Lactobacillus buchneri strain for improving gaba content and sleep and application thereof

Abstract

This invention, entitled "A Strain of *Lactobacillus brucellosis* Improving GABA Levels and Sleep and Its Application," belongs to the field of microbial technology. The technical problem to be solved is to provide a strain of *Lactobacillus brucellosis* that regulates GABA levels and improves sleep. The key technical solution provided is a strain of *Lactobacillus brucellosis*, with accession number CGMCC No. 37544. This invention relates to *Lactobacillus brucellosis*. (Lentilactobacillus buchneri) JZ-QX01 has the ability to regulate host GABA levels and improve sleep, and has great application potential in the preparation of products that regulate host GABA levels and improve sleep.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus brucellosis that improves GABA levels and sleep, and its applications. Background Technology

[0002] Currently, mental and neurological health problems, such as anxiety, depression, insomnia, and cognitive decline, are becoming increasingly prominent. One of the common neurobiological bases of these problems is a relative or absolute deficiency of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain, leading to an imbalance between neural excitation and inhibition. Therefore, supplementing or promoting endogenous GABA synthesis has become an important strategy for intervening in related diseases.

[0003] Currently, while direct supplementation with exogenous GABA or the use of its receptor agonists have some effect, they also have limitations. Exogenous GABA has difficulty effectively crossing the blood-brain barrier, limiting its direct effect on the central nervous system; while synthetic drugs may be accompanied by side effects such as tolerance, dependence, or excessive central nervous system depression. Therefore, finding new ways to safely, persistently, and synergistically regulate GABA in the body has become an important direction for development in this field. Utilizing specific probiotic strains to produce or regulate GABA is a highly promising biotechnological strategy. Many probiotics, such as lactic acid bacteria and bifidobacteria, naturally possess glutamate decarboxylase (GAD), which can efficiently convert glutamate in fermentation media into GABA. The gut is known as the "second brain," communicating bidirectionally with the central nervous system through the vagus nerve, immune, endocrine, and metabolic pathways (such as short-chain fatty acids) (i.e., the "gut-brain axis"). Oral administration of probiotics that can colonize the gut and produce GABA can provide a continuous supply of GABA or related signaling molecules, indirectly and stably influencing the function of the central GABAergic system through multiple pathways of the gut-brain axis, thus overcoming the barrier of the blood-brain barrier.

[0004] This patent, published in Japan (JP7492208B2) on August 30, 2021, discloses a novel lactic acid bacteria that produces large amounts of GABA and ornithine. The invention provides a novel lactic acid bacteria that simultaneously produces high levels of both GABA and ornithine, as well as a method for producing foods and beverages high in GABA and ornithine using this bacteria. The lactic acid bacteria belongs to the Lactobacillus brucelli strain, strain number NITE P-03111. Patent JP7492208B2 focuses only on producing ordinary foods and beverages containing GABA and ornithine, failing to provide a Lactobacillus brucelli that simultaneously produces high levels of GABA and improves sleep.

[0005] Therefore, there is an urgent need to provide a strain that can enhance GABA function and improve sleep disorders. Summary of the Invention

[0006] The purpose of this invention is to provide: A strain of *Lactobacillus brucellosis* JZ-QX01 that regulates GABA levels and improves sleep, its applications, and related technologies, to solve technical problems such as providing a strain of *Lactobacillus brucellosis* that regulates GABA levels and improves sleep, or a combination thereof.

[0007] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0008] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0009] Definitions of standard chemical terms can be found in the references *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Science Press, 4th edition, 2017; and *Microbiology Experiments*, Higher Education Press, 4th edition, 2016.

[0010] Unless otherwise stated, conventional methods within the scope of the art, such as sequencing, strain culture, strain fermentation, gavage, etc., shall be used.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] The term “CFU (colony forming unit)” used in this article refers to colony forming unit.

[0013] The term "culture medium" used in this article refers to the nutrient substrate for the growth and reproduction of microorganisms. Based on the different contents of the solidifying agent, it can be divided into three types: solid culture medium, semi-solid culture medium (semi-liquid culture medium), and liquid culture medium.

[0014] In a first aspect, the present invention provides: a strain of *Lactobacillus brucellosis* JZ-QX01, with accession number CGMCC No. 37544, and classified as *Lactobacillus brucellosis* (…). Lentilactobacillus buchneri It was deposited at the China General Microbiological Culture Collection Center on January 26, 2026.

[0015] This includes technical features such as 16S sequence and growth morphology.

[0016] The 16S rDNA sequence, a key technical feature, is shown in SEQ ID NO:1.

[0017] Among them, the technical characteristic growth morphology is as follows: the growth morphology of Lactobacillus brucellosis JZ-QX01 on MRS medium is round, milky white, smooth and raised colonies. Select milky white raised colonies.

[0018] Secondly, the present invention provides a microbial agent comprising the aforementioned *Lactobacillus brucellosis* JZ-QX01.

[0019] Specifically, the bacterial agent includes one or more of the following: Lactobacillus brucellosis JZ-QX01 cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0020] Preferably, the bacterial agent includes the fermentation broth of Lactobacillus brucellosis JZ-QX01 or the lyophilized powder of Lactobacillus brucellosis JZ-QX01.

[0021] The microbial agent also includes nutritionally acceptable nutrient additives.

[0022] The nutritional additives mentioned include, but are not limited to, dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0023] Thirdly, the present invention provides a preparation of Lactobacillus brucellosis JZ-QX01, comprising: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, and exosomes.

[0024] Specifically, the fermentation broth of Lactobacillus brucellis JZ-QX01 is a mixed liquid system obtained by culturing Lactobacillus brucellis JZ-QX01 in a culture medium under artificially controlled fermentation conditions. It contains the bacteria themselves, intracellular and extracellular metabolites, unused culture medium components, and fermentation byproducts.

[0025] Specifically, the precipitate of Lactobacillus brucellosis JZ-QX01 fermentation broth is the solid phase component separated from the fermentation broth of the strain after treatment such as standing, centrifugation or filtration. It mainly includes live / dead cells of the strain, cell fragments and insoluble substances produced in the fermentation system.

[0026] Specifically, the supernatant of Lactobacillus brucellis JZ-QX01 fermentation broth is a clear liquid phase component containing extracellular metabolites of the strain, soluble culture medium residues, and soluble fermentation by-products obtained after the fermentation broth of the strain has been allowed to stand, centrifuged, or filtered to remove solid phase precipitates such as bacterial cells.

[0027] Specifically, the live Lactobacillus brucellosis JZ-QX01 is a bacterium with normal physiological activity, capable of carrying out life activities such as metabolism and reproduction.

[0028] Specifically, inactivated Lactobacillus brucellosis JZ-QX01 refers to bacteria that have lost their metabolic and reproductive activities but whose overall structure has been basically preserved after being treated by physical, chemical or other means.

[0029] Specifically, Lactobacillus brucellosis JZ-QX01 freeze-dried powder refers to a solid powder that retains the original active components, obtained by freeze-drying liquid materials such as strain fermentation broth, fermentation supernatant, and bacterial suspension to remove moisture.

[0030] Specifically, Lactobacillus brucellosis JZ-QX01 lysate refers to the mixture of intracellular substances formed after live or inactivated Lactobacillus brucellosis JZ-QX01 cells are broken down by physical, chemical or enzymatic methods, releasing intracellular substances.

[0031] Specifically, Lactobacillus brucellosis JZ-QX01 lysate refers to a mixed system containing intracellular active components and fragmented bacterial cells formed after live or inactivated Lactobacillus brucellosis JZ-QX01 bacteria are ruptured through physical, chemical, enzymatic, or biological lysation methods to release all intracellular substances.

[0032] Specifically, the secondary metabolites of Lactobacillus brucellosis JZ-QX01 refer to various compounds produced by microorganisms such as the strain during their stable growth phase that are not essential for their own growth and reproduction and often possess specific biological activities such as anti-inflammatory and metabolic regulation.

[0033] Specifically, the exosomes of Lactobacillus brucellis JZ-QX01 refer to extracellular vesicles encapsulated by nanoscale lipid bilayer membranes that are actively secreted during the growth and metabolism of Bifidobacterium lactis or released after cell lysis.

[0034] Fourthly, the present invention provides the use of the above-mentioned Lactobacillus brucellosis JZ-QX01 or the above-mentioned bacterial agent or the above-mentioned preparation in the preparation of products that increase the content of GABA in vivo.

[0035] The product in question is a drug.

[0036] Furthermore, the drug can increase GABA levels in at least one of the following tissue sites: serum, colon, hippocampus, and hypothalamus.

[0037] Furthermore, the drug includes any one or more of the following applications: (1) Increase GABA production; (2) Increase serum GABA levels; (3) Promotes GABA secretion in intestinal tissue; (4) Promotes GABA secretion in the hippocampus; (5) Promotes GABA secretion in the hypothalamus.

[0038] Specifically, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL).

[0039] According to some embodiments of the present invention, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL) - 1.0 × 10⁻⁶ 12 CFU / g (CFU / mL), specifically: 1.0 × 10⁻⁶ 9 CFU / g (CFU / mL), 1.0×10 10 CFU / g (CFU / mL), 1.0×10 11 CFU / g (CFU / mL), 1.0×10 12 The CFU / g (CFU / mL) value and any intermediate value range can be selected.

[0040] Preferably, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL).

[0041] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0042] More preferably, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0043] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, and mannitol.

[0044] Specifically, the stabilizer is selected from at least one of agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0045] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0046] Specifically, the adhesive is selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0047] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0048] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0049] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0050] Fifthly, the present invention provides the use of the above-mentioned *Lactobacillus brucellosis* JZ-QX01 or the above-mentioned bacterial agent or the above-mentioned preparation in the preparation of products for improving sleep.

[0051] The products mentioned include pharmaceuticals.

[0052] The products mentioned include health food products.

[0053] Specifically, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is not less than 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL).

[0054] According to some embodiments of the present invention, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL) - 1.0 × 10⁻⁶ 12 CFU / g (CFU / mL), specifically: 1.0 × 10⁻⁶ 9 CFU / g (CFU / mL), 1.0×10 10 CFU / g (CFU / mL), 1.0×10 11 CFU / g (CFU / mL), 1.0×10 12 The CFU / g (CFU / mL) value and any intermediate value range can be selected.

[0055] Preferably, the viable count of *Lactobacillus brucellosis* JZ-QX01 in the product is 1.0 × 10⁻⁶. 9 CFU / g (CFU / mL).

[0056] The product is said to improve insomnia caused by stress, mood disorders, and / or abnormal short-chain fatty acid metabolism.

[0057] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0058] More preferably, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0059] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, and mannitol.

[0060] Specifically, the stabilizer is selected from at least one of agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0061] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0062] Specifically, the adhesive is selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0063] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0064] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0065] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0066] Furthermore, the health food also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0067] Furthermore, the health food products mentioned include emulsion products, solution products, powder products, and solid products.

[0068] Specifically, the product has at least one of the following functions: (1) Prolong sleep time; (2) Increase the content of 5-HT, DA and NE; (3) Increase the content of short-chain fatty acids.

[0069] Specifically, increasing the content of short-chain fatty acids means increasing the content of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and isobutyric acid.

[0070] In a sixth aspect, the present invention provides a product that increases the level of GABA in the body or improves sleep, the product comprising the above-mentioned *Lactobacillus brucellosis* JZ-QX01 or the above-mentioned bacterial agent or the above-mentioned preparation.

[0071] When the function of the product is to regulate the GABA content in the body, the product is a medicine.

[0072] When the function of the product is to improve sleep, the product is a medicine.

[0073] When the function of the product is to improve sleep, the product is a health supplement.

[0074] Preferably, the drug comprises a pharmaceutically acceptable carrier.

[0075] More preferably, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0076] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0077] Furthermore, the health food also includes conventional excipients for health foods, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0078] Furthermore, the health food products mentioned include emulsion products, solution products, powder products, and solid products.

[0079] The present invention has at least the following beneficial effects: The present invention contains *Lactobacillus brevicornu*. (Lentilactobacillus buchneri) JZ-QX01 has the ability to regulate host GABA levels and improve sleep, specifically in the following ways: (1) It has the ability to produce high levels of GABA in vitro; (2) Increase serum GABA levels in stressed mice; (3) Increase the GABA level in the colon tissue of stressed mice; (4) Increase the level of GABA in the hippocampus of stressed mice; (5) Increase the level of GABA in the hypothalamus of stressed mice; (6) Prolong sleep time; (7) Restore the levels of neurotransmitters such as DA, NE, and 5-HT in the brain tissue of stressed mice; (8) Promotes the content of short-chain fatty acids in stressed mice.

[0080] Lactobacillus brevicornu (Lentilactobacillus buchneri) JZ-QX01 has great application potential in the preparation of products that regulate host GABA content and promote sleep.

[0081] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: This invention provides a method for regulating GABA levels in the body or improving sleep, which involves administering a therapeutically effective amount of the aforementioned *Lactobacillus brucellosis* JZ-QX01, the aforementioned bacterial agent, or the aforementioned preparation to a subject.

[0082] Preservation Instructions Preserved strain: Lactobacillus brucellosis JZ-QX01; Classification and nomenclature: Lactobacillus brucellosis Lentilactobacillus buchneri ; Accession number: CGMCC No. 37544; Preservation period: January 26, 2026; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0083] Figure 1 This is a colony diagram of Lactobacillus brucellosis JZ-QX01.

[0084] Figure 2 The in vitro GABA production capacity of Lactobacillus brucellosis JZ-QX01 was measured.

[0085] Figure 3 The effect of Lactobacillus brucellosis JZ-QX01 on serum GABA levels in stressed mice is shown in the figure. "*" indicates a statistically significant difference from the model group (P<0.05), and "**" indicates a statistically significant difference from the model group (P<0.01).

[0086] Figure 4 The effect of Lactobacillus brucellosis JZ-QX01 on GABA content in the intestinal tissue of stressed mice was investigated. "*" indicates a statistically significant difference from the model group (P<0.05), and "**" indicates a statistically significant difference from the model group (P<0.01).

[0087] Figure 5 The effect of Lactobacillus brucellosis JZ-QX01 on GABA content in hippocampal tissue of stressed mice is shown. "*" indicates a statistically significant difference from the model group (P<0.05), and "**" indicates a statistically significant difference from the model group (P<0.01).

[0088] Figure 6 The effect of Lactobacillus brucellosis JZ-QX01 on GABA content in the hypothalamus of stressed mice was investigated. "*" indicates a statistically significant difference from the model group (P<0.05), and "**" indicates a statistically significant difference from the model group (P<0.01).

[0089] Figure 7 The effect of Lactobacillus brucellosis JZ-QX01 on sleep time in stressed mice is shown in the figure. "*" indicates a statistically significant difference from the model group (P<0.05), and "ns" indicates no significant difference from the model group (P>0.05).

[0090] Figure 8 The effects of Lactobacillus brucellosis JZ-QX01 on the levels of 5-HT, DA, and NE in the brain tissue of stressed mice were investigated. "**" indicates a statistically significant difference from the model group (P<0.01), and "***" indicates a statistically significant difference from the model group (P<0.001).

[0091] Figure 9 The effect of Lactobacillus brucellosis JZ-QX01 on the content of short-chain fatty acids in the intestine of stressed mice is shown in the figure. "ns" indicates no significant difference, "*" indicates a statistically significant difference from the model group (P<0.05), "**" indicates a statistically significant difference from the model group (P<0.01), and "***" indicates a statistically significant difference from the model group (P<0.001). Detailed Implementation

[0092] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0093] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0094] Data analysis and statistical analysis were performed using professional data processing software. The t-test was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0095] The culture media involved in the following examples are as follows: MRS liquid culture medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diamine hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, cysteine ​​hydrochloride 0.5 g / L, Tween-80 1.0 g / L, pH 5.7±0.2.

[0096] MRS solid medium: Add 1.2%-1.5% agar powder to the above MRS liquid medium.

[0097] MRS liquid medium containing glutamate: Add 10 g / L sodium glutamate to the above MRS liquid medium.

[0098] Example 1: Screening and Identification of Strains This embodiment provides a strain of *Lactobacillus brucellosis* JZ-QX01, which was obtained through the following screening steps: 1. Preliminary screening The samples were derived from healthy human breast milk. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, the samples were mixed, and 0.5 mL of the sample was added to 4.5 mL of physiological saline. Serial dilutions were performed, and appropriate serial dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus brucellosis* were picked and streaked onto MRS solid medium for purification. Single colonies were transferred to MRS liquid medium for enrichment and preserved in 50% glycerol to obtain *Lactobacillus brucellosis* JZ-QX01. Typical colonies of *Lactobacillus brucellosis* JZ-QX01 were round, milky white, and smooth with raised surfaces. Milky white, raised colonies were selected. The results are as follows: Figure 1 As shown.

[0099] 2. Identification The genome of *Lactobacillus brunetti* JZ-QX01 was extracted, and the 16S rDNA of strain JZ-QX01 was amplified and sequenced (performed by Shanghai Saiheng Biotechnology Co., Ltd.). The nucleotide sequence of the amplified 16S rDNA of JZ-QX01 was compared with the nucleic acid sequence in NCBI. The results showed that the strain was *Lactobacillus brunetti*, and it was named *Lactobacillus brunetti*. Lentilactobacillus buchneri )JZ-QX01.

[0100] Example 2 Screening of GABA-producing strains (1) High performance liquid chromatography conditions This study used an Agilent 1260 high-performance liquid chromatography (HPLC) system to determine the GABA content in the fermentation supernatant. The chromatographic conditions were as follows: column: Agilent Zorbax Eclipse XDB-C18 (4.6*150mm, 5µm); column temperature: 40℃; injection volume: 20µL; mobile phase A: 25 mmol / L anhydrous sodium acetate aqueous solution; mobile phase B: pure acetonitrile; flow rate: 1 mL / min; detection wavelength: 250 nm. The elution gradient was: 0-30 min, B increased from 10% to 60%; 30-35 min, B remained at 60%; 35-40 min, B decreased from 60% to 10%; 40-45 min, B remained at 10%.

[0101] (2) HPLC detection of derivatization reaction Take 200 μL of the GABA sample to be tested, 200 μL of acetonitrile, 200 μL of NaHCO3 (0.06 g / mL), 200 μL of water, and 100 μL of dansyl chloride (10 g / L). Mix well and react in the dark at 60℃ for 1 h, then add 100 μL of acetic acid to stop the derivatization. After derivatization, filter through a 0.22 μm organic filter membrane and immediately inject for analysis. Strictly control the entire derivatization operation time.

[0102] (3) Standard Curve Accurately weigh 10 mg of GABA standard and dissolve it in water in a 10 mL volumetric flask. Make up to volume to obtain a stock solution with a mass concentration of 1000 mg / L. This stock solution is then sequentially diluted to prepare a series of standard solutions with concentrations of 125 mg / L, 100 mg / L, 80 mg / L, 50 mg / L, 25 mg / L, and 10 mg / L. Plot a standard curve with GABA concentration on the x-axis and peak area on the y-axis.

[0103] (4) Strains culture and detection The bacterial strain preserved at -80℃ was inoculated into 4.5 mL of MRS liquid medium at 0.5 mL and incubated statically at 37℃ for 24 h. Then, 4% of the inoculum was inoculated into a glutamate-containing medium and incubated statically at 37℃ for 96 h. After centrifugation at 4000 r for 15 min, the supernatant was collected, and the GABA production was determined according to the derivatization method. *Lactobacillus brucellosis* (accession number CICC 6080) was used as a control. The strain was purchased from the China Industrial Microbiological Culture Collection Center. A high-GABA-producing *Lactobacillus brucellosis* strain JZ-QX01 was obtained. Figure 2 It can be seen that its GABA production is 243.38 mg / L.

[0104] Example 3: Effect of Lactobacillus brevicornu JZ-QX01 on serum GABA levels in mice under chronic unpredictable stress (CUMS). 1. Animal husbandry and grouping Thirty SPF-grade male C57BL / 6J mice, aged 6-8 weeks and weighing 18g-22g, were purchased and fed for one week to allow them to acclimatize before the study. Initial body weight was measured before the experiment, and the animals were randomly divided into three groups (n=10 per group): a normal control group, a model group, and a *Lactobacillus brucellosis* JZ-QX01 group. The grouping was set as follows: Normal group: administered physiological saline by gavage, without modeling; Model group: Orally administered physiological saline to establish a chronic unpredictable stress (CUMS) model; Lactobacillus brucellosis JZ-QX01 group: 0.2 ml of Lactobacillus brucellosis JZ-QX01 was administered by gavage, at a dose of 1.0 × 10⁻⁶. 9 CFU / ml; Conduct chronic unpredictable stress (CUMS) modeling.

[0105] Mice were administered 0.2 ml of medication via gavage once daily for 6 weeks. Weight changes and mouse condition were recorded periodically. The experimental environment was kept at a suitable temperature, quiet and well-ventilated, with 12-hour light-dark cycles, and free access to food and water.

[0106] 2. The modeling method for the mouse chronic unpredictable stress (CUMS) model is as follows: One or two stimuli were randomly selected each day, and the timing of the stimulation was randomized to avoid circadian rhythms. This was carried out for 30 days. To prevent the mice from developing stress adaptation, the same stimulus was not repeated within 7 days.

[0107] Stimulating factors include: fasting for 24 hours; damp bedding for 24 hours; restraint for 4 hours; tail clamping for 3 minutes; continuous light for 24 hours; no bedding for 24 hours; swimming in cold water for 5 minutes; water restriction for 24 hours; crowding for 24 hours; isolation for 24 hours; sleep deprivation for 4 hours; reversed day and night for 24 hours; tilted cage for 24 hours; noise stimulation for 3 hours; and stroboscopic stimulation for 2 hours.

[0108] 3. Serum GABA detection Mice were euthanized by dislocation after blood was collected from the orbital cavity. Blood samples were allowed to stand at room temperature for 2 hours, and then centrifuged at 4°C and 3500 rpm for 15 minutes to obtain serum. The GABA content in the serum was detected using an ELISA kit purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml001894.

[0109] Depend on Figure 3 It was found that, compared with the normal group, the serum GABA content in the model group mice was significantly reduced (p < 0.01), confirming the successful establishment of the CUMS model and the resulting inhibition of the GABAergic system in vivo, leading to a decrease in serum GABA content. Compared with the model group, the serum GABA content in the JZ-QX01 intervention group mice was significantly increased (p < 0.05), and the content recovered to normal levels. These results demonstrate that JZ-QX01 has a significant effect on increasing serum GABA content.

[0110] Serum GABA levels are an important indicator of the functional status of central and peripheral GABAergic neurons. This example demonstrates that JZ-QX01 can effectively increase serum GABA levels in model animals and correct functional defects in the GABAergic system.

[0111] 4. Colon tissue sample processing and GABA detection Colon tissue was weighed and homogenized with 9 volumes of pre-cooled PBS. The GABA content in the colon tissue was determined by ELISA. The GABA ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number ml001894.

[0112] Depend on Figure 4 It was found that, compared with the normal control group, the GABA content in the colonic tissue of the model group mice was significantly reduced (p < 0.01), indicating that the model successfully induced local GABA loss in the colon. Compared with the model group, the GABA content in the colonic tissue of the JZ-QX01 strain intervention group mice was significantly increased (p < 0.05), and its content returned to near normal levels. These results fully demonstrate that strain JZ-QX01 can effectively increase the GABA content in colonic tissue.

[0113] Gastrointestinal cells take up glutamate via a specific transporter protein. L-glutamate undergoes irreversible decarboxylation within the cell, consuming one proton to produce GABA. It is then excreted by the GABA antitransporter protein.

[0114] In the gastrointestinal system, GABA also functions as a neurotransmitter; approximately 5-8% of enteric ganglion neurons exhibit high affinity for GABA, indicating its gabatagenic properties. Furthermore, mucosal endocrine cells can synthesize, store, and secrete GABA as an endocrine substance, regulating gastrointestinal motility, digestive secretion, and immune cell activity associated with intestinal inflammation. These findings suggest that GABA is a key participant in gut-brain communication, and its regulation may have significant implications for the treatment of various gastrointestinal and neuropsychiatric disorders. Although the blood-brain barrier is impermeable to GABA, oral administration of GABA still affects the function of nerve cells in the brain. In this case, GABA-activated enteric cells induce exocytosis to activate nerve cells.

[0115] 5. Detection of GABA content in hippocampal tissue Brain tissue was dissected and removed, and the hippocampus was rapidly separated on ice. The hippocampal tissue was weighed, homogenized with 9 volumes of pre-cooled PBS, and the supernatant was collected. The GABA content in the hippocampal tissue was detected by ELISA. The GABA ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number ml001894.

[0116] Depend on Figure 5 It was found that, compared with the normal group, the GABA content in the hippocampus of the model group was significantly reduced (p < 0.01), confirming that the model successfully induced the depletion of GABA neurotransmitters in key central brain regions. Compared with the model group, after intervention with the JZ-QX01 strain, the GABA content in the hippocampus of the animals was significantly increased (p < 0.05), and its level recovered to the normal state. This result directly proves that oral administration of the JZ-QX01 strain can effectively increase the GABA content in the hippocampus of the brain.

[0117] GABA, as a major inhibitory neurotransmitter, works with glutamate in the hippocampus to maintain the excitation-inhibition balance of neural networks. Furthermore, insufficient GABA levels are a crucial neurochemical basis for negative emotional states such as anxiety and depression; increasing hippocampal GABA levels directly helps improve cognitive function and stabilize mood.

[0118] In vitro experiments have confirmed that JZ-QX01 is a high-GABA-producing strain. After oral administration, the GABA produced and released in situ in the intestine, although it cannot directly cross the blood-brain barrier in large quantities, may act as a key signaling molecule, initiating a series of cascade reactions through the neuro-humoral pathway, ultimately upregulating the synthesis of endogenous GABA in the hippocampus.

[0119] 6. Increases GABA content in hypothalamic tissue Hypothalamic tissue was weighed and homogenized with 9 volumes of pre-cooled PBS. The supernatant was collected and the GABA content in the hypothalamic tissue was detected by ELISA. The GABA ELISA kit was purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml001894.

[0120] Depend on Figure 6 It was found that, compared with the normal control group, the GABA content in the hypothalamus of rats in the chronic stress model group was significantly reduced (p < 0.01), confirming that the model successfully induced the inhibition of GABAergic neurotransmission in the core brain region of the stress response. Compared with the model group, the GABA content in the hypothalamus of rats in the JZ-QX01 intervention group was significantly increased (p < 0.05), indicating that JZ-QX01 can effectively reverse stress-induced hypothalamic GABA depletion. This result directly proves that oral administration of the JZ-QX01 strain can specifically increase the GABA content in the hypothalamus region of the brain.

[0121] The hypothalamus is a key target for receiving and integrating signals from the gut-brain axis. Increased GABA levels in the hypothalamus provide direct evidence that ascending signals from the gut-brain axis have been successfully translated into central functional changes. This example demonstrates that orally administered strain JZ-QX01 significantly increases GABA levels in the hypothalamus of animals with a chronic unpredictable stimulation model. This not only confirms the efficacy of JZ-QX01 at the highest regulatory level of the neuroendocrine and autonomic nervous systems but also organically links previously discovered local effects in the colon, changes in peripheral blood markers, and effects in key brain regions of the limbic system. This provides a complete, in-depth, and mutually corroborating chain of evidence for the application of strain JZ-QX01 in the prevention or treatment of stress-related diseases, endocrine disorders, and mood disorders.

[0122] 7. Sleep Improvement Experiment The mice were subjected to direct sleep experiments, prolonged sodium pentobarbital sleep time experiments, and subthreshold sodium pentobarbital hypnotic experiments, in accordance with the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)".

[0123] (1) Direct sleep experiment After daily gavage, mice in each group were observed to see if they experienced direct sleep. The disappearance of the righting reflex was used as the indicator of sleep onset. When a mouse was placed in a dorsal position, it should be able to immediately right itself. If it could not right itself for more than 30-60 seconds, the righting reflex was considered to have disappeared, and the mouse had entered sleep. The recovery of the righting reflex was considered as awakening. The number of animals that fell asleep and the sleep time in each group were recorded.

[0124] Within 30 minutes after daily gavage, no sleep phenomenon was observed in mice in the normal group, model group, and Lactobacillus brucellosis JZ-QX01 group, indicating that Lactobacillus brucellosis JZ-QX01 has no direct hypnotic effect on mice.

[0125] (2) Experiment on prolonging sodium pentobarbital sleep time Thirty minutes after the last gavage, mice were injected intraperitoneally with sodium pentobarbital (40 mg / kg BW). The duration of sleep in the mice was calculated based on the time interval between the disappearance and recovery of the righting reflex.

[0126] Results of prolonged sodium pentobarbital sleep time in each group of mice are as follows: Figure 7 As shown, compared with the model group, the Lactobacillus brevicornu JZ-QX01 group significantly prolonged the sodium pentobarbital-induced sleep time in mice to 75.2 min.

[0127] (3) Subthreshold dose hypnotic experiment with sodium pentobarbital Mice were injected intraperitoneally with sodium pentobarbital (19 mg / kg BW) 30 minutes after the last gavage. Mice whose righting reflex disappeared for more than 60 seconds entered a sleep state. The number of animals that fell asleep within 30 minutes was recorded.

[0128] The results of the subthreshold dose experiment of sodium pentobarbital in each group of mice are shown in Table 1. The sleep rate of mice in the normal group was 40%, while the sleep rate of mice in the model group was the lowest, at 18.18%, indicating that mice under chronic unpredictable stimuli (CUMS) will have sleep problems. The sleep rate of mice in the Lactobacillus brucellosis JZ-QX01 group was 30%. Compared with the model group, Lactobacillus brucellosis JZ-QX01 improved the sleep rate of mice.

[0129] Table 1. Effects of Lactobacillus brevicornu JZ-QX01 on sleep rate induced by sodium pentobarbital in mice.

[0130] 8. Effects on neurotransmitters in mice Hippocampal tissue was weighed and homogenized with 9 volumes of pre-cooled PBS. The supernatant was collected, and the levels of 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in the hippocampal tissue were detected by ELISA. The 5-HT ELISA kit was purchased from Wuhan Elabscience (Catalog No. E-EL-0033), the DA ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Catalog No. ml002024A), and the NE ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Catalog No. ml063805A).

[0131] To elucidate the neurochemical basis of sleep regulation mediated by *Lactobacillus brucellosis* JZ-QX01, this study quantified key sleep-related neurotransmitters 5-HT, DA, and NE in the brain. Among them, L-tryptophan (Trp) can be metabolized into 5-hydroxytryptophan (5-HTP), which is then converted into serotonin (5-HT); L-DOPA is a precursor to dopamine (DA), which is further metabolized into norepinephrine (NE). These neuromodulators are key in coordinating the sleep-wake transition.

[0132] Depend on Figure 8 The results showed that, compared with the normal control group, the levels of three monoamine neurotransmitters, 5-HT, DA, and NE, in the hippocampus of mice in the model group were significantly reduced (p < 0.01), confirming that the CUMS model successfully induced functional inhibition of the hippocampal monoaminergic system. Compared with the model group, the levels of 5-HT, DA, and NE in the hippocampus of mice in the JZ-QX01 intervention group were significantly increased (p < 0.01), and their levels returned to the normal range. These results demonstrate that oral administration of the JZ-QX01 strain can effectively reverse the depletion of key monoamine neurotransmitters in the hippocampus and fully restore their levels.

[0133] In conjunction with the GABA-boosting effect of JZ-QX01 in the aforementioned embodiments, this embodiment further reveals the restorative ability of JZ-QX01 to monoamine neurotransmitters.

[0134] 9. Effects on short-chain fatty acids in mice Weigh the contents of the mouse cecum, add 200 μL of acetonitrile solution and two small steel balls, grind in a grinder (45 Hz, 2 min), extract by sonication in an ice-water bath for 10 min, centrifuge for 10 min (4℃, 12000 rpm), collect the supernatant and dilute it 5-fold with acetonitrile, and transfer 80 μL of the diluted supernatant to a vial. Then add 40 μL of 200 mM 3-NPH (prepared in 50% acetonitrile aqueous solution, v / v) and 40 μL of 120 mM EDC-6% pyridine (prepared in 50% acetonitrile aqueous solution, v / v), and react at 40℃ for 30 min to complete the derivatization reaction, which is then used for sample detection.

[0135] The contents of six short-chain fatty acids, namely acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and isobutyric acid, were qualitatively and quantitatively determined using UPLC-ESI-MS / MS. The specific analytical conditions and methods are as follows: Chromatographic conditions: Injection volume: 1 μL; Flow rate: 0.35 mL / min; Mobile phase: A (0.1% formic acid-water solution), B (acetonitrile / methanol = 2:1); Gradient elution procedures: 0 min A / B (75:25, V / V), 2 min A / B (75:25, V / V), 11 min A / B (45:55, V / V), 12 min A / B (75:25, V / V), 13 min A / B (75:25, V / V).

[0136] Mass spectrometry conditions: Curtain gas: 35 (psi); Collision-induced dissociation (CAD) parameters: medium; Negative ion spray voltage: -4500V; Ion source temperature: 450℃; Column temperature: 40℃; Spray gas (Gas1): 50 (psi); Auxiliary heating gas (Gas2): 50 (psi).

[0137] The results are as follows Figure 9 As shown, compared with the control group, the levels of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and isobutyric acid in the model group mice were reduced. After intervention with Lactobacillus brucellosis JZ-QX01, the short-chain fatty acid content in CUMS mice was significantly regulated, and the levels of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, and isobutyric acid were significantly increased. This indicates that intervention with Lactobacillus brucellosis JZ-QX01 can effectively improve sleep in mice by increasing the content of short-chain fatty acids.

[0138] Abnormal metabolism of short-chain fatty acids (SCFAs) (insufficient production or imbalance) is a crucial pathological link in various sleep disorders, and its disordered patterns are closely related to disease phenotypes. Butyrate, as the most significant SCFA component in regulating sleep, can directly activate GABAergic neurons in the hypothalamus, the core center for sleep initiation. Butyrate activates the PI3K-AKT signaling pathway via GPR41 receptors on the neuronal surface, promoting the expression of GABA synthase and increasing GABA secretion by 30%-40%. Simultaneously, butyrate inhibits the activity of GABA transporters, reducing GABA reuptake in the synaptic cleft and prolonging its duration of action. After GABA binds to GABA receptors on the surface of neurons in the sleep-wake promotion zone, it inhibits the excitability of these areas, promoting the body's transition from wakefulness to sleep.

[0139] In summary, the Lactobacillus brucellosis JZ-QX01, which has a high GABA-producing capacity obtained through in vitro screening, can improve sleep by promoting the secretion of GABA in the body, restoring the balance of neurotransmitters in the brain, and working synergistically with short-chain fatty acids.

[0140] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus buchneri (L. buchneri) JZ-QX01, characterized in that, Lentilactobacillus buchneri ) JZ-QX01, characterized in that, Its accession number is CGMCC No.37544.

2. The Lactobacillus buchneri JZ-QX01 according to claim 1, characterized in that, The 16S rDNA sequence is shown in SEQ ID NO:

1.

3. A microbial agent, characterized in that, The bacterial agent includes *Lactobacillus brucellosis* JZ-QX01 as described in any one of claims 1-2.

4. The preparation of *Lactobacillus brucellosis* JZ-QX01 according to claim 1, characterized in that, include: Fermentation broth, live bacteria, or freeze-dried powder.

5. The use of Lactobacillus brucellosis JZ-QX01 as described in any one of claims 1-2, or the bacterial agent as described in claim 3, or the preparation as described in claim 4, in the preparation of products that increase the content of GABA in vivo.

6. The application according to claim 5, characterized in that, The product has at least one of the following functions: (1) Increase serum GABA levels; (2) Promotes GABA secretion in intestinal tissues; (3) Promotes GABA secretion in the hippocampus; (4) Promotes GABA secretion in the hypothalamus.

7. The application according to claim 5, characterized in that, The viable cell number of the Lactobacillus buchneri JZ-QX01 in the product is not less than 1.0×10 9 CFU / g or 1.0×10 9 CFU / mL.

8. The use of Lactobacillus brucellosis JZ-QX01 as described in any one of claims 1-2, or the bacterial agent as described in claim 3, or the preparation as described in claim 4, in the preparation of products for improving sleep.

9. The application according to claim 8, characterized in that, The product in question is a medicine.

10. The application according to claim 8, characterized in that, The product in question is a health food product.

11. The application according to claim 8, characterized in that, The viable cell number of the Lactobacillus buchneri JZ-QX01 in the product is not less than 1.0×10 9 CFU / g or 1.0×10 9 CFU / mL.

12. The application according to claim 8, characterized in that, The product described can improve insomnia caused by stress, mood disorders, and / or abnormal short-chain fatty acid metabolism.

13. The application according to claim 9, characterized in that, The drug includes a pharmaceutically acceptable carrier.

14. The application according to claim 10, characterized in that, The health food also includes conventional excipients for health food, which are selected from one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

15. The application according to claim 8, characterized in that, The product has at least one of the following functions: (1) Prolong sleep time; (2) Increase the levels of serotonin, dopamine, and norepinephrine; (3) Increase the content of short-chain fatty acids.

16. A product that increases GABA levels in the body or improves sleep, characterized in that, The product includes Lactobacillus brucellosis JZ-QX01 as described in any one of claims 1-2, or the bacterial agent as described in claim 3, or the preparation as described in claim 4.

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