Lactobacillus plantarum for treating anxiety disorder and depressive disorder and application thereof

Abstract

The application provides a lactobacillus plantarum HLP0107 and application of the strain or a fermentation product thereof in preventing and / or improving depression and anxiety.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a strain of Lactobacillus plantarum, and the application of the strain or its fermentation products in the prevention and / or improvement of depression and anxiety. Background Technology

[0002] Anxiety disorders and depressive disorders are prevalent mental illnesses worldwide. They are characterized by prolonged course, high relapse rates, and significant impairment of patients' occupational functioning and quality of life, and are recognized as one of the leading causes of disability globally. According to estimates from the World Health Organization (WHO) and authoritative epidemiological studies, hundreds of millions of people worldwide suffer from these two types of illnesses, and the incidence rate continues to rise. [1] With the fast pace of modern life, the incidence of these diseases remains high and shows a trend towards affecting younger people. Current clinical guidelines typically recommend interventions based on psychotherapy and pharmacological treatment. Psychotherapy is an important approach for mild to moderate cases, while in moderate to severe depression or anxiety disorders, antidepressants, especially SSRIs (Selective Serotonin Reuptake Inhibitors) and SNRIs (Serotonin-Norepinephrine Reuptake Inhibitors), are often used as first-line or combination therapies. [2] However, while existing medications are effective in relieving acute symptoms, there are still unmet needs in clinical practice regarding long-term management and prognosis improvement. Firstly, drug-related adverse reactions are a major bottleneck limiting long-term adherence. Existing antidepressants, while working by regulating neurotransmitters, often have widespread off-target effects. Studies indicate that even one month after starting SSRIs, a significant proportion of patients still experience persistent problems such as sexual dysfunction, abnormal weight gain, gastrointestinal discomfort, and emotional blunting. [3] These adverse reactions not only directly reduce patients' quality of life, but are also a common reason for patients to discontinue medication or interrupt treatment on their own. Secondly, long-term medication can lead to changes in the sensitivity of nerve receptors; sudden or too rapid interruption of treatment can trigger physical and psychological symptoms such as dizziness, paresthesia, and rebound anxiety. [4] .

[0003] Given the limitations of existing drug treatments, exploring alternative or adjunctive strategies that are safer, suitable for long-term use, and can effectively improve anxiety and depression outcomes has become an urgent clinical need. Summary of the Invention

[0004] In recent years, with the in-depth elucidation of the mechanisms of the gut-brain axis...[5] This has opened up a completely new perspective for the treatment of mental illnesses. Meanwhile, numerous studies have demonstrated that the gut microbiota does not exist in isolation, but rather maintains real-time communication with the central nervous system through a complex bidirectional communication network. This network encompasses multiple parallel and coupled pathways, such as neural, immune, endocrine, and metabolic pathways. The gut microbiota can communicate through these complex bidirectional communication networks. [6] Utilizing the vagus nerve to directly transmit neural signals, it regulates immune inflammation by inducing cytokine release and deeply participates in the hypothalamic-pituitary-adrenal axis to reshape stress sensitivity. Simultaneously, it synthesizes key metabolites such as short-chain fatty acids and neurotransmitter precursors (e.g., GABA). Through the parallel and coupled involvement of neural, immune, endocrine, and metabolic pathways, it achieves systematic regulation of central nervous system function and emotional states such as anxiety and depression. Based on the elucidation of the gut-brain axis mechanism, the concept of psychobiotics was proposed. Unlike traditional probiotics that only focus on digestive health, psychobiotics refer to microecological interventions that can influence mood, stress response, or cognitive function through the gut-brain axis. Studies have shown that when adequate intake is achieved, by regulating the bidirectional communication network of the gut-microbe axis, it can bring mental health benefits to hosts suffering from depressive disorders, anxiety disorders, or under high stress. [7] .

[0005] Lactobacillus plantarum has become an important candidate strain for developing probiotics for mental health due to its acid and bile salt tolerance in the human gastrointestinal environment, its potential for residence and colonization through interaction with the intestinal mucosa / mucus layer, and its metabolic diversity in carbon source utilization, amino acid conversion and synthesis of various small molecules.

[0006] Through extensive screening and activity studies, the inventors of this application isolated a strain of *Lactobacillus plantarum* HLP0107 from healthy volunteers. Behavioral testing in a mouse model demonstrated that *Lactobacillus plantarum* HLP0107 exhibits significantly superior dual antidepressant and anti-anxiety activities, and its efficacy surpasses that of currently commercially available mainstream probiotics for mental health—*Lactobacillus plantarum* strains PS128 and UA1290. Furthermore, during the research process leading to this invention, the inventors observed no toxic side effects related to the administration of strain HLP0107. Therefore, strain HLP0107 shows broad application prospects in improving symptoms of depression and anxiety.

[0007] Therefore, in one respect, this application provides *Lactobacillus plantarum* (… Lactiplantibacillus plantarum HLP0107 was deposited on May 21, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number 34612.

[0008] On the other hand, this application provides progeny of *Lactobacillus plantarum* HLP0107 as described above.

[0009] In some embodiments, the progeny of *Lactobacillus plantarum* HLP0107 is a passaged culture of *Lactobacillus plantarum* HLP0107, for example, a passaged culture of up to 5, 10, 20, 30, or 40 generations.

[0010] In some embodiments, the progeny of *Lactobacillus plantarum* HLP0107 has at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or at least 99.99% sequence identity with the genome of *Lactobacillus plantarum* HLP0107.

[0011] In some embodiments, the progeny of *Lactobacillus plantarum* HLP0107 retain the anxiolytic and / or antidepressant activities of *Lactobacillus plantarum* HLP0107.

[0012] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0013] On the other hand, this application provides fermentation products of *Lactobacillus plantarum* HLP0107 or its progeny as described above.

[0014] As used herein, the term "fermentation product" refers to all substances synthesized and / or secreted by a specific bacterial strain through metabolic pathways during its growth and reproduction, when the strain is inoculated into a suitable culture medium and cultured under certain fermentation conditions (such as temperature, pH, dissolved oxygen, time, etc.). This includes primary metabolites, secondary metabolites, and other extracellular products. It also covers products obtained after processing fermentation broth or its components containing the above substances through conventional post-processing procedures such as centrifugation, filtration, concentration, extraction, and drying. In some cases, fermentation products may also contain bacterial cells or their cellular components.

[0015] On the other hand, this application provides the use of *Lactobacillus plantarum* HLP0107 or its progeny as described above, or the fermentation product of *Lactobacillus plantarum* HLP0107 or its progeny as described above, in the preparation of a product for the prevention and / or improvement of depression and / or anxiety in subjects.

[0016] In some implementations, the product is used in subjects for the prevention and / or treatment of depressive disorders and / or anxiety disorders.

[0017] In some implementations, the depressive disorder is selected from: disruptive mood disorders, major depressive disorder (also commonly referred to as "depression," the two terms are used interchangeably), persistent depressive disorder, premenstrual dysphoric disorder, perinatal depressive disorder, substance or drug-induced depressive disorder, depressive disorder caused by other physical illnesses, and any combination thereof.

[0018] In some implementations, the anxiety disorder is selected from: generalized anxiety disorder (also commonly referred to as "anxiety disorder," the two terms are used interchangeably), panic disorder, agoraphobia, social anxiety disorder (social phobia), separation anxiety disorder, selective mutism, anxiety disorder caused by other physical illnesses, anxiety disorder caused by substances or drugs, and any combination thereof.

[0019] According to the World Health Organization's International Classification of Diseases, Eleventh Revision (ICD-11) and the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), depressive disorders are a group of mood disorders characterized by a significant and persistent depressed mood. The main clinical manifestations include depressed mood (such as feeling sad, irritable, empty), loss of interest or pleasure (anhedonia), and decreased energy. They are often accompanied by poor concentration, low self-esteem or feelings of worthlessness, excessive guilt or self-blame, pessimism about the future, changes in sleep or appetite, psychomotor retardation or agitation, and recurrent thoughts of death or suicide. These symptoms usually persist for most of the day for at least two consecutive weeks and have a significant impact on an individual's daily social functioning and occupational abilities.

[0020] As used in this article, "anxiety disorder" is a group of common mental disorders characterized by excessive fear, anxiety, and related behavioral disturbances. It is systematically defined in authoritative international diagnostic criteria such as the International Classification of Diseases (11th edition) (ICD-11) and the Diagnostic and Statistical Manual of Mental Disorders (5th edition) (DSM-5). The basic characteristics of this type of disorder are: patients experience excessive fear and anxiety in response to perceived or anticipated threats that are disproportionate to the objective situation, accompanied by maladaptive behavioral changes such as avoidance behavior; fear usually refers to an emotional response to an immediate and pressing threat, while anxiety manifests as persistent worry and vigilance about anticipated future threats. Symptoms of anxiety disorders are persistent, usually lasting at least several months (most commonly more than six months), and are accompanied by autonomic dysfunction (such as palpitations, sweating, and difficulty breathing), muscle tension, difficulty concentrating, sleep disturbances, and other physical and mental symptoms; the severity of the symptoms is sufficient to cause significant clinical distress or impairment in other important areas of functioning, such as social or occupational health.

[0021] In some implementations, the product is a medicine, health product, or food.

[0022] In some implementations, the product is used for non-disease prevention and / or treatment purposes, such as for preventing and / or improving depressive or anxious moods, the severity, duration, and symptom clusters of which do not meet medical diagnostic thresholds (e.g., do not meet the criteria for depressive or anxiety disorders as described above), and the product is a health supplement or food.

[0023] In some embodiments, the product is used for disease prevention and / or treatment purposes (e.g., for the prevention and / or treatment of depressive disorders and / or anxiety disorders), and the product is a medicine.

[0024] In some implementations, the product is administered to the subject orally.

[0025] In some implementations, the product is a microbial agent.

[0026] In some embodiments, the microbial agent comprises *Lactobacillus plantarum* HLP0107 and / or its progeny in live cell form.

[0027] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 8 CFU / g (e.g., at least 5 × 10⁻⁶) 8 CFU / g, at least 1×10 9 CFU / g, at least 5×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×1011 CFU / g, at least 3×10 11 CFU / g, at least 1×10 12 The plant lactobacillus HLP0107 and / or its progeny (CFU / g).

[0028] In some embodiments, the microbial agent further comprises strains selected from Lactobacillus reuteri, Bifidobacterium, Lactobacillus casei, and any combination thereof.

[0029] In some embodiments, the product is administered in combination with additional active ingredients (e.g., pharmaceutically active agents) that have the effect of preventing and / or improving depression and / or anxiety, for example, separately or in combination, simultaneously or sequentially.

[0030] On the other hand, this application provides a method for preventing and / or improving depression and / or anxiety in a subject, comprising administering to the subject in need an effective amount of *Lactobacillus plantarum* HLP0107 or its progeny as described above, or a fermentation product of *Lactobacillus plantarum* HLP0107 or its progeny as described above.

[0031] In some implementations, the method is used to prevent and / or treat depressive disorders and / or anxiety disorders in subjects.

[0032] In some embodiments, the depressive disorder is selected from: disruptive mood disorders, major depressive disorder, persistent depressive disorder, premenstrual dysphoric disorder, perinatal depressive disorder, substance or drug-induced depressive disorder, depressive disorder caused by other physical illnesses, and any combination thereof.

[0033] In some implementations, the anxiety disorder is selected from: generalized anxiety disorder, panic disorder, agoraphobia, social anxiety disorder (social phobia), separation anxiety disorder, selective mutism, anxiety disorder caused by other physical illnesses, anxiety disorder caused by substances or drugs, and any combination thereof.

[0034] In some implementations, the method is used for non-disease prevention and / or treatment purposes, such as for preventing and / or improving depressive or anxious moods, the severity, duration, and symptom clusters of which do not meet medical diagnostic thresholds (e.g., do not meet the criteria for depressive or anxiety disorders as described above).

[0035] In some implementations, the method is used for disease prevention and / or treatment purposes (e.g., for the prevention and / or treatment of depressive disorders and / or anxiety disorders).

[0036] In some embodiments, the *Lactobacillus plantarum* HLP0107 or its progeny, and the fermentation product are administered to the subject orally.

[0037] In some embodiments, the *Lactobacillus plantarum* HLP0107 or its progeny is administered to the subject in the form of a bacterial agent.

[0038] In some embodiments, the microbial agent comprises *Lactobacillus plantarum* HLP0107 and / or its progeny in live cell form.

[0039] In some embodiments, the microbial agent contains at least 1 × 10⁻⁶ 8 CFU / g (e.g., at least 5 × 10⁻⁶) 8 CFU / g, at least 1×10 9 CFU / g, at least 5×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g, at least 3×10 11 CFU / g, at least 1×10 12 The plant lactobacillus HLP0107 and / or its progeny (CFU / g).

[0040] In some embodiments, the microbial agent further comprises strains selected from Lactobacillus reuteri, Bifidobacterium, Lactobacillus casei, and any combination thereof.

[0041] In some embodiments, the *Lactobacillus plantarum* HLP0107 or its progeny, the fermentation product, and other active ingredients (e.g., pharmaceutically active agents) that have the effect of preventing and / or improving depression and / or anxiety are administered in combination, for example, separately or in combination, simultaneously or sequentially.

[0042] On the other hand, this application provides a microbial agent comprising live cell form of *Lactobacillus plantarum* HLP0107 and / or its progeny as described above.

[0043] In some embodiments, the microbial agent comprises at least 1 × 10⁻⁶. 8 CFU / g (e.g., at least 5 × 10⁻⁶) 8 CFU / g, at least 1×10 9 CFU / g, at least 5×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g, at least 3×10 11 CFU / g, at least 1×10 12 The plant lactobacillus HLP0107 and / or its progeny (CFU / g).

[0044] In some embodiments, the microbial agent further comprises strains selected from Lactobacillus reuteri, Bifidobacterium, Lactobacillus casei, and any combination thereof.

[0045] In another aspect, this application provides a pharmaceutical composition comprising *Lactobacillus plantarum* HLP0107 or its progeny as described above, or a fermentation product of *Lactobacillus plantarum* HLP0107 or its progeny as described above.

[0046] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0047] In some embodiments, the pharmaceutical composition further comprises additional active ingredients (e.g., pharmaceutically active agents) that prevent and / or improve depression and / or anxiety.

[0048] On the other hand, this application provides the use of Lactobacillus plantarum HLP0107 or its progeny as described above in the construction of engineered strains for the prevention and / or improvement of depression and / or anxiety.

[0049] In some embodiments, the engineered strain is used to prevent and / or treat depressive disorders and / or anxiety disorders in subjects.

[0050] Beneficial effects of the invention

[0051] The *Lactobacillus plantarum* strain HLP0107 provided in this application has demonstrated effects in improving depression and anxiety across multiple behavioral dimensions in a mouse model. Therefore, strain HLP0107 possesses unique technical value in the prevention and improvement of anxiety and depression, and shows significant potential for clinical application.

[0052] Furthermore, through parallel comparative studies, the inventors of this application found that, compared with the currently commercially available popular probiotic strains of the same genus *Lactobacillus plantarum*, PS128 and UA1290, the strain HLP0107 of this application showed significant superiority in improving multiple symptoms related to anxiety and depression.

[0053] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0054] Instructions on the Preservation of Biological Materials

[0055] This invention relates to the following biological materials that have been deposited at the China General Microbiological Culture Collection Center (CGMCC): Lactobacillus plantarum ( Lactiplantibacillus plantarum HLP0107, which has accession number 34612 and accession date of May 21, 2025. Attached Figure Description

[0056] Figure 1 : Statistical graphs of behavioral test results for each group of mice (n=11). Among them, (a) is a statistical graph of immobility time for each group of mice in the forced swimming test (FST); (b) is a statistical graph of sugar water preference rate for each group of mice in the sugar water preference test (SPT); (c) is a statistical graph of immobility time for each group of mice in the tail suspension test (TST).

[0057] Figure 2 : Statistical graph of open field test results for each group of mice (n=11). Among them, (a) is the total distance moved by each group of mice in the open field test, which is used to assess the mice's voluntary movement ability; (b) is the percentage of time each group of mice spent in the center area in the open field test, which is used to assess the mice's anxiety level.

[0058] Figure 3 : Statistical graph of the results of the elevated cross maze test for each group of mice (n=11). Among them, (a) is the number of open arm entries; (b) is the percentage of time the mouse spent on the open arm. Detailed Implementation

[0059] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the invention.

[0060] Example 1: Screening of *Lactobacillus plantarum* (also referred to herein as *Lactobacillus plantarum*) in this application

[0061] 1. Fecal sample processing

[0062] Healthy human feces were used. The anaerobic chamber was filled with an anaerobic gas mixture (85% N2 / 10% CO2 / 5% H2) the day before the experiment. Fecal samples stored at -80°C were removed and transferred to the pre-prepared anaerobic chamber as soon as possible. After two hours of anaerobic treatment, the fecal samples were dissolved and suspended in pre-anaerobic YCFA medium. To prevent clogging of the microfluidic chip, the bacterial suspension was filtered through a 40 μm sterile membrane to remove large food residues and particles.

[0063] 2. Single-cell encapsulation

[0064] Processed fecal samples from healthy volunteers were diluted with PBS, and the diluted bacterial suspension was used as the dispersion phase, while light mineral oil served as the continuous phase. After evaluating and optimizing parameters such as flow rate and cell loading density, the system achieved stable and uniform droplet formation and single-cell encapsulation. Droplets were collected in Eppendorf tubes filled with mineral oil to prevent droplet breakage. Microfluidic operations were performed in an anaerobic chamber.

[0065] The concentration of the droplets in the collection tube was measured using a microscope and adjusted to approximately 6.0 × 10⁻⁶ per microliter using mineral oil. 4 Droplets. Since approximately 1% of the droplets were single-cell droplets, and the remainder were empty droplets, 10 µL of droplet / oil solution was removed from the Eppendorf tube and plated onto an agar plate, then incubated in an anaerobic incubator for 72 hours. Similarly, 10 µL of 6.0 × 10⁻⁶ droplets / oil solution was... 5 Cell solutions of live cells / mL were spread in parallel on agar plates and incubated in an anaerobic chamber for 72 hours. YCFA plates were used, with three replicates for each condition.

[0066] 3. Identifying probiotics with potential functions

[0067] 3.1 Preservation of microbial strains

[0068] Remove the agar plate from the 37°C incubator, pick a single colony from the agar plate, transfer it to 4 mL of YCFA liquid medium, and incubate at 37°C for 24 hours to enrich the colony. Label the colony. Then remove the cryovial, add 300 μL of 70% glycerol and 700 μL of the enriched bacterial solution, label the colony, and store it at -80°C for subsequent experiments. The remaining enriched bacterial solution will be used for 16S rRNA sequencing to determine the bacterial strain.

[0069] 3.2 16S rRNA gene sequencing

[0070] The 16S rRNA sequencing of the isolated bacteria was performed by Suzhou Genewiz Biotechnology Co., Ltd. The preserved bacterial cultures were sequenced for 16S rRNA to identify the bacterial strains. Genomic DNA was extracted from each single-cell colony using the TIANamp Bacteria DNA Kit, and then polymerase chain reaction was performed using universal primers 27F (5′-AGAGTTGATCCTGGCTCAG-3′, SEQ ID NO: 1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO: 2) to amplify the full-length 16S rRNA gene fragment.

[0071] PCR reactions were performed in a 50 μL system containing 2 μL upstream primer, 2 μL downstream primer, 25 μL 2x Phanta Max Buffer, 17 μL ddH2O, 1 μL Phanta Max Super-Fidelity DNA polymerase, 1 μL dNTP Mix, and 2 μL DNA template. PCR amplification was performed as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C (30 s), 57.3°C (1 min), and 72°C (30 s), with a final extension at 72°C for 5 min. The target PCR product was confirmed by agarose gel electrophoresis, purified using the QIAquick Gel Extraction Kit, and sequenced using Sanger sequencing to obtain the gene sequence. Finally, the 16S rRNA sequence of the strain was compared with sequences in the GenBank database using the NCBI database, and species identification was performed using BLAST. The strain with the highest sequence similarity was selected for identification. When the gene sequence of the tested strain has more than 99% homology with the gene sequence in the database, it can be considered to be the same species.

[0072] 3.3 A highly active strain of Lactobacillus plantarum HLP0107 was screened using the above methods. It is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 34612.

[0073] Example 2: Lactobacillus plantarum of this application for the prevention and treatment of chronic unpredictable mild stress (CUMS)

[0074] Objective: To observe the effects of the probiotic HLP0107 of this invention on depressive-like behavior, activity level, and anxiety-like condition in mice with chronic unpredictable mild stress-induced depression, and to compare it with existing probiotics such as Lactobacillus plantarum PS128 and UA1290 under the same conditions.

[0075] Experimental animals and grouping: Healthy SPF-grade C57BL / 6J mice (7 weeks old, male) were purchased from Zhishan Medical Research Institute, catalog number: ZS-C57BL / 6J-2m; they were randomly divided into the following groups, and all mice were acclimatized for 7 days in a standard environment (temperature 22±2℃, humidity 50±5%, 12h light-dark cycle) before the experiment began: Normal control group (WT group): No modeling was performed, and an equal volume of sterile PBS was given; Model control group (CUMS group): Only depression model was established, and the same volume of sterile PBS was administered; Control group (PS128 group): Lactobacillus plantarum PS128 was administered simultaneously with modeling (dose was 1×10⁻⁶). 9 CFU / dose / day, dissolved in sterile PBS); Control group (UA1290 group): Lactobacillus plantarum UA1290 was administered simultaneously with modeling (dose was 1×10⁻⁶). 9 CFU / dose / day, dissolved in sterile PBS); Treatment group (HLP0107 group): Lactobacillus plantarum HLP0107 was administered simultaneously with model establishment (dose was 1×10⁻⁶). 9 CFU / dose / day, dissolved in sterile PBS).

[0076] The strains PS128 and UA1290 used in this embodiment are both commercially available Lactobacillus plantarum probiotics. PS128 was purchased from Shanghai Lianya Probiotics Technology Co., Ltd., catalog number: 51326012701; UA1290 was purchased from Sichuan Fuyuan Energy Biotechnology Co., Ltd., catalog number: 669ELO11.

[0077] Lactobacillus plantarum PS128 is a recognized "psychological probiotic" strain with outstanding probiotic effects. Existing research has shown that in germ-free mouse models, PS128 enhances open-field activity and exhibits less anxiety-like behavior in the elevated cruciate maze; furthermore, in the brain, it increases striatal 5-HT and DA levels. [8] In early stress (ELS) and normal adult mice, PS128 exhibited situation-dependent behavioral effects: it reduced anxiety-like behaviors (elevated cross maze) in normal adult mice and significantly reduced depression-like behaviors in ELS mice. Administration of PS128 to neurotransmitters increased dopamine levels in the prefrontal cortex (PFC) of both groups of mice; serotonin levels increased in the PFC of normal mice, while serotonin metabolic turnover returned to normal in ELS mice. [9] Furthermore, PS128 has demonstrated its potential in treating depressive and anxiety disorders in other animal models. Therefore, PS128 can serve as a benchmark control strain for evaluating the efficacy of Lactobacillus plantarum in treating depressive and anxiety disorders.

[0078] Depression Model Construction and Drug Administration: Chronic unpredictable mild stress (CUMS) model: The modeling groups (CUMS group, PS128 group, UA1290 group, HLP0107 group) were randomly assigned mild stressors (1-2 stressors were randomly selected daily to avoid predictability), and the modeling and drug administration were carried out continuously for 4 weeks at a dose of 1×10 9CFU / time / day; normal control mice were not subjected to any stress, but were given PBS simultaneously.

[0079] Administration method: All treatment groups were administered the drug once daily via gavage, with a volume of 0.2 mL per animal, containing a bacterial agent concentration of 5 × 10⁻⁶. 9 CFU / mL, the normal control group and the model control group were given the same volume of sterile PBS, and the process was carried out simultaneously with the modeling process for 4 weeks.

[0080] Behavioral testing and evaluation: After modeling and drug administration, behavioral tests were performed sequentially at 24-hour intervals to evaluate the depressive-like behavior of mice and the treatment effect.

[0081] (1) The following statistics were collected regarding the stressors: A) Inclined cage: Purpose: To disrupt the animal's balance and comfort, causing mild but persistent discomfort.

[0082] Operating procedures: Elevate one side of a standard feeding cage so that the bottom of the cage is at approximately a 45° angle to the horizontal. Keep the cage door secure to prevent the animal from falling out. Tilt the feed rack and water bottle along with the cage to ensure the animal can still reach them, but this will slightly increase the difficulty of feeding. Continue this for 12 hours.

[0083] B) Damp bedding: Objective: To induce stress through uncomfortable, damp environments.

[0084] Operating steps: Add warm water to 2 / 3 of the cage volume, wetting it until it is noticeably damp but without excessive standing water. Spread the wet bedding evenly and leave it for 12 hours.

[0085] C) Swimming in cold water (4℃)

[0086] Objective: To induce transient cold stimulation and exercise stress.

[0087] Procedure: Prepare a cylindrical or rectangular container (deep enough so the animal cannot touch the bottom to prevent escape). Mix ice and water, adjusting the water temperature to 4°C. Gently place the animal in the water, forcing it to swim and float. After the designated time, quickly dry it with a towel and return it to a dry, warm cage for 5 minutes.

[0088] D) Tail-clamping stress

[0089] Purpose: Transient pain stress.

[0090] Operating steps: Use a tail clip to hold the tail tip about 1 cm away for 5 minutes.

[0091] E) Day and night reversed

[0092] Objective: To disrupt biological rhythms

[0093] Operating procedures: Keep the animal room lights off from 8:00 to 20:00 to create a dark environment; keep the animal room lights on from 20:00 to 8:00 the next day to create a daytime environment.

[0094] F) Food deprivation

[0095] Objective: Starvation stress

[0096] Operating procedures: At 8:00 AM, remove all feed from the cage, leaving only water. Resume feeding on schedule 24 hours later.

[0097] G) Deprivation of drinking water

[0098] Objective: Thirst stress

[0099] Operating procedures: At 8:00 AM, remove all water from the cages, leaving only feed. Restore water supply 24 hours later.

[0100] H) Vibration stress

[0101] Objective: To induce restlessness and mild physical stress through continuous, slight vibrations.

[0102] Operating procedures: Place the animal, cage and all, on the vibrating plate. Set to low-frequency, gentle vibration (80 rpm) and provide continuous stimulation for 20 minutes.

[0103] I) Empty cage exposed (no bedding, no sheltered environment)

[0104] Purpose: To deprive individuals of potential comfort and security, which constitutes mild environmental stress.

[0105] Procedure: Transfer the animal to a clean, empty cage without bedding or nesting material. Leave the water bottle in place for 12 hours.

[0106] J) Thermal high-temperature stress

[0107] Objective: To induce physical stress through appropriate high-temperature stimulation.

[0108] Procedure: Transfer the animal to an environment of 42°C for 20 minutes.

[0109] During implementation, the aforementioned stressors should be administered at most once a week, without repetition.

[0110] (2) Behavioral testing scheme

[0111] Tail Suspension Test (TST): Despair-like behavioral assessment was conducted using a mouse tail suspension device. The experimental setup consisted of a suspension bracket, fixing tape / clamps, and a video acquisition and data analysis system, and was used to detect immobile behavior in mice in situations where escape was impossible.

[0112] A) Adaptation and placement: Secure the mouse to the suspension beam with tape about 1–2 cm from the tail end, with a suspension height of about 30–50 cm, ensuring that its forelimbs are off the ground and do not touch surrounding objects.

[0113] B) Formal test: A single mouse was suspended for 6 minutes, and its immobile behavior (slight swaying with gravity and no active struggle) was recorded.

[0114] Record the following indicators: time spent immobile within four minutes of being suspended, percentage of immobile time, and incubation period before immobility begins; record the frequency of struggling if necessary.

[0115] Forced swimming test (FST): Despair-like behaviors were assessed using a transparent cylindrical water tank. The experimental setup consisted of a water tank (approximately 10–20 cm in diameter, 15–20 cm in depth, and approximately 23–25 °C in temperature) and a video acquisition and data analysis system.

[0116] A) Pretreatment: Conduct a pre-test as needed (e.g., place the tank in the tank for 10–15 min the day before) to reduce the effects of novelty stress.

[0117] B) Formal test: Each mouse was placed in a water tank for 6 minutes, and its behavior in the last 4 minutes was recorded.

[0118] Record metrics: cumulative immobility time, immobility initiation latency, swimming / climbing (struggling) time; optional total number of behavior transitions.

[0119] Sugar Water Preference Test (SPT): The two-bottle selection paradigm was used to assess anhedonia-like behavior. The experimental setup consisted of a single-cage dual-drinking system, an electronic balance, and a data recording system.

[0120] A) Adaptation phase: Provide a 1% (mass fraction) sucrose solution for 24 hours to allow the body to adapt to the sweetness, followed by two bottles (sucrose solution and water) for 24 hours, with the left and right positions switched every 12 hours to eliminate lateral bias. If necessary, withhold water for 12 hours before the formal test.

[0121] B) Formal test: Provide sucrose solution and water simultaneously for 24 hours, weigh the liquid mass / volume before and after, and switch the liquids once.

[0122] Record indicators: sucrose intake, water intake, total intake; Sugar water preference rate = sucrose intake ÷ (sucrose + water intake) × 100%.

[0123] Open Field Test (OFT): A square open-field box was used to assess spontaneous activity and anxiety-related behaviors. The experimental setup consisted of a bounded square box (e.g., 50 cm × 50 cm × 40 cm, with the bottom divided into a central area and a peripheral area) and a video acquisition and trajectory analysis system.

[0124] A) Adaptation and cleanup: Each mouse was placed in a standby cage for 2–5 minutes before testing, and the cage was wiped with 75% ethanol between individuals.

[0125] B) Formal test: Gently place the mouse in a fixed corner / center of the box and test continuously for 5–10 minutes.

[0126] Record indicators: total walking distance, average speed, time spent in the central area and number of times the hair is entered, number of times the hair is raised and groomed, etc.

[0127] Elevated Cross Maze Test (EPM): An elevated cross maze was used to assess anxiety-related behaviors. The experimental setup consisted of two open arms and two closed arms (perpendicular to each other, with an arm length of about 30–50 cm; the open arms were unenclosed, while the closed arms were enclosed on three sides), an elevated platform (about 50 cm above the ground), and a video acquisition and data analysis system.

[0128] A) Adaptation and Placement: Maintain constant lighting in the laboratory (e.g., ~100–200 lx), place the mice in the central area with their heads facing any of the closed arms.

[0129] B) Formal test: Observe continuously for 5 minutes.

[0130] Recording metrics: number of times the open arm is entered, dwell time in the open arm, number of times and dwell time in the closed arm, and dwell time in the central area; risk assessment behaviors (such as probing) can also be recorded.

[0131] Experimental results: Effects on depressive-like behavior and anhedonia in mice with depressive disorders: Forced swimming, tail suspension, and sucrose preference tests verified that the engineered bacterium HLP0107 of this invention exhibited significant antidepressant and anhedonia-improving abilities. The HLP0107 treatment group not only showed significant reversals in all behavioral indicators compared to the model group (reduced immobility time, increased sucrose preference), but also demonstrated significantly better improvement than the PS128 and UA1290 treatment groups. Figure 1 This result fully demonstrates the significant efficacy and potential advantages of the engineered probiotic HLP0107 in treating depressive disorders.

[0132] To eliminate the interference of altered motor abilities in mice on the results of forced swimming and tail suspension tests, and to further assess anxiety in mice, an open field test (OFT) was performed. As shown in Figure 2a, there was no significant difference (ns) in the total distance traveled in the open field between the WT group and the CUMS group, and no significant differences were found between the drug administration groups (PS128, UA1290, and HLP0107) and the model group. This result indicates that the CUMS modeling treatment and the bacterial strain gavage intervention did not impair or alter the basic motor abilities of mice. It also confirms that the reduced immobility time in the aforementioned behavioral tests (FST and TST) was due to an improvement in depressive-like states, rather than motor excitement or hyperactivity. As shown in Figure 2b, the percentage of time spent in the central region by mice in the CUMS model group was significantly lower than that in the WT group, exhibiting significant anxiety-like behavior. After the intervention of the strain, the time spent in the central region of mice in the HLP0107 group was significantly increased compared with that in the CUMS group (P < 0.001), and this improvement was statistically significantly better than that of the positive control strains UA1290 and PS128 (P < 0.05).

[0133] Finally, an elevated cross maze was used to assess anxiety levels in mice by utilizing the conflict between their fear of open, elevated environments and their exploratory instincts. Figure 3 As shown, compared with the WT group, the number of times mice entered the open arm (a) and the percentage of time spent in the open arm (b) were significantly reduced in the CUMS group (P < 0.001), confirming that the model induced anxiety-like behavior. After HLP0107 intervention, the mice's exploration behavior in the open arm was significantly restored. In terms of the number of entries, the HLP0107 group was significantly higher than the CUMS group (P < 0.001), restoring it to a level similar to the WT group. In addition, the percentage of time spent in the open arm in the HLP0107 group was also significantly higher than that in the CUMS group (P < 0.0001).

[0134] As mentioned above, PS128 has been reported in numerous existing technical documents to improve depression and anxiety in mouse models. However, as confirmed by the experimental data of this application, the *Lactobacillus plantarum* HLP0107 screened in this application significantly outperforms PS128 in multiple behavioral dimensions. Furthermore, compared to another commercially available *Lactobacillus* strain, UA1290 (also known as CCFM1290), the strain HLP0107 of this application also shows significantly superior antidepressant and anti-anxiety effects. This functional advantage of the strain HLP0107 cannot be expected from a simple strain replacement, demonstrating the unique technical value of the strain of this invention.

[0135] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

[0136] References

[0137] [1] World Health Organization. Depressive disorder (depression)[EB / OL]. 2025-08-29.

[0138] [2] Al-Harbi KS. Treatment-resistant depression: therapeutic trends, challenges, and future directions[J]. Patient Prefer Adherence, 2012.

[0139] [3] Hirschfeld RMA. Long-term side effects of SSRIs: sexualdysfunction and weight gain. J Clin Psychiatry. 2003;64 Suppl 18:20–24.

[0140] [4] Young Sook, Roh, Hyun Soo, Chung, Boeun, Kwon, Giyon, Kim. Association between depression, patient scar assessment and burn-specific health inhospitalized burn patients. [J]. Burns: journal of the International Society for Burn Injuries, 2012, 38(4): 506-12.

[0141] [5] Cryan JF, et al. The Microbiota-Gut-Brain Axis. PhysiologicalReviews. 2019.

[0142] [6] Foster JA, Cryan JF. Stress & the gut-brain axis: Regulation bythe microbiome. Neurobiology of Stress. 2017;7:124-136.

[0143] [7] Messaoudi M, Lalonde R, Violle N, et al. Assessment ofpsychotropic-like properties of a probiotic formulation (Lactobacillushelveticus R0052 and Bifidobacterium longum R0175) in rats and humansubjects. British Journal of Nutrition. 2011;105(5):755–764.

[0144] [8] Liu WH, Chuang HL, Huang YT, et al. Alteration of behavior andmonoamine levels attributable to Lactobacillus plantarum PS128 in germ-freemice. Behavioural Brain Research. 2016;298:202–209. DOI: 10.1016 / j.bbr.2015.10.046.

[0145] [9] Liu YW, Liu WH, Wu CC, et al. Psychotropic effects ofLactobacillus plantarum PS128 in early life-stressed and naïve adult mice.Brain Research. 2016;1631:1–12. DOI: 10.1016 / j.brainres.2015.11.018。

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum HLP0107 was deposited on May 21, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number 34612.

2. The progeny of Lactobacillus plantarum HLP0107 of claim 1.

3. Fermentation products of Lactobacillus plantarum HLP0107 of claim 1 or progeny of claim 2.

4. The use of the Lactobacillus plantarum HLP0107 of claim 1, the progeny of claim 2, or the fermentation product of claim 3 in the preparation of a product for the prevention and / or improvement of depression and / or anxiety in subjects; Preferably, the product is used in subjects for the prevention and / or treatment of depressive disorders and / or anxiety disorders; Preferably, the depressive disorder is selected from: disruptive mood disorders, major depressive disorder, persistent depressive disorder, premenstrual dysphoric disorder, perinatal depressive disorder, substance or drug-induced depressive disorder, depressive disorder caused by other physical diseases, and any combination thereof. Preferably, the anxiety disorder is selected from: generalized anxiety disorder, panic disorder, agoraphobia, social anxiety disorder (social phobia), separation anxiety disorder, selective mutism, anxiety disorder caused by other physical illnesses, anxiety disorder caused by substances or drugs, and any combination thereof; Preferably, the product is a drug, health product, or food.

5. The use of claim 4, wherein, The product is administered to subjects orally.

6. The use according to claim 4 or 5, wherein, The product is a microbial agent; Preferably, the microbial agent comprises *Lactobacillus plantarum* HLP0107 in live cell form and / or its progeny; Preferably, the microbial agent contains at least 1×10 8 CFU / g (e.g., at least 5 × 10⁻⁶) 8 CFU / g, at least 1×10 9 CFU / g, at least 5×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g, at least 3×10 11 CFU / g, at least 1×10 12 The plant lactobacillus HLP0107 and / or its progeny (CFU / g); Preferably, the microbial agent further comprises strains selected from: Lactobacillus reuteri, Bifidobacterium, Lactobacillus casei, and any combination thereof.

7. The use according to any one of claims 4-6, wherein, The product is administered in combination with other active ingredients (e.g., pharmaceutically active agents) that have the effect of preventing and / or improving depression and / or anxiety.

8. A microbial agent comprising, in live cell form, *Lactobacillus plantarum* HLP0107 of claim 1 and / or progeny of claim 2; Preferably, the microbial agent contains at least 1×10 8 CFU / g (e.g., at least 5 × 10⁻⁶) 8 CFU / g, at least 1×10 9 CFU / g, at least 5×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g, at least 3×10 11 CFU / g, at least 1×10 12 The plant lactobacillus HLP0107 and / or its progeny (CFU / g); Preferably, the microbial agent further comprises strains selected from: Lactobacillus reuteri, Bifidobacterium, Lactobacillus casei, and any combination thereof.

9. A pharmaceutical composition comprising Lactobacillus plantarum HLP0107 of claim 1, the progeny of claim 2, or the fermentation product of claim 3; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional active ingredients (e.g., pharmaceutically active agents) that prevent and / or improve depression and / or anxiety.

10. Use of the Lactobacillus plantarum HLP0107 of claim 1 or the progeny of claim 2 in the construction of engineered strains for the prevention and / or improvement of depression and / or anxiety.

Images