Lactobacillus plantarum and its use in preventing or treating tourette syndrome

Abstract

The application discloses a plant lactobacillus and application of the plant lactobacillus in a composition for preventing or treating a tic disorder, and belongs to the technical field of microorganisms. The technical problem to be solved is to provide a plant lactobacillus LP496 isolated from healthy breast milk, wherein the plant lactobacillus LP496 can significantly reduce a tic behavior, improve a stereotypic symptom, improve motor coordination and repair muscle strength damage, realize double neurotransmitter regulation by down-regulating dopamine and up-regulating serotonin, and has a significant protective effect on brain neuron damage. A technical solution point is that the plant lactobacillus LP496 has a preservation number of CGMCC No. 33499. The application further discloses a microbial agent containing the strain or a preparation of the strain, and application of the strain and the microbial agent in preparation of a composition for preventing or treating a tic disorder and related compositions, and provides a safe, effective and mild new microecological intervention scheme for the tic disorder.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus plantarum* and its application in compositions for the prevention or treatment of tic disorders. Background Technology

[0002] Tic disorders (TD) are a group of neuropsychiatric disorders that begin in childhood and adolescence. Their core clinical manifestations are involuntary, repetitive, rapid muscle tics and / or vocal tics in one or more areas of the body. They are often accompanied by comorbidities such as inattention, hyperactivity, compulsive behaviors, and thought disorders, severely impacting patients' learning, social interactions, and overall mental and physical well-being. The pathogenesis of this disease is complex and not yet fully understood. Current research suggests that its occurrence is closely related to the interaction of multiple factors, including genetic factors, imbalances in central neurotransmitters (especially striatal dopamine overactivity or postsynaptic dopamine receptor hypersensitivity), psychosocial factors, environmental factors, and gut microbiota dysbiosis.

[0003] Based on clinical characteristics and duration of illness, tic disorders can be divided into three types: transient tic disorder, chronic tic disorder, and Tourette syndrome (also known as multiple tic disorder). Among them, Tourette syndrome is relatively severe, accompanied by motor and vocal tics, with a course of illness lasting more than one year. It has a significant impact on the patient's social functioning, and some patients may also experience emotional instability, sleep disorders, self-harming behaviors, etc., which further increases the complexity of the disease and the difficulty of treatment.

[0004] Current treatments for Tourette syndrome primarily include medication and psychotherapy. Medication, mainly consisting of dopamine receptor antagonists and central alpha-2 agonists, is used to alleviate tic symptoms and related comorbidities. However, these medications have significant limitations: firstly, they cannot fundamentally address the core mechanisms of the disease, only temporarily controlling symptoms and failing to provide a cure; relapse is also common after discontinuation. Secondly, long-term use of these medications can cause various adverse reactions, such as drowsiness, weight gain, cognitive impairment, and gastrointestinal discomfort. Some medications may even induce excessive growth of intestinal pathogens, further disrupting the gut microbiota balance and affecting treatment efficacy. While psychotherapy can help improve patients' psychological state and behavioral performance, its symptom relief effect is limited for moderate to severe Tourette syndrome and cannot be used as a primary treatment. Therefore, developing safe, effective, and minimally invasive preventative and therapeutic methods is an urgent need in the clinical treatment of Tourette syndrome.

[0005] Relevant patent documents retrieved: The country of origin is China, publication number CN110023485A, publication date December 5, 2023. This document discloses a subspecies of *Lactobacillus plantarum* registered under the DSMZ accession number DSM 28632. Lactobacillus plantarum subsp.plantarum PS128 is used to prepare medications for the treatment or prevention of Parkinson's disease or tic-related disorders induced by abnormally elevated dopamine signaling in individuals. Tic disorders include Tourette syndrome, DOI-induced tic-related disorders, chronic motor disorders, or vocal tic disorders.

[0006] Relevant non-patent literature retrieved: The literature, titled "Study on the Effects of Lactobacillus plantarum PS128 on Behavioral and Serum IL-6 Levels in a Rat Model of Tourette Syndrome," was published in March 2022. This literature discloses that Lactobacillus plantarum PS128 has a certain effect on improving the behavioral changes in a rat model of Tourette Syndrome, and this effect is dose-dependent. Lactobacillus plantarum PS128 effectively inhibits IL-6 levels in a rat model of Tourette Syndrome (TD).

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: No existing technology discloses a plant lactobacillus that can effectively treat tic disorders. Providing a plant lactobacillus with specific probiotic functions and applying it to a composition for the prevention or treatment of tic disorders to achieve effective prevention and treatment of tic disorders is a technical problem that urgently needs to be solved. Summary of the Invention

[0008] The purpose of this invention is to provide: A strain of *Lactobacillus plantarum* and its application in a composition for the prevention or treatment of tic disorders, and related technologies, to address the technical problems of providing a strain of *Lactobacillus plantarum* LP496, which can improve stereotyped behaviors, enhance motor coordination, repair muscle strength damage, reduce dopamine levels, increase serum serotonin levels, and reduce the number of damaged neurons, or combinations thereof.

[0009] 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.

[0010] 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.

[0011] Definitions of standard chemical terms can be found in the references "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy, Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01"; "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Science Press, 4th Edition, 2017; and "Microbiology Experiments", Higher Education Press, 4th Edition, 2016.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as growth curves, cell adhesion ability, acid resistance tests, antioxidant capacity tests, and adhesion ability tests, shall be used.

[0013] 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.

[0014] The term "IDPN" used in this article refers to a neurotoxin commonly used to induce tic disorders and ataxia models in animals.

[0015] The term "haloperidol" used in this article refers to a classic antipsychotic drug, primarily used to treat schizophrenia, mania, tic disorders, etc. It works by blocking dopamine receptors to exert sedative effects and control hallucinations, delusions, and tic symptoms.

[0016] The term "Lactobacillus plantarum" used in this article refers to a type of lactic acid bacteria belonging to the genus Lactobacillus. It is a Gram-positive bacterium with multiple functions, including regulating intestinal flora, enhancing immunity, and improving health.

[0017] In a first aspect, the present invention provides: a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The Lactobacillus plantarum LP496 mentioned above has the accession number CGMCC No. 33499.

[0018] Among them are technical features such as Lactobacillus plantarum LP496.

[0019] Among them, the 16S sequence of Lactobacillus plantarum LP496 is as shown in SEQ ID NO:1 or has at least 90% sequence identity with the sequence shown in SEQ ID NO:1.

[0020] Among them, the 16S sequence of Lactobacillus plantarum LP496 is as shown in SEQ ID NO:1 or has at least 95% sequence identity with the sequence shown in SEQ ID NO:1.

[0021] Among them, the 16S sequence of Lactobacillus plantarum LP496 is as shown in SEQ ID NO:1 or has at least 99% sequence identity with the sequence shown in SEQ ID NO:1.

[0022] The preferred 16S sequence of *Lactobacillus plantarum* LP496 is as shown in SEQ ID NO:1.

[0023] The colony characteristics of *Lactobacillus plantarum* LP496 on MRS medium are as follows: white or light yellow colonies, opaque, round, with a smooth and moist surface, neat edges, and a raised center.

[0024] The *Lactobacillus plantarum* LP496 described therein exhibits rod-shaped cells on MRS liquid culture medium, does not produce spores, has no flagella, and is non-motile.

[0025] Among them, the plant lactobacillus LP496 can utilize 25 kinds of carbon sources.

[0026] Furthermore, the 25 carbon sources include L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, methyl-αD-mannopyranoside, N-acetylglucosinolate, amygdalin, arbutin, aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-merinobiose, D-sucrose, D-trehalose, D-melatotriose, D-raffinose, D-gentiobiose, D-merinodiol, and gluconate.

[0027] Among them, Lactobacillus plantarum LP496 has a high antioxidant capacity; Furthermore, the *Lactobacillus plantarum* LP496 exhibits high DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and total antioxidant capacity.

[0028] Furthermore, the *Lactobacillus plantarum* LP496 is non-pathogenic.

[0029] Furthermore, the *Lactobacillus plantarum* LP496 described therein can inhibit pathogens.

[0030] Furthermore, the pathogens mentioned include Cronobacter sakazakii, Escherichia coli, Shigella flexneri, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0031] Furthermore, the *Lactobacillus plantarum* LP496 exhibits a high survival rate in gastric and intestinal fluids.

[0032] Furthermore, the *Lactobacillus plantarum* LP496 described herein exhibits no resistance to antimicrobial drugs.

[0033] Furthermore, the antibacterial drugs mentioned include ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol, and vancomycin.

[0034] Secondly, the present invention provides a microbial inoculant, wherein the microbial inoculant includes the above-mentioned *Lactobacillus plantarum* LP496 or a preparation of *Lactobacillus plantarum* LP496.

[0035] Specifically, the microbial agent also includes nutritionally acceptable nutrient additives.

[0036] Preferably, the nutritional additives include any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0037] The preparations of *Lactobacillus plantarum* LP496 include: live bacteria, inactivated bacteria, lysates, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder, lysates, secondary metabolites, and exosomes.

[0038] Specifically, live Lactobacillus plantarum LP496 is a bacterial cell with normal physiological activity, capable of carrying out life activities such as metabolism and reproduction.

[0039] Specifically, inactivated Lactobacillus plantarum LP496 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.

[0040] Specifically, Lactobacillus plantarum LP496 lysate refers to the mixture of intracellular substances and cell fragments formed after live or inactivated Lactobacillus plantarum LP496 cells are broken down by physical, chemical or enzymatic methods, releasing intracellular substances.

[0041] Specifically, the fermentation broth of *Lactobacillus plantarum* LP496 is a mixed liquid system obtained by culturing *Lactobacillus plantarum* LP496 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.

[0042] Specifically, the supernatant of the fermentation broth of Lactobacillus plantarum LP496 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.

[0043] Specifically, the precipitate of Bacillus plantarum LP496 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.

[0044] Specifically, Lactobacillus plantarum LP496 freeze-dried powder refers to a solid powder that retains the original active components, obtained by removing moisture from liquid materials such as strain fermentation broth, fermentation supernatant, and bacterial suspension through a freeze-drying process.

[0045] Specifically, Lactobacillus plantarum LP496 lysate refers to a mixed system containing intracellular active components and fragmented bacterial cells formed after live or inactivated Lactobacillus plantarum LP496 bacteria have had their cell walls and cell membranes ruptured through physical, chemical, enzymatic, or biological lysation methods, releasing all intracellular substances.

[0046] Specifically, secondary metabolites of Lactobacillus plantarum LP496 refer to various compounds produced by microorganisms such as the strain during their stable growth period, which are not essential for their own growth and reproduction, and often possess specific biological activities such as anti-inflammatory and metabolic regulation.

[0047] Specifically, *Lactobacillus plantarum* LP496 exosomes refer to extracellular vesicles encapsulated by nanoscale lipid bilayer membranes that are actively secreted during the growth and metabolism of *Bifidobacterium animalis* subsp. *major* or released after cell lysis.

[0048] Thirdly, the present invention provides the use of the above-mentioned *Lactobacillus plantarum* LP496 or the above-mentioned microbial agent in the preparation of compositions for the prevention or treatment of tic disorders.

[0049] Composition: a product comprising an active ingredient and an inert component (pharmaceutically acceptable excipient) constituting a carrier, and any product obtained directly or indirectly from a combination, complexation or aggregation of two or more components, or from the decomposition of one or more components, or from other types of reactions or interactions of one or more components.

[0050] The composition described herein has any one or more of the following functions: (1) Improved stereotyped behaviors, reduced the frequency of tic behaviors, and no adverse reactions in the motor system; (2) Improve motor coordination; (3) Repair muscle strength damage; (4) Improves dopamine levels and increases serum serotonin levels; (5) Reduce the number of damaged neurons.

[0051] Furthermore, the composition has any one or more of the following functions: (1) Relieves symptoms of tic disorders without adverse reactions in the motor system; (2) Protect motor coordination ability; (3) Relieve tic symptoms by regulating the dopamine system; (4) Inhibits the dopamine system; (5) Improve neuronal damage caused by tic disorders.

[0052] The aforementioned "tic disorder" has the following characteristics: (1) Increased stereotyped behaviors; (2) The number of tics increases; (3) Decreased motor coordination and muscle strength; (4) Increased dopamine levels; (5) Serum serotonin levels are decreased; (6) The number of damaged neurons increases.

[0053] Specifically, the tic disorders include, but are not limited to, transient tic disorders, persistent motor or vocal tic disorders, and Tourette syndrome.

[0054] Furthermore, the aforementioned tic disorder is Tourette syndrome.

[0055] The composition contains 1×10⁶ Lactobacillus plantarum LP496 bacteria. 8 CFU-1×10 12 CFU.

[0056] According to some embodiments of the present invention, the bacterial content of *Lactobacillus plantarum* LP496 may include 1 × 10⁻⁶. 8 CFU, 2×10 8 CFU, 3×10 8 CFU, 4×10 8 CFU, 5×10 8 CFU, 6×10 8 CFU, 7×10 8 CFU, 8×10 8 CFU, 9×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU, 1×10 11 CFU, 1×10 12 CFU.

[0057] The composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants.

[0058] The composition may be in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule or aerosol.

[0059] Fourthly, the present invention provides a composition comprising the above-mentioned *Lactobacillus plantarum* LP496 or the above-mentioned microbial agent, the composition having the function of preventing or treating tic disorders.

[0060] Composition: a product comprising an active ingredient and an inert component (pharmaceutically acceptable excipient) constituting a carrier, and any product obtained directly or indirectly from a combination, complexation or aggregation of two or more components, or from the decomposition of one or more components, or from other types of reactions or interactions of one or more components.

[0061] The composition described herein has any one or more of the following functions: (1) Improved stereotyped behaviors, reduced the frequency of tic behaviors, and no adverse reactions in the motor system; (2) Improve motor coordination; (3) Repair muscle strength damage; (4) Improves dopamine levels and increases serum serotonin levels; (5) Reduce the number of damaged neurons.

[0062] Furthermore, the composition has any one or more of the following functions: (1) Relieves symptoms of tic disorders without adverse reactions in the motor system; (2) Protect motor coordination ability; (3) Relieve tic symptoms by regulating the dopamine system; (4) Inhibits the dopamine system; (5) Improve neuronal damage caused by tic disorders.

[0063] The aforementioned "tic disorder" has the following characteristics: (1) Increased stereotyped behaviors; (2) The number of tics increases; (3) Decreased motor coordination and muscle strength; (4) Increased dopamine levels; (5) Serum serotonin levels are decreased; (6) The number of damaged neurons increases.

[0064] Specifically, the tic disorders include, but are not limited to, transient tic disorders, persistent motor or vocal tic disorders, and Tourette syndrome.

[0065] Furthermore, the aforementioned tic disorder is Tourette syndrome.

[0066] The composition contains 1×10⁶ Lactobacillus plantarum LP496 bacteria. 8 CFU-1×10 12 CFU.

[0067] According to some embodiments of the present invention, the bacterial content of *Lactobacillus plantarum* LP496 may include 1 × 10⁻⁶. 8 CFU, 2×10 8 CFU, 3×10 8 CFU, 4×10 8 CFU, 5×10 8 CFU, 6×10 8 CFU, 7×10 8 CFU, 8×10 8 CFU, 9×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU, 1×10 11 CFU, 1×10 12 CFU.

[0068] The composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants.

[0069] The composition may be in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule or aerosol.

[0070] The present invention has at least the following beneficial effects: This invention provides a strain of *Lactobacillus plantarum* LP496, with accession number CGMCC No. 33499. This *Lactobacillus plantarum* LP496 can utilize 25 carbon sources, has high in vitro antioxidant capacity, is non-pathogenic, has strong tolerance to gastrointestinal fluid, and shows good inhibitory effects on five pathogenic bacteria. The adhesion index is 24.92%, the adhesion rate is 1.75%, the self-aggregation power is 7.55%, and it has no resistance to nine antibacterial drugs (ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol, and vancomycin).

[0071] The *Lactobacillus plantarum* LP496 of this invention has a good effect on improving tic disorders, can improve stereotyped behaviors, reduce the frequency of tic behaviors, and alleviate the core symptoms of tic disorders. No side effects such as bradykinesia have been observed, and its safety is superior to that of drugs. It improves motor coordination and repairs muscle strength damage associated with tic disorders; it reduces dopamine levels and relieves tic symptoms by regulating the dopamine system; it forms a dual neurotransmitter regulation mechanism of "dopamine downregulation + serotonin upregulation", which is more comprehensive and gentler; and it has a significant protective effect against neuronal damage.

[0072] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for preventing or treating tic disorders, comprising administering to a subject an effective amount of *Lactobacillus plantarum* LP496 or the above-described bacterial agent or the above-described preparation or the above-described composition.

[0073] The term "subject" includes living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0074] Specifically, the method has any one or more of the following functions: (1) Improved stereotyped behaviors, without bradykinesia or decreased activity; (2) The frequency of tic behaviors decreased; (3) Improve motor coordination; (4) Repair muscle strength damage; (5) Reduce dopamine levels; (6) Increase serum serotonin levels; (7) The number of damaged neurons is reduced.

[0075] Preservation Instructions Preserved strain: Lactobacillus plantarum LP496; Classification and nomenclature: Lactobacillus plantarum Lactiplantibacillus plantarum ; Accession number: CGMCC No. 33499; Preservation period: January 23, 2025; 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

[0076] Figure 1 Photograph of Lactobacillus plantarum LP496 plate.

[0077] Figure 2 Gram-stained microscopic image of Lactobacillus plantarum LP496.

[0078] Figure 3 Blood plate image of Lactobacillus plantarum LP496.

[0079] Figure 4 This is a growth curve of Lactobacillus plantarum LP496.

[0080] Figure 5 This is the experimental timeline.

[0081] Figure 6 For tic behavior counts, ns indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001.

[0082] Figure 7 The time of residence of the rotator is ns, where ns indicates no significant difference, **p<0.01, ***p<0.001.

[0083] Figure 8 The maximum grip strength of the forelimb is given by ns, where ns indicates no significant difference, ***p<0.001, ****p<0.0001.

[0084] Figure 9 The values ​​represent serum dopamine levels. *p < 0.05, **p < 0.01, ****p < 0.0001.

[0085] Figure 10 The value represents serum serotonin levels. ns indicates no significant difference. *p < 0.05.

[0086] Figure 11 Nissl staining of the brain striatum.

[0087] Figure 12 The number of normal neurons per unit area of ​​the striatum is given by ns, where ns indicates no significant difference. *p<0.05, ***p<0.001, ****p<0.0001. Detailed Implementation

[0088] 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.

[0089] 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.

[0090] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0091] Example 1: Isolation and Identification of Lactobacillus plantarum LP496 The *Lactobacillus plantarum* LP496 of this invention was isolated from healthy breast milk. The 16S rRNA sequencing results are shown in SEQ ID NO:1.

[0092] Example 2 Characterization of Lactobacillus plantarum LP496 2.1 Morphological and colony observation, hemolytic characteristics Pure bacterial culture was evenly spread onto a glass slide and fixed in the outer flame of an alcohol lamp. After Gram staining, the slide was slowly rinsed with deionized water to remove excess staining solution. After the slide dried, it was observed and photographed under an oil immersion microscope. One loopful of bacterial culture was streaked onto MRS agar medium and incubated at 37°C for 48 hours. The colony morphology was then observed.

[0093] The microbiological characteristics of Lactobacillus plantarum LP496 are as follows: (1) Colony morphology: such as Figure 1 As shown, the colonies grown in MRS agar medium are white or light yellow, opaque, round, with a smooth and moist surface, neat edges, and a raised center.

[0094] (2) Gram staining morphology under microscopy: such as Figure 2 As shown, after staining, the bacteria are rod-shaped, do not produce spores, have no flagella, and are non-motile.

[0095] (3) Colony characteristics on Columbia blood agar plates: such as Figure 3 No hemolysis was observed.

[0096] 2.2 Utilization of different carbohydrates by Lactobacillus plantarum LP496 Preparation of colony plates: The strain is streaked on MRS agar plates. After colonies grow, single colonies are picked and streaked again until single colonies grow.

[0097] Preparation of bacterial test solution: Pick the colonies on the plate into 2 mL of physiological saline, shake and mix well, take an appropriate amount of bacterial solution (v) into 5 mL of physiological saline, measure the OD value, and then take (2v) from the original bacterial solution into 10 mL of API-matched culture medium and mix well.

[0098] Incubation reaction: Add sterile deionized water to the bottom plate of the incubation box to ensure a humid environment. Take out the test strips (0-19, 20-39, 40-49) from the packaging bag, separate them, place them in the bottom plate of the incubation box, and gently tilt them forward.

[0099] Using a pipette, draw approximately 115 μL of bacterial culture, placing the pipette tip against the edge of the cup to add it, avoiding air bubbles. Fill only the top of the tube (cup) completely, then seal the top with sterile liquid paraffin to maintain an anaerobic environment. Incubate the tube at 37°C, observing the color changes of the reagent strips after 24 and 48 hours. The results are shown in Table 1, indicating that *Lactobacillus plantarum* LP496 can utilize 25 carbon sources.

[0100] Table 1. Identification results of Lactobacillus plantarum LP496 and API-50.

[0101] Note: + indicates that the color is different from the control color, and - indicates that the color is the same as the control color.

[0102] 2.3 Growth curve determination of Lactobacillus plantarum LP496 After activating Lactobacillus plantarum LP496 for two generations at a 1% inoculum in 96-well plates, the third generation was inoculated at a 1% inoculum and the growth curve was measured at regular intervals using an ELISA reader.

[0103] Specific procedures: The test strain preserved in glycerol was inoculated into sterile MRS liquid medium at a 1% inoculum and anaerobically cultured at 37°C for 16 h. 1% was then transferred to a sterile 96-well plate containing 200 µl of MRS broth. After purging with nitrogen, the plate was cultured at 37°C for 8 h. 1% was then transferred to a new 96-well plate. Each strain was replicated in 3 wells. After sealing, the plates were cultured at 37°C with shaking. OD600 was measured every 1 h for 48 h.

[0104] The results are as follows Figure 4 The results showed that *Lactobacillus plantarum* LP496 entered the logarithmic growth phase at 4 hours and the stationary phase at 21 hours, with an OD600 value of approximately 1.18 at the stationary phase.

[0105] 2.4 In vitro antioxidant capacity of Lactobacillus plantarum LP496 Lactobacillus plantarum LP496 was activated and cultured for three generations. Cell lysates were prepared by sonication (100W, 10 min) and the supernatant obtained by centrifugation (10000g, 5 min) and filtration through a 0.22 μm filter. The DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and total antioxidant capacity of the cell lysates and fermentation broth supernatant were tested using the Nanjing Jiancheng FRTP kit (catalog number: A015-3-1). The experimental procedures followed the kit instructions. The results are shown in the table below.

[0106] Table 2 Antioxidant capacity of Lactobacillus plantarum LP496

[0107] 2.5 Virulence and Safety Testing of Lactobacillus plantarum LP496 The pathogenicity test method for food-grade bacteria in Appendix A of GB 31615.2-2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" was used to test Lactobacillus plantarum LP496. The test animals showed no abnormalities or deaths, and their body weight was not statistically significant compared with the control group (p>0.05), indicating that this strain is non-pathogenic.

[0108] 2.6 Detection of ability to inhibit pathogenic bacteria Preparation of fermentation supernatant of the test bacteria: After three consecutive generations of activation, the third generation fermentation broth was centrifuged at 6000×g for 10min, the supernatant was collected, filtered through a 0.22μm micromembrane, and stored at -20℃ for later use.

[0109] After activating the pathogenic bacteria (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, Shigella flexneri CICC 21534, and Cronobacter sakazakii CICC 21560) three times in liquid culture medium, the third-generation culture medium was adjusted to a suitable absorbance value, so that the bacterial suspension concentration was 1×10⁻⁶. 8 CFU / mL ~5×10 8 CFU / mL.

[0110] Preparation of test plates: Heat and dissolve the prepared NA medium, cool to 45℃~50℃, add the prepared indicator bacterial suspension to the NA medium at an addition rate of 1%, mix thoroughly, measure 20mL and pour into a sterile Petri dish, gently shake the Petri dish to spread it evenly, and wait for it to solidify before use.

[0111] Place 4–6 Oxford cups at equal intervals on a test plate containing indicator bacteria, press gently, and slowly add 200 μL of the fermentation supernatant of the test bacteria into the Oxford cups. Repeat each treatment three times. Place the plates in a refrigerator at 4–6°C for pre-diffusion for 4–10 hours. Remove the plates and place them in a constant temperature incubator at 36°C ± 1°C, incubating upright until the inhibition zone is clear. Measure the diameter of the inhibition zone using calipers or an inhibition zone measuring instrument. Measure each inhibition zone three times along different directions and record the average value. Results are shown in Table 3.

[0112] Table 3. Diameter of the outer inhibition zone of LP496 strain against the pathogen.

[0113] The results showed that Lactobacillus plantarum LP496 had a good inhibitory effect on all five pathogenic bacteria.

[0114] 2.7 Resistance of Lactobacillus plantarum LP496 to artificial gastrointestinal fluid Preparation of artificial gastric fluid: Measure 0.2 mL of hydrochloric acid, add 0.8 mL of water and mix well to obtain dilute hydrochloric acid. Weigh 0.2 g of NaCl and 0.35 g of pepsin, add 100 mL of water and dissolve completely. Adjust the pH to 2.5 with dilute hydrochloric acid, mix well, and filter under sterile conditions using a 0.22 μm filter membrane.

[0115] Preparation of artificial intestinal fluid: Dissolve 0.68g of potassium dihydrogen phosphate in 50mL of water, adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution, and dissolve 1g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions, dilute with water to 100mL, mix well, and filter under sterile conditions using a 0.22μm filter membrane.

[0116] Activation of the strain: 2% of the strain cryopreservation solution was inoculated into 1.5 mL of MRS broth and incubated overnight at 37°C. The next day, 5% of the bacterial solution was inoculated into 1.5 mL of MRS broth and incubated at 37°C for 7 hours. 2% of the activated second-generation bacterial solution was inoculated into 15 mL of MRS broth and incubated overnight at 37°C.

[0117] Bacterial culture treatment: Take 10 mL of culture medium, centrifuge at 6000 g for 10 min, discard the supernatant, wash the bacteria twice with sterile physiological saline, resuspend the bacterial sludge in 3 mL of artificial gastric fluid, mix well, and then bring the volume to 10 mL with artificial gastric fluid. Take 100 μL and perform 10-fold serial dilutions, and use MRS plate counting to determine the initial viable count. After anaerobic static incubation at 37℃ for 3 h, take 100 μL and perform serial dilutions, and use MRS plate counting to determine the viable count after 3 h of gastric fluid treatment. Bacterial culture with artificial intestinal fluid treatment: The treatment method is the same as that for bacterial culture with artificial gastric fluid treatment, only the artificial gastric fluid in the system is replaced with artificial intestinal fluid. The results are shown in Table 4 below.

[0118] Table 4. Statistical analysis of viable bacterial counts of strain LP496 in simulated gastric and intestinal fluids after 3 hours.

[0119] 2.8 Determination of Adhesion Ability of Lactobacillus plantarum LP496 Strain preparation: LP496 strain was activated, cultured for three generations, centrifuged at 10000g for 5 min in 0.01M pBS (pH 7.2-7.4), washed twice, and the bacterial concentration was adjusted to approximately 1×10⁻⁶ in DMEM medium. 6 CFU / mL.

[0120] Cell preparation: Resuscitate and culture Caco-2 cells to passage 3, digest the cells and plate them into 24-well cell culture plates. The Caco-2 cells should form a monolayer with a confluence of 80-90%. Wash the cells with 0.01M pBS, 1 mL / well, twice. The washing process should be gentle to avoid adhering cells detaching.

[0121] Adhesion experiment: After cell washing, add 1 mL of solution containing 1×10⁻⁶ strains. 6Incubate at CFU / mL in a 37℃, 5% CO2 incubator for 2 h. Use the washed bacterial suspension as a reference for co-incubation. After incubation, discard the cell culture supernatant containing unadhered strains. Wash cells twice with 0.01M pBS (1 mL / well). Digest cells with Trypsin-EDTA (0.25%) for 15 min (0.5 mL / well). Count the cells in each well of a 24-well cell culture plate. Dilute the digested bacterial suspension with physiological saline and perform plate counting. Record the number of colonies formed by the strains on MRS plates before and after adhesion, and calculate the adhesion index and adhesion rate.

[0122] Adhesion index = Number of viable bacteria after adhesion / Number of cells in negative control well; Adhesion rate (%) = Number of viable bacteria after adhesion / Number of viable bacteria before adhesion.

[0123] The adhesion experiment results of strain LP496 are shown in Table 5 below.

[0124] Table 5 Adhesion ability of LP496 strain

[0125] 2.9 Determination of strain self-polymerization ability Take the fermentation broth of the experimental strain, centrifuge the broth for 10 min (4℃, 6000 r / min), collect the precipitate, wash the cells twice with sterile physiological saline, and finally resuspend the cells with physiological saline. Measure the OD600 and record it as A0. Take the supernatant after the broth has stood for 2 h and measure the OD600, record it as At. Calculate the self-polymerization ability. Self-polymerization ability (%) = (1-At / A0)*100. The results are shown in Table 6.

[0126] Table 6 Adhesion ability of LP496 strain

[0127] 2.10 Drug susceptibility testing of strains The method of determining the resistance of food-grade bacterial strains to antimicrobial agents (micro-broth dilution method) in Appendix E of GB31615.2-2025 "National Food Safety Standard - Procedure for Safety Evaluation of Food-grade Microbial Strains" was used to test *Lactobacillus plantarum* LP496. The results are shown in the table below. *Lactobacillus plantarum* LP496 showed no resistance to the following antimicrobial agents.

[0128] Table 7

[0129] Example 3: Improvement of Tic Disorders by Lactobacillus plantarum LP496 1. After constructing an animal model of tic disorder using IDPN (3,3'-iminodipropionitrile), the effects of the strain on alleviating tic disorder were evaluated by differentiating the stereotyped behavior scores, tic performance, and motor coordination ability of the experimental animals.

[0130] See experimental timeline information Figure 5 .

[0131] Strain preparation: Activate the strain, culture for three generations, and concentrate to 1×10⁻⁶. 9 CFU / mL, aliquot and store at 4℃.

[0132] Animal modeling drug: IDPN was purchased from SIGMA, catalog number: 317306; Animal therapeutic drug: Haloperidol was purchased from SIGMA, catalog number: H1512; Animal testing instruments: Rotary bar fatigue tester was purchased from Jiangsu Saions Biotechnology Co., Ltd., model: SA102; Grip force tester was purchased from Jiangsu Saions Biotechnology Co., Ltd., model: SA415.

[0133] Eight-week-old male BALB / c mice, weighing 20-24g, were housed in a specific pathogen-free (SPF) environment for one week. They were kept under cyclical conditions of 12 hours of light and 12 hours of darkness. Each group consisted of eight mice with free access to food and water. A seven-day acclimatization period was given to the mice before the experiment. Temperature, humidity, and noise levels in the animal housing were controlled within specified limits, and the housing was cleaned regularly each day.

[0134] The experiment included a blank control group, a model group, a treatment group, an LP496 group, and a TS005 group (the TS005 strain was isolated from healthy breast milk and, like LP496, is a plant-based Lactobacillus strain, serving as an experimental control strain).

[0135] Model group: IDPN modeling, with saline gavage; Treatment group: IDPN modeling, haloperidol administered by gavage, i.e., positive control group; LP496 group: IDPN modeling, LP496 gavage; TS005 group: DPN modeling, TS005 gavage.

[0136] Gavage dose 1×10 8 CFU / animal / day (i.e., 100 μl / animal), administered via gavage for 28 days. See details of the experimental procedure below. Figure 5 .

[0137] IDPN working solution (79.45 mg / mL) formulation: 8.8% IDPN + 91.2% physiological saline (autoclaved). For use, administer intraperitoneally at a dose of 350 mg / kg.

[0138] Haloperidol working solution (0.454 mg / mL) formulation: After weighing haloperidol, add solvents in the following order to dissolve it: 2% DMSO + 30% PEG300 + 1% Tween80 + 67% physiological saline.

[0139] Animal behavioral tests need to be conducted in a specific order, progressing from most difficult to least difficult, based on the level of stress the animal experiences. First, stereotyped behavior tests are performed, followed by the rotating bar test, and finally the grip strength test. Before the behavioral tests, the animals are allowed 30 minutes to acclimatize to the testing environment to minimize the interference of stress on the test results.

[0140] 2. Scoring of stereotyped behaviors and tic performance in laboratory animals Stereotyped behaviors in mice refer to a series of repetitive, fixed, and purposeless movement patterns. These behaviors are often considered responses to environmental stress, abnormal brain function, or specific drugs (such as stimulants), resulting in a loss of the flexibility and purposefulness of normal behavior. Tic disorders in mice manifest as an increase in stereotyped behaviors, with the key behavioral phenotype being an increase in tics, accompanied by a certain degree of excessive grooming, circling, and head shaking. A stereotyped behavior rating scale for mice is shown in Table 8; higher scores indicate more severe symptoms.

[0141] Observers placed mice in boxes and recorded 30 minutes of video for each mouse. The boxes used for the stereotyped behavior test were mouse cages (cleaned before testing, sprayed with 75% alcohol, and wiped clean with paper towels), with the camera placed on top of the cage. Lighting conditions during the observation period were LED lights (consistent with the lighting in the mice's daily environment), and these conditions were maintained throughout the experiment. Each animal was observed for 1 minute every 5 minutes, for a total of 6 cycles. Behaviors meeting the corresponding level were assigned a score according to a rating scale, serving as an objective indicator of behavioral change.

[0142] Table 9 shows the stereotyped behavior scores of mice after drug administration and modeling. The model group had the highest stereotyped behavior scores. The treatment group had lower stereotyped behavior scores than the model group, indicating a therapeutic effect. However, most mice in the treatment group showed decreased activity and slowed movement. The LP496 group had lower stereotyped behavior scores than the model group, indicating a relieving effect on the core symptoms of tic disorders. Moreover, no side effects such as bradykinesia were observed in the treatment group, indicating that its safety was superior to the drug. Meanwhile, although TS005 is homologous to LP496 and is also a type of Lactobacillus plantarum, it did not show any effect on improving tic disorders, indicating that the LP496 strain has a unique effect on improving tic disorders.

[0143] Key indicators of stereotyped behavior in mice, such as tic counts, can be found in [link to relevant documentation]. Figure 6 Compared with the model group, the LP496 group showed a significant decrease in the number of tics (P < 0.05) and a significant relief of symptoms.

[0144] Table 8. Scoring Table for Stereotyped Behaviors in Mice

[0145] Note: a. For example, remaining still (the animal does not make any obvious movements); b. For example, rotating clockwise / counterclockwise, leaving a loose circular track at the bottom of the cage; c. For example, lowering its head to sniff (nose touching the ground), grooming its fur (self-cleaning with its mouth or paws), washing its face (front paws moving back and forth from the ears to the nose and mouth).

[0146] Table 9. Scores of stereotyped behavior in mice after drug administration and modeling.

[0147] 3. Motor coordination and muscle strength performance of experimental animals To assess the coordination and balance of the mice, they were placed individually on a rotating bar device and trained for 5 minutes daily for 3 days prior to the formal rotating bar test at a speed of 4 revolutions per minute (rpm) to learn how to use the device. The initial speed of the rotating bar was 4 rpm for 1 minute, then accelerated to 40 rpm over 5 minutes. The mice were held by their tails and placed on the rotating bar, facing the opposite direction of rotation. The time from the start of acceleration to the mouse falling was recorded. Each mouse underwent three tests, with at least 5 minutes between each test, and the average time was calculated.

[0148] Start with a stationary spinner, allowing the mouse to learn to maintain balance on the spinner and adapt to the structure and surrounding environment (1 minute of adaptation). Gradually introduce low-speed rotation (e.g., 4 revolutions per minute), requiring the mouse to maintain balance at this speed for at least 60 seconds. This phase typically lasts 1-3 days, with 5-minute training sessions, conducted in batches to avoid interference. If a mouse falls off during training, it should be immediately placed back on the spinner to reinforce adaptation to continuous movement.

[0149] Mouse rotator bar test after drug administration and model establishment; rotator dwell time is shown in [reference needed]. Figure 7 Mice in the IDPN-treated model group all exhibited decreased motor coordination and muscle strength, indicating that IDPN causes widespread damage to the brain or body, which haloperidol cannot effectively alleviate. There was no significant difference in rotarod dwell time between the LP496 group and the blank group (P>0.05), suggesting its enhancing effect on motor and muscle strength, and also indicating that LP496 has a protective effect on motor coordination.

[0150] To assess forelimb muscle strength and motor coordination in mice, grip strength tests were performed. The procedure was performed according to the manufacturer's instructions for the grip strength meter (Jiangsu Saions Biotechnology Co., Ltd., model: SA415), and the statistical indicator was grip strength (gf). The mouse was placed on a grip bar connected to a force sensor, which was then connected to a peak amplifier. When the mouse grasped the grip bar, it was pulled away from the base by grabbing its tail. Maximum grip strength was measured over 10 seconds.

[0151] To improve data reliability, each mouse is typically measured three times consecutively, and the average value (or the average value after removing outliers) is taken as the final result. Mice must rest for 1-2 minutes between measurements to avoid muscle fatigue that could lead to data bias. The pulling speed should be uniform, completed within 1-2 seconds, avoiding sudden force, ensuring the mouse's forepaws fully grasp the test rod, and the tail is pulled horizontally. Testing should be conducted in a quiet environment with moderate lighting to minimize external interference.

[0152] The maximum grip strength test of the forelimbs of mice after drug administration and modeling is shown in the figure. Figure 8 The model successfully induced a decrease in grip strength in the forelimbs of mice, indicating that IDPN caused damage to the brain or body. Both the treatment group and the LP496 group showed significant improvement in grip strength, with the LP496 group showing better results.

[0153] 4. After the experimental animals were administered the medication by gavage, their serum and brain tissue were collected. Neurotransmitter detection was performed on the serum and brain tissue homogenate supernatant. Nissl staining images of the brain tissue were prepared and observed for analysis.

[0154] After gavage administration to the experimental animals, blood was collected from the eyeballs. The blood was left at room temperature for 1 hour, then centrifuged at 1000g for 10 minutes. The supernatant serum was collected and subjected to enzyme-linked immunosorbent assays (ELISA) according to the instructions of the Dopamine Competitive ELISA Kit (Thermo, catalog number: EEL144) and the Serotonin / 5-HT (ST / 5-HT) ELISA Kit (Shanghai Sangon Biotech, catalog number: D751013). Brain tissue was also collected for Nissl staining, image preparation, and observation and analysis.

[0155] 4.1 Detection of dopamine in serum of animals in each experimental group Results of dopamine in biological samples as follows Figure 9 Serum dopamine levels were significantly elevated in the model group mice. The treatment group, LP496, and TS005 significantly reversed this abnormally elevated dopamine level, with the treatment group and LP496 showing better results. In the model group, serum dopamine levels were abnormally elevated, and LP496 significantly reversed this phenomenon, with a regulatory effect comparable to the positive control group, indicating that LP496 can alleviate tic symptoms by regulating the dopamine system.

[0156] 4.2 Detection of serotonin in the serum of animals in each experimental group serotonin results in biological samples as follows Figure 10 There were differences in serum serotonin levels among the mouse groups, with the LP496 group showing significantly higher levels than the model group (P < 0.05). This suggests that serotonin may indirectly affect the tic symptoms of tic disorder by inhibiting the dopamine system.

[0157] 4.3 Nissl staining of the striatum of animals in each experimental group After animal euthanasia, the brain tissue was quickly obtained on ice and bisected along the sagittal plane. One side was fixed in 4% paraformaldehyde fixative for 3 days, followed by routine paraffin embedding. Serial sections were prepared using a Leica Biocut pathological sectioning machine. The sections were dewaxed in xylene (twice, 5 min each time), hydrated in a gradient of ethanol (5 min in anhydrous ethanol, 2 min each in 95%, 80%, and 70% ethanol), rinsed with distilled water for 2 min, and then stained in Nissl tar purple staining solution (Solarbio, G1430) at 56°C for 1 h. After staining, the sections were rinsed with deionized water and then transferred to Nissl differentiation solution for differentiation for a few seconds to 2 min, monitored under a microscope in real time. Differentiation was terminated when the background of the sections was nearly colorless. Subsequently, the sections were rapidly dehydrated in anhydrous ethanol, cleared in xylene, mounted with neutral resin (Solarbio, G8590), and finally treated with 3DHISTECH P250 FLASH. A panoramic scanner completes panoramic imaging of slices for morphological observation and distribution characteristic analysis of Nissl bodies in neurons.

[0158] Neuronal states in the striatum region, such as Figure 11 In the control group, neurons had plump, round or polygonal cell bodies, were numerous, had clear nucleoli, and abundant, dark blue granular Nissl bodies in the cytoplasm, evenly distributed. In the model group, neurons had atrophied and shrunken cell bodies, deeply stained nuclei, and Nissl bodies aggregated into darkly stained clumps in the cytoplasm, with decreased neuronal density, indicating late-stage neuronal injury. In the treatment group, neurons had plump cell bodies, basically normal morphology, clear nucleoli, lighter cytoplasmic staining, and low Nissl body content. Neurons in the LP496 group were mostly plump and round, with clear nucleoli, abundant, dark blue granular Nissl bodies in the cytoplasm, evenly distributed, and of moderate density. This indicates that the LP496 strain has a protective effect against neuronal damage in mice with tic disorders. The striatal neurons in the LP496 group were plump and had abundant Nissl bodies, close to the normal morphology of the control group, significantly better than the neuronal atrophy and Nissl body aggregation observed in the model group.

[0159] The number of normal neurons per unit area in the striatum of a mouse brain is as follows: Figure 12The number of neurons in the model group was significantly lower than that in the blank group (P < 0.0001); the number of neurons in the treatment group increased significantly (P < 0.0001), but was still lower than that in the blank group; the number of neurons in the LP496 group was significantly higher than that in the model group (P < 0.001), suggesting that LP496 produced active substances and played a protective role for neurons; at the same time, there was no statistically significant difference between the treatment group and the LP496 group, indicating that LP496 was comparable to the drug in terms of protecting neurons.

[0160] 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 plantarum* ( Lactiplantibacillus plantarum LP496, characterized in that, The preservation number of the Lactobacillus plantarum LP496 is CGMCC No. 33499.

2. A microbial inoculant, characterized in that, The microbial agent includes the preparation of *Lactobacillus plantarum* LP496 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The preparations of *Lactobacillus plantarum* LP496 include: live bacteria, inactivated bacteria, lysates, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder, lysates, secondary metabolites, and exosomes.

4. The use of the *Lactobacillus plantarum* LP496 of claim 1 or the microbial agent of claim 2 in the preparation of a composition for the prevention or treatment of tic disorders.

5. The application according to claim 4, characterized in that, The composition has any one or more of the following functions: (1) Improved stereotyped behaviors, reduced the frequency of tic behaviors, and no adverse reactions in the motor system; (2) Improve motor coordination; (3) Repair muscle strength damage; (4) Improves dopamine levels and increases serum serotonin levels; (5) Reduce the number of damaged neurons.

6. The application according to claim 4, characterized in that, The aforementioned "tic disorder" has the following characteristics: (1) Increased stereotyped behaviors; (2) The number of tics increases; (3) Decreased motor coordination and muscle strength; (4) Increased dopamine levels; (5) Serum serotonin levels are decreased; (6) The number of damaged neurons increases.

7. The application according to claim 4, characterized in that, The bacterial content of *Lactobacillus plantarum* LP496 in the composition is 1×10⁻⁶. 8 CFU-1×10 12 CFU.

8. The application according to claim 4, characterized in that, The composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants.

9. The application according to claim 4, characterized in that, The composition may be in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule or aerosol.

10. A composition comprising *Lactobacillus plantarum* LP496 as described in claim 1 or the microbial agent as described in claim 2, characterized in that, The composition described herein has the function of preventing or treating tic disorders.

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