mg / L lysozyme.

[0022] Figure 6 shows the hemolysis results of Lactobacillus plantarum JN9 in the embodiments of this application.

[0023] Figure 7 shows the cytotoxicity results of *Lactobacillus plantarum* JN9 against HT-29 intestinal epithelial cells in the embodiments of this application. Data are expressed as Mean ± Sem, n = 6.

[0024] Figure 8 shows the results of cell adhesion of Lactobacillus plantarum JN9 in the embodiments of this application.

[0025] Figure 9 shows the results of GABA production capacity of *Lactobacillus plantarum* JN9 in the embodiments of this application. (a) is a PCR amplification diagram of gadB; (b) is a mass spectrum of the GABA standard and the fermentation broth of *Lactobacillus plantarum* JN9.

[0026] Figure 10 shows the detection of the acetaldehyde dehydrogenase gene (aldH) and alcohol dehydrogenase gene (adh) of *Lactobacillus plantarum* JN9 in the embodiments of this application. (a) is the amplification map of the adh gene, and (b) is the amplification map of the aldh gene. (Detailed description follows.)

[0027] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood as equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with lower or preferred values, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0028] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0029] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0030] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0031] Currently, most probiotic products on the market focus on gastrointestinal regulation, women's physiological health, and pet health, with no leading brands or best-selling products specifically targeting emotional health and alleviating anxiety and depression. This application aims to develop strains with antidepressant effects for individuals suffering from insomnia, anxiety, depression, and other emotional problems. It screens for probiotics capable of converting monosodium glutamate (MSG), a common seasoning in daily diets, into GABA. This provides the possibility of discovering sleep-improving and antidepressant probiotics and complements existing antidepressant probiotic product combinations. Furthermore, *Lactobacillus plantarum* JN9, in addition to its high GABA conversion capacity, also exhibits strong alcohol tolerance and high aldehyde dehydrogenase activity, making this strain potentially applicable not only in the sleep improvement and anxiety relief market but also in the alcohol tolerance and post-drinking discomfort relief market.

[0032] This application provides a strain of *Lactiplantibacillus plantarum* JN9, which is classified as *Lactiplantibacillus plantarum* and has the accession number CCTCC NO: M 20241647. This strain is the first to be isolated from fermented food, and whole-genome sequencing indicates that it is a *Lactiplantibacillus plantarum* strain not previously reported in NCBI.

[0033] On the other hand, this application also provides a formulation prepared from Lactobacillus plantarum JN9 as described herein.

[0034] In some implementations, the formulation is selected from one or more of the following:

[0035] 1) Inoculum of *Lactobacillus plantarum* JN9; 2) Inoculum suspension of *Lactobacillus plantarum* JN9; 3) Metabolites of *Lactobacillus plantarum* JN9; 4) Metabolites of *Lactobacillus plantarum* JN9; and 5) Fermented extract of *Lactobacillus plantarum* JN9.

[0036] In some embodiments, *Lactobacillus plantarum* JN9, as described herein, is fermented and then used to prepare the various formulations described herein. The methods for preparing the various formulations described herein are known to those skilled in the art.

[0037] In another aspect, this application also provides a probiotic composition comprising Lactobacillus plantarum JN9 as described herein or a formulation as described herein.

[0038] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the following: Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Weizmannia, Mammaliicoccus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis. subtilis).

[0039] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the following: *Bifidobacterium adolescentis*, *Bifidobacterium animalis* subsp. *animalis*, *Bifidobacterium animalis* subsp. *lactis*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum* subsp. *longum*, *Bifidobacterium longum* subsp. *infantis*, *Lactobacillus acidophilus*, *Lactobacillus crispatus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, and *Lactobacillus delbrueckii* subsp. *lactis*. Lactobacillus subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, and Lactobacillus kefiranofaciens subsp.Lactobacillus kefiranofaciens, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar. The following bacteria are listed: *Lactococcus cremoris*, *Propionibacterium freudenreichii* subsp. *shermanii*, *Acidipropionibacterium acidipropionici*, *Leuconostoc mesenteroides* subsp. *mesenteroides*, *Pediococcus acidilactici*, *Pediococcus pentosaceus*, *Weizmannia coagulans*, *Mammaliicoccus vitulinus*, *Staphylococcus xylosus*, *Staphylococcus carnosus*, *Kluyveromyces marxianus*, and *Bacillus subtilis* DE111. .

[0040] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of medicaments for reducing stress, improving mood, treating depression, improving sleep quality, and / or lowering blood pressure.

[0041] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of medicaments for liver protection and / or prevention of alcohol hangovers.

[0042] In some implementations, the drug is administered orally.

[0043] On the other hand, this application also provides the use of the Lactobacillus plantarum JN9, the formulations described herein, or the probiotic compositions described herein in the preparation of food or health products.

[0044] In some implementations, the food or health product is used to reduce stress, improve mood, combat depression, improve sleep quality, lower blood pressure, and / or enhance alcohol tolerance.

[0045] In some implementations, the food or health product is a dairy product, soy product, meat product, fruit and vegetable product, beverage, or snack. In some implementations, the food or health product also contains edible excipients.

[0046] In some embodiments, the food is a health food; or the food includes dairy products, soy products, meat products, or fruit and vegetable products; or the food is a beverage or snack. In some embodiments, the food contains *Lactobacillus plantarum* JN9 and edible additives as described herein.

[0047] On the other hand, this application also provides the use of *Lactobacillus plantarum* JN9 described herein in the production of GABA.

[0048] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may substitute for any other feature in any other embodiment.

[0049] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0050] Furthermore, in describing representative embodiments, the specification may have presented methods and / or processes as a specific sequence of steps. However, the method or process should not be limited to the specific order of steps described herein, to the extent that it does not depend on such a specific order. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Moreover, the claims concerning the method and / or process should not be limited to the steps performed in the written order, and those skilled in the art will readily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0051] Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. Unless otherwise defined or stated, all technical and scientific terms used in this application have the same meaning as those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this application.

[0052] Example 1: Obtaining and Identifying Lactobacillus plantarum JN9

[0053] The *Lactobacillus plantarum* JN9 strain provided in this application was isolated from a type of pickled vegetable from Sichuan, China.

[0054] 1. Sequencing of 16S rRNA of Lactobacillus plantarum JN9

[0055] Fresh *Lactobacillus plantarum* JN9 cells were collected after culturing at 37℃ for 16 h, and DNA was extracted using a DNA extraction kit (9763, Takara, Japan). Genomic DNA was amplified by 16S rRNA PCR, purified, and then sequenced. The 16S rRNA amplification primers were universal primers 27F: 5'-AGAGTTTTGATCCTGTCCAG-3' (SEQ ID NO:1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:2). The PCR reaction conditions were: 94℃ denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 1 min, 72℃ extension for 1 min, for a total of 30 cycles, followed by a 10-min extension at 72℃. The PCR product was purified and subjected to first-generation sequencing. The obtained 16S rRNA sequence is shown in SEQ ID NO:3.

[0056] Sequencing of the 16S rRNA of Lactiplantibacillus plantarum JN9 revealed a 99.86% similarity to the standard strain SRCM100442. Based on the physiological and biochemical indicators, morphological characteristics, and molecular biological identification results of strain Lactiplantibacillus plantarum JN9, we named it Lactiplantibacillus plantarum JN9 and deposited it at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO: M 20241647.

[0057] 2. Whole genome sequencing of Lactobacillus plantarum JN9

[0058] For genome sequencing, *Lactobacillus plantarum* JN9 was cultured in MRS broth under anaerobic conditions at 37°C for 24 hours. Single colonies were cultured overnight in MRS broth. The broth was centrifuged at 8000×g, 4°C, for 5 min. The precipitate was sent to GENEWIZ Sequencing (China) for sequencing, assembly, annotation, and bioinformatics analysis.

[0059] Whole-genome sequencing was performed on an Illumina PE150 platform and a PacBio Sequel system. For PacBio sequencing libraries, 5–10 μg of genomic DNA was cleaved into 10–15 kb fragments using a g-TUBE device. Then, [the sequencing was] performed using [the appropriate methods]. The Express Template Preparation Kit 2.0 was used to construct the library. In short, DNA fragments were cut and subjected to single-strand overhang removal, DNA damage repair, end repair, A-tailing, and ligation with barcode-labeled overhang adapters. The library was quantified using a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA) and its size was checked using an Agilent 2100 bioanalyzer system. Subsequent steps were performed according to the manufacturer's instructions to prepare the SMRTbell library. The library was sequenced using the PacBio Sequel platform. PacBio reads were assembled using Hifiasm / Canu. The genome was then recalibrated using previous Illumina data using Pilon software. Prodigal / Augustus gene search software was used to locate coding genes. Transfer RNA (tRNA) was detected in the genome using the tRNAscan-SE program with default parameter settings. rRNA was identified using Barrnap. Other RNAs were identified using the rfam database. Coding genes were annotated using the National Center for Biotechnology Information (NCBI) NR database via Diamond. Gene function was then annotated using the GO (Gene Ontology) database, and pathways were annotated using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Proteins encoded by the genes were phylogenetically classified using the COG (Clusters of Orthologous Groups) database. Protein sequences with E < 1e-5 were retrieved using Diamond from the CAZy, Swiss_Prot, Pfam, CARD, VFDB, or DFVF databases.

[0060] The genome sequence of *Lactobacillus plantarum* JN9 was assembled and analyzed. Basic genome information is shown in Table 1. The genome length is 3187478 bp, containing 3151 genes, including 3029 protein-coding genes. A genome circle map displaying gene, ncRNA, GC content, and repetitive sequence information was created using Circos (version 0.69) software, as shown in Figure 1.

[0061] Table 1. Basic genomic information of Lactobacillus plantarum JN9

[0062] 3. Identification of Lactobacillus plantarum JN9

[0063] The genus and species of *Lactobacillus plantarum* JN9 were determined by calculating the best hit (one-way ANI) and the best mutual hit (two-way ANI) between the genome sequence of *Lactobacillus plantarum* JN9 and the standard strain GCA_009913655.1 (ASM991365v1).

[0064] A search for the complete genome or genome draft of *Lactobacillus plantarum* in NCBI (accessed on February 20, 2024) yielded 1215 *Lactobacillus plantarum* genome sequences. A database of these 1215 *Lactobacillus plantarum* genomes was created using Makeblastdb software. The complete genome of *Lactobacillus plantarum* JN9 was compared with the database using Blastn, and mismatched regions and their numbers were generated for each comparison entry, assuming 1000 mismatches.

[0065] The ANI value of the *Lactobacillus plantarum* JN9 genome sequence compared to the reference strain GCA_009913655.1, obtained using an online ANI calculator, was 99.10%, indicating that *Lactobacillus plantarum* JN9 is indeed *Lactobacillus plantarum*. Furthermore, a comparison of the *Lactobacillus plantarum* JN9 genome with 1215 *Lactobacillus plantarum* genomes using Blasten revealed no completely identical or at most 1000 mismatched references, indicating that *Lactobacillus plantarum* JN9 is a strain never before reported in NCBI.

[0066] Example 2. Characterization of Lactobacillus plantarum JN9

[0067] Fermentation culture was performed on Lactobacillus plantarum JN9, and the following characteristics of the strain were determined.

[0068] 1) Morphological observation and sugar fermentation test of Lactobacillus plantarum JN9

[0069] Colony morphology of Lactobacillus plantarum JN9 was observed, and single colonies were picked for Gram staining and examined under a 100x microscope.

[0070] The sugar fermentation assay of *Lactobacillus plantarum* JN9 was performed using API 50CH (50300, API, France). The procedure was as follows: After culturing *Lactobacillus plantarum* JN9 at 37°C for 16 hours, fresh bacterial cells were collected. The cells were washed twice with sterile PBS and resuspended in sterile PBS, with the OD adjusted to 0.3. 1 mL of the resuspended bacterial solution was added to 10 mL of 50CH medium, mixed thoroughly, and then 150 μL was added to each well of a 50CH test strip. The mixture was incubated at 37°C for 48 hours. Results were determined by color change; a yellow color compared to the control group indicated a positive result (with a dark red color for esculin positive), while no color change indicated a negative result.

[0071] The results showed that *Lactobacillus plantarum* JN9 colonies grown on MRS plates were milky white, round, with neat edges, smooth and moist surfaces, and a colony diameter of approximately 1-2 mm (Figure 2(a)). Gram staining of single colonies revealed them to be Gram-positive bacteria, appearing as rod-shaped bacteria (Figure 2(b)).

[0072] Figure 3 shows the results of carbohydrate utilization by *Lactobacillus plantarum* JN9: *Lactobacillus plantarum* JN9 can metabolize D-ribose (5), D-galactose (10), D-glucose (11), D-fructose (12), D-mannitol (13), mannitol (18), methyl-α-D-mannopyranoside (20), methyl-α-D-glucopyranoside (21), N-acetylglucosamine (22), amygdalin (23), arbutin (24), aescin (25), salicin (26), D-cellobiose (27), D-maltose (28), D-lactose (29), D-micobiose (30), D-sucrose (31), D-trehalose (32), D-minotriose (34), D-gentiobiose (39), D-thulene (40) and potassium gluconate (47) to produce acid through fermentation.

[0073] 2) Determination of the growth curve of Lactobacillus plantarum JN9

[0074] Growth curves of *Lactobacillus plantarum* JN9 were plotted using a Growth Profiler 960 (Enzyscreen BV, Heemstede, Netherlands). The specific procedure was as follows: *Lactobacillus plantarum* JN9 was cultured in MRS for 16 hours and then inoculated into fresh MRS broth, with the final OD600 adjusted to 0.1. The OD-adjusted bacterial culture was added to sterile 96-well plates, ensuring 250 μL per well, for a total of 11 replicates (A1-B9). The plates were incubated aerobicly at 200 rpm and 37°C for 48 hours, with OD values ​​recorded every 30 minutes. MRS liquid medium was used as a blank control group.

[0075] Lactobacillus plantarum JN9 with an initial OD of 0.1 was cultured in MRS liquid medium at 37°C and 200 rpm. It reached the exponential growth phase within 16 hours and then entered the stationary phase within the next 48 hours. Figure 4 shows the growth curve of Lactobacillus plantarum JN9 fitted by Growth Profiler 960.

[0076] 3) Tolerance of Lactobacillus plantarum JN9

[0077] Acid and bile salt resistance

[0078] Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C for 16 h. Fresh bacterial culture was collected by centrifugation at 12000 rpm for 2 minutes at 4°C. The bacterial cells were washed twice with sterile PBS buffer, resuspended in PBS (pH=7), and the bacterial concentration was adjusted to 10-1. 8 CFU / mL. The resuspended bacterial culture was inoculated at a 5% addition rate into MRS broth at pH 3.0 or containing 0.3% bile salts (w / v, Sigma, USA) and incubated at 37°C for 3 hours. The viable counts of *Lactobacillus plantarum* JN9 in different culture media were recorded before incubation (N0) and after 3 hours of incubation (N1).

[0079] Lysozyme resistance

[0080] Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C for 16 h. Fresh bacterial culture was collected by centrifugation at 12000 rpm for 2 min at 4°C. The bacterial cells were washed twice with sterile PBS buffer and resuspended in 2 mL Ringer's solution (8.5 g / L NaCl, 0.4 g / L KCl, 0.34 g / L CaCl2 hydrate) to a final volume of 10 mL. 8 -10 9 CFU / mL. The bacterial suspension was inoculated into sterile electrolyte solution (SES) (0.22 g / L CaCl2, 6.2 g / L NaCl, 2.2 g / L KCl, 1.2 g / L NaHCO3), and lysozyme (Sigma, USA) was added to a final concentration of 100 mg / L. The mixture was incubated at 37°C for 30 minutes or 1 hour. A bacterial suspension in SES without added lysozyme served as a negative control. The viable counts of *Lactobacillus plantarum* JN9 before incubation (N0) and after incubation (N1) were recorded.

[0081] Tolerance to gastrointestinal fluids

[0082] To test the gastrointestinal tolerance of *Lactobacillus plantarum* JN9, fresh single colonies were picked and cultured in MRS broth at 37°C for 16 h. 5 mL of the bacterial suspension was centrifuged at 12000 rpm for 2 minutes at 4°C. The cells were washed with sterile PBS (pH=7) and resuspended, and the cell concentration was adjusted to 10⁻⁶. 7CFU / mL. Take 100 μL of bacterial suspension and add it to 900 μL of simulated gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3, and 3 g / L pepsin; pH adjusted to 3.0 with HCl) and incubate at 37°C. After 3 hours, take 250 μL of the gastric fluid mixture and add it to 6 mL of simulated intestinal fluid (45 mM NaCl, 1 g / L trypsin, 3 g / L bile salts; pH adjusted to 8.0 with NaOH), and continue incubation at 37°C for 3 hours. The number of viable cells before incubation in the gastric and intestinal fluids is recorded as N0, and the number of viable cells after 6 hours of incubation multiplied by the dilution factor of 25 is recorded as N1.

[0083] The survival rate of *Lactobacillus plantarum* JN9 under acid, bile salt, lysozyme, and gastrointestinal treatments was calculated using the following formula:

[0084] Survival rate (%) = (N1 ÷ N0) × 100%

[0085] N1 is the live cell count after treatment in MRS (pH 3.0 or 0.3% bile salts) for 3 hours, in SES (100 mg / L lysozyme) for 30 minutes or 1 hour, or in gastrointestinal fluid for 6 hours; N0 is the live cell count of Lactobacillus plantarum JN9 after treatment under different conditions for 0 hours.

[0086] Tolerance assessment of Lactobacillus plantarum JN9

[0087] The survival rates of *Lactobacillus plantarum* JN9 in MRS at pH 3.0 and MRS containing 0.3% bile salts were 47.87% and 75.11%, respectively (Figure 5(a)). After incubation in sterile electrolyte solution containing 100 mg / L lysozyme for 30 min and 1 h, the survival rates were 87.18% and 73.42%, respectively (Figure 5(b)). After digestion in simulated gastric and intestinal fluids for 6 hours, the viable cell count of *Lactobacillus plantarum* JN9 decreased to 12.50% of the pre-treatment count.

[0088] 4) Safety of Lactobacillus plantarum JN9

[0089] Antibiotic sensitivity and MIC

[0090] Antibiotic susceptibility analysis was performed on the bacterial strain using seven commonly used clinical antibiotics: gentamicin, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, and clindamycin. Stock solutions containing 256 mg / L of each antibiotic were prepared and serially diluted twofold, adding 100 μL to each well of a 96-well plate. Fresh bacterial culture after 16 h of incubation was diluted to an OD of 0.0002 and added to each well of the antibiotic plate at 100 μL, and incubated at 37°C for 24 h. The minimum inhibitory concentration (MIC) of *Lactobacillus plantarum* JN9 against each antibiotic was determined and compared with the critical values ​​recommended by the European Food Safety Authority (EFSA) for microbial antibiotics suitable for use in feed additives or production.

[0091] The MICs of ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol against *Lactobacillus plantarum* JN9 are shown in Table 2. The results showed that the MIC values ​​of all seven antibiotics were lower than the antibiotic threshold values ​​specified in the EFSA guidelines, indicating that this strain is sensitive to these seven antibiotics and possesses microbial antibiotic safety.

[0092] Table 2. MIC (mg / L) of different antibiotics against Lactobacillus plantarum JN9

[0093] hemolytic activity

[0094] The absence of hemolytic activity and antibiotic resistance are considered safety prerequisites for selecting probiotic strains (FAO / WHO, 2002).

[0095] Two generations of activated Lactobacillus plantarum JN9 was streaked onto Columbia blood agar medium containing 5% defibrinated sheep blood (3400071, Haibo, China) and incubated at 37°C for 48 hours. The appearance of a grass-green hemolytic ring indicated α-hemolysis; the appearance of a colorless and transparent hemolytic ring indicated β-hemolysis; and the absence of a hemolytic ring indicated γ-hemolysis. Lactobacillus rhamnosus LGG was used as a negative control.

[0096] As shown in Figure 6, after culturing *Lactobacillus plantarum* JN9 on blood agar, no hemolytic zone appeared around the colonies, indicating γ-hemolysis. This demonstrates that *Lactobacillus plantarum* JN9 has no hemolytic ability.

[0097] Determination of D-lactic acid and L-lactic acid production by Lactobacillus plantarum JN9

[0098] Fresh single colonies were picked and incubated in MRS broth at 37°C for 16 hours. The D / L-lactic acid content of the culture supernatant was analyzed using an enzymatic method according to the product instructions using a commercially available D / L-lactic acid quantitative kit (Jingmei Co., China).

[0099] The results showed that after culturing Lactobacillus plantarum JN9 in MRS broth at 37℃ for 16 hours, the concentrations of D-lactic acid and L-lactic acid in the supernatant were 2.28 nmol / L and 8.99 nmol / L, respectively. The L-lactic acid / D-lactic acid ratio was 3.94.

[0100] Detection of the ability to produce amines

[0101] After activation, *Lactobacillus plantarum* JN9 was cultured overnight in MRS liquid medium. It was then added to MRS liquid medium containing 0.1 g / L histidine, tyrosine, ornithine, lysine, and 0.05 g / L pyridoxal-5-phosphate until the final concentration reached OD 0.01, and passaged. Subculture was performed five times, with a 2% inoculum every 24 hours. The same liquid medium was then inoculated at a 2% inoculum into media containing 0.1% histidine, tyrosine, ornithine, and lysine, and cultured for 72 hours. The color change of the medium was observed. A purple color indicated a positive result, while a yellow color indicated a negative result. *Lactobacillus rhamnosus* LGG was used as a negative control, and *Escherichia coli* as a positive control.

[0102] The results showed that *Lactobacillus plantarum* JN9 turned yellow after three days of growth in four different biogenic amine detection media, indicating that the strain does not produce putrescine, cadaverine, histamine, or tyramine.

[0103] Cytotoxicity

[0104] The CCK-8 assay is a universal method for detecting cell proliferation and cytotoxicity. HT-29 cells are a human intestinal epithelial cell line, cultured in RPMI 1640 medium containing 10% FBS and 1% P / S (penicillin / streptomycin). Cell proliferation activity was detected using the Beyotime CCK-8 assay kit. HT-29 cells were cultured in a 37°C incubator containing 5% CO2 for approximately 36-72 hours until the cell density reached about 90%. After trypsin digestion and passage, cells were seeded at an appropriate density into 96-well plates. After cell attachment, 100 μL of a 10% concentration of [a specific chemical compound] was added to each well. 8 CFU / mL Lactobacillus plantarum JN9 was co-cultured with cells for 18 h. The cell culture medium was washed off with sterile PBS and 1 / 10 of the total volume of CCK-8 solution was added. The cells were incubated in the dark at 37°C for 2 h. After removing air bubbles, the absorbance at 450 nm was measured using a microplate reader.

[0105] To assess the potential cytotoxic effects of *Lactobacillus plantarum* JN9 on HT-29 intestinal epithelial cells, a CCK-8 assay was performed after 18 hours of bacterial cell co-culture. The fold-infection index (MOI) for this assay was approximately 1:200. As shown in Figure 7, at this MOI, the effect of *Lactobacillus plantarum* JN9 on cell viability was not significantly different from the control group (p>0.05), indicating that *Lactobacillus plantarum* JN9 had no negative impact on the survival of HT-29 intestinal epithelial cells.

[0106] 5) Intestinal adhesion of Lactobacillus plantarum JN9

[0107] HT-29 cells were used at a rate of 5 × 10 5 Seeds were transferred from culture flasks to 24-well plates at a concentration of 10 cells / mL. The plates were then cultured in antibiotic-free medium until the cells were fully adhered. Before adding bacteria, the cells were washed twice with sterile PBS, and then 500 μL of a 10-1 concentration was added to each well. 8 CFU / mL (V0) of bacteria were collected. The 24-well plates were transferred to a 37°C, 5% CO2 incubator and incubated for 4 h to allow adhesion. Each well was washed 5 times with PBS to remove unadhered bacteria and metabolic secretions. 200 μL of 1% Triton X-100 was added to each well for digestion, and the solution from each well was then collected, serially diluted, and counted (V1). The adhesion rate (%) was calculated as follows:

[0108] Adhesion rate (%) = (V1 / V0) × 100%

[0109] To assess the cell adhesion of *Lactobacillus plantarum* JN9 to HT-29 intestinal epithelial cells, adhesion was measured after 4 hours of bacterial cell co-culture. The fold-infection ratio (MOI) for this assay was approximately 1:200. The results showed that 15.98% of *Lactobacillus plantarum* JN9 adhered to HT-29 cells after 4 hours of co-culture, and its adhesion efficiency was approximately 1.5 times that of LGG (10.50%) (Figure 8).

[0110] Example 3. Fermentation and powder production of Lactobacillus plantarum JN9

[0111] After activation, *Lactobacillus plantarum* JN9 was inoculated into MRS broth and cultured at 37°C for 18 hours. After two subculturings, the culture was transferred to a bioreactor. Fermentation was carried out in Eppendorf. The total fermentation liquid volume was 6L. After 12 hours of fermentation, the bacterial cells were collected at 4°C, 10,000 rpm, and 10 minutes. The obtained bacterial cells were mixed with a protectant (1-10 g / L polysaccharide, 20-50 g / L disaccharide, 1-20 g / L vitamin C or its salts, and 1-10 g / L peptone) at a dry weight ratio of 1:1 and pre-frozen at -80°C for 24 hours. The bacterial powder was freeze-dried and pulverized into powder using a vacuum freeze dryer (PO14416, Telstar LyoQuest-55plus), and then vacuum-packed in aluminum foil bags.

[0112] The viable cell count of *Lactobacillus plantarum* JN9 was 3.23 × 10⁻⁶ cells after 12 hours of fermentation in a bioreactor. 9 -5.20×10 9 CFU / mL. 6 L of bacterial culture was centrifuged at 10000 rpm and 4℃ for 10 min, yielding 120-127 g of bacterial cells. The cells were mixed with a cryoprotectant and freeze-dried to obtain 50-60 g of bacterial powder with a viable cell count of 2.81 × 10⁻⁶ cells / mL. 11 -3.53×10 11 CFU / g.

[0113] Example 4. GABA production capacity of Lactobacillus plantarum JN9

[0114] Detection of the gadB gene in Lactobacillus plantarum JN9

[0115] Fresh cells of *Lactobacillus plantarum* JN9 were collected after culturing at 37℃ for 16 h, and DNA was extracted according to the instructions of the DNA extraction kit (9763, Takara, Japan). Primers for the amplification of the glutamate decarboxylase B gene (gadB gene) are shown in Table 3. PCR reaction system (25 μL): 2×Premix Taq TM 12.5 μL of (R004Q, Takara), 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of template (10 ng), and nuclease-free water to a final volume of 25 μL. PCR reaction conditions: 94℃ denaturation for 5 min, 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 30 cycles, followed by a 72℃ extension for 7 min. The formation and size of PCR bands were detected by gel electrophoresis: 1 μL of the above PCR product was added to 5 μL of DNA loading buffer, mixed well, and then the sample was added to the wells. Electrophoresis was performed at 100V for 40 min, and the bands were observed using a gel imaging system afterward.

[0116] Table 3 Primer sequences (SEQ ID NO: 4-9)

[0117] Determination of GABA production capacity of Lactobacillus plantarum JN9

[0118] *Lactobacillus plantarum* JN9 (experimental group) and *Lactobacillus plantarum* SG5 (GDMCC 60020), a commercial control strain capable of producing GABA, were activated and then single colonies were inoculated into MRS liquid supplemented with 20 g / L L-glutamate and cultured at 37 °C for 48 h. The fresh bacterial culture was centrifuged at 12000 rpm for 1 minute at 4 °C, and 200 μL of the supernatant was transferred to a centrifuge tube. 400 μL of 4.2% sodium bicarbonate solution and 200 μL of 1% 2,4-dinitrofluorobenzene solution were added, and the mixture was incubated at 60 °C for 1 h. After cooling, 9.2 mL of 0.136% potassium dihydrogen phosphate solution was added, and the mixture was shaken well. The mixture was filtered through a 0.22 μm filter and analyzed by LC-MS. LC conditions: C18 column, column temperature 35 °C ± 5 °C, gradient elution, mobile phase A was 0.41% anhydrous sodium acetate solution, and mobile phase C was acetonitrile. Flow rate: 0.8 mL / min. Detection wavelength: 360 nm. Injection volume: 10 μL. Mass spectrometry conditions: ESI positive ion mode, mass range: 20-2000 m / z. ESI source conditions: capillary voltage 3.5 kV, desolvation gas temperature 400 °C, cone voltage 30 V, desolvation gas flow rate 700 L / h, cone gas flow rate 50 L / h, collision energy 6 / 20 V.

[0119] The results showed that gene amplification revealed the presence of the gadB gene in the genome of *Lactobacillus plantarum* JN9 (Figure 9(a)). LC-MS analysis of the fermentation broth of *Lactobacillus plantarum* JN9 confirmed the production of GABA during fermentation (Figure 9(b)), and the GABA yield increased with prolonged fermentation time. After 24 hours of fermentation in MRS liquid medium supplemented with 20 g / L L-glutamate, the GABA yield of *Lactobacillus plantarum* JN9 was 10.53 ± 1.70 g / L, while the GABA yield of the commercially available control group *Lactobacillus plantarum* SG5 was 0.42 ± 0.08 g / L. After 48 hours of fermentation, the GABA yield of *Lactobacillus plantarum* JN9 increased to 13.54 ± 0.80 g / L, while the GABA yield of the commercially available control group *Lactobacillus plantarum* SG5 was 0.56 ± 0.03 g / L.

[0120] It is evident that the genome of *Lactobacillus plantarum* JN9 contains the gadB gene, indicating a high GABA production capacity, making it suitable for GABA production. Furthermore, due to its good safety profile and alcohol tolerance, it can be used to prepare sleep-improving and antidepressant drugs, as well as a range of functional health products or foods (antidepressant, sleep-improving, liver-protecting, immune-boosting, and / or hangover prevention).

[0121] Example 5. Alcohol tolerance of Lactobacillus plantarum JN9

[0122] Detection of acetaldehyde dehydrogenase gene (aldH) and alcohol dehydrogenase gene (adh)

[0123] Fresh cells of *Lactobacillus plantarum* JN9 were collected after culturing at 37℃ for 16 h, and DNA was extracted according to the instructions of the DNA extraction kit (9763, Takara, Japan). Primers for aldH and adh genes are shown in Table 3. PCR reaction system (25 μL): 2×Premix Taq TM 12.5 μL of (R004Q, Takara) primer, 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of template (10 ng), and nuclease-free water to a final volume of 25 μL. PCR conditions: 98℃ denaturation for 4 min, 98℃ denaturation for 10 s, 56℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 32 cycles, with a final extension of 72℃ for 7 min. PCR product band formation and size were detected by electrophoresis: 1 μL of the PCR product was added to 5 μL of DNA loading buffer, mixed well, and then added to the sample wells. Electrophoresis was performed at 100V for 40 min, and the bands were observed using a gel imaging system afterward.

[0124] Evaluation of the tolerance of Lactobacillus plantarum JN9 to different concentrations of alcohol.

[0125] After activation, *Lactobacillus plantarum* JN9 was inoculated into MRS broth and cultured at 37°C for 16 hours. The bacterial concentration was then adjusted to 10⁻⁶. 8 CFU / mL, bacterial suspension was added to MRS liquid medium containing anhydrous ethanol at a ratio of 1:1000 to a final ethanol concentration of 3%, 7%, 12% and 15%, and cultured at 37°C for 24 h. The growth of the strain was detected by viable cell counting.

[0126] Enzyme activity detection of acetaldehyde dehydrogenase in Lactobacillus plantarum JN9

[0127] Lactobacillus plantarum JN9 (experimental group), Lactobacillus rhamnosus LGG (control 1), and commercially available Lactobacillus plantarum YLA1 (CCTCC NO: M2020289, control 2) with acetaldehyde dehydrogenase activity were activated and inoculated into MRS broth. After static incubation at 37℃ for 16 h, the bacterial cells were collected by centrifugation, washed twice with sterile PBS buffer, and resuspended. The OD was adjusted to 1. The cells were then sonicated on ice (300 W, 5 s sonication, 7 s interval, total time 15 min) to obtain the test samples. PBS was used as a blank control. The enzyme activity reaction systems of Lactobacillus plantarum JN9, LGG, YLA1, and the blank sample were prepared according to Table 4 and incubated at 37℃ for 30 min. Record the absorbance values ​​A1 and A2 of the sample before and after incubation at 340 nm. Calculate ΔAtest tube = A2test tube - A1test tube, ΔAblank tube = A2blank tube - A1blank tube, and ΔA = ΔAtest tube - ΔAblank tube. Enzyme activity is defined as 1 μmol of NADH generated per milliliter of sample per minute.

[0128] ALDH enzyme activity (U / mL) = ΔA ÷ (ε × d) × 10 6 ×V_total ÷V_sample ÷T

[0129] ε: NADH molar extinction coefficient, 6.22 × 10³ L / mol / cm; d: optical path length of the 96-well plate, 0.6 cm; Vreaction_total: total volume of the reaction system; Vsample: volume of the sample in the reaction system; T: reaction time, 30 min; 10 6 Unit conversion factor, 1 mol = 10 6 μmol

[0130] Table 4 Reaction system for enzyme activity assay

[0131] The adh and aldh genes of *Lactobacillus plantarum* JN9 were amplified. The electrophoresis results of the PCR amplification products are shown in Figure 10. Both gene bands were single, with sizes of approximately 500 bp and 1400 bp, respectively, consistent with the expected sizes. This indicates that the genome of *Lactobacillus plantarum* JN9 contains aldh and adh. Alcohol tolerance experiments showed that *Lactobacillus plantarum* JN9 exhibited good growth ability in MRS media containing 3%, 7%, and 12% ethanol, and could tolerate MRS media containing 15% ethanol. After 16 hours of incubation, viability testing revealed a bacterial survival rate of 16.67%. The acetaldehyde dehydrogenase activity assay showed that the acetaldehyde dehydrogenase activity of *Lactobacillus plantarum* JN9 strain was 5.29 μmol / mL. The commercially available control group *Lactobacillus plantarum* YLA (control 2) contained the adh and aldh genes and its acetaldehyde dehydrogenase activity was 3.22 μmol / mL. *Lactobacillus rhamnosus* LGG (control 1) did not contain the aldh gene and no acetaldehyde dehydrogenase activity was detected.

[0132] It is evident that the genome of Lactobacillus plantarum strain JN9 contains the aldehyde dehydrogenase gene (aldH) and the alcohol dehydrogenase gene (adh). The strain exhibits high aldehyde dehydrogenase activity and strong alcohol tolerance, making it suitable for preparing drugs to protect the liver and / or prevent alcohol hangovers.

Claims

A plant-derived Lactobacillus JN9, wherein, The plant lactobacillus JN9 is classified as *Lactiplantibacillus plantarum*, with accession number CCTCC NO: M 20241647. The formulation prepared from Lactobacillus plantarum JN9 as described in claim 1. According to claim 2, the formulation wherein, The formulation is selected from one or more of the following: 1) Inoculum of Lactobacillus plantarum JN9; 2) A bacterial suspension of Lactobacillus plantarum JN9; 3) The metagene of Lactobacillus plantarum JN9; 4) Metabolites of *Lactobacillus plantarum* JN9; and 5) Fermentation extract of Lactobacillus plantarum JN9. A probiotic composition comprising Lactobacillus plantarum JN9 as described in claim 1 or the formulation as described in claim 2 or 3. According to claim 4, the probiotic composition, wherein, The probiotic composition further comprises one or more probiotics selected from the following: Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Weizmannia, Mammaliicoccus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis. Use of the *Lactobacillus plantarum* JN9 of claim 1, the formulation of claim 2 or 3, or the probiotic composition of claim 4 or 5 in the preparation of a medicament for reducing stress, improving mood, antidepressant effects, improving sleep quality, and / or lowering blood pressure. Use of the Lactobacillus plantarum JN9 of claim 1, the formulation of claim 2 or 3, or the probiotic composition of claim 4 or 5 in the preparation of a medicament for liver protection and / or prevention of alcohol hangovers. The use according to claim 6 or 7, wherein, The drug is administered orally. The use of Lactobacillus plantarum JN9 as described in claim 1, the preparation as described in claim 2 or 3, or the probiotic composition as described in claim 4 or 5 in the preparation of food or health products. According to the use described in claim 9, wherein, The food or health product mentioned is intended to reduce stress, improve mood, combat depression, improve sleep quality, lower blood pressure, and / or enhance alcohol tolerance. The use according to claim 9 or 10, wherein, The food or health products mentioned are dairy products, soy products, meat products, fruit and vegetable products, beverages, or snacks. The use according to any one of claims 9-11, wherein, The food or health product also includes edible ingredients. The use of Lactobacillus plantarum JN9 as described in claim 1 in the production of GABA.

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