Lactobacillus gasseri and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus gasseri and application thereof. The lactobacillus gasseri 198-01 is obtained through wide screening, the preservation number of the lactobacillus gasseri 198-01 is GDMCC No: 67302, DPP4 enzyme inhibition tests and anxiety disorder animal model tests find that the lactobacillus gasseri 198-01 can control blood sugar rise and stimulate endogenous 5-hydroxytryptamine level and GABA level, then the brain emotion and psychological state of a host is adjusted, and the brain function of the host is improved. The composition can prevent and relieve anxiety disorder and inhibit inflammation of nervous systems in the body and brain, and has multiple biological effects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular, this invention relates to a strain of Lactobacillus gasseri and its applications. Background Technology

[0002] Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus gasseri is a probiotic found in the human gut, mouth, and vagina, and studies have shown it to have various potential health benefits. For example, Lactobacillus gasseri can regulate gut microbiota, maintain intestinal balance, improve diarrhea, constipation, irritable bowel syndrome (IBS), reduce intestinal permeability, and prevent "leaky gut syndrome." Lactobacillus gasseri has also been reported for use in weight management and blood glucose control. CN202111199953.1 discloses that Lactobacillus gasseri has a good inhibitory effect on dipeptidyl peptidase-4 and α-glucosidase, but it does not disclose its inhibitory effect on anxiety-related behaviors.

[0003] Anxiety disorder is a common chronic mental illness characterized by high mortality and relapse rates, severely impacting work and quality of life and imposing a significant economic burden on individuals and society. Anxiety disorder is primarily characterized by excessive anxiety, manifesting as restlessness and insomnia. Widely used anti-anxiety medications, such as buspirone hydrochloride and fluoxetine hydrochloride, operate on the principle of specifically regulating the serotonin neurotransmitter system, thereby improving neurological function. While these medications play a crucial role in the treatment of anxiety disorder, their side effects are also significant, commonly including headaches, dizziness, nausea, diarrhea, sexual dysfunction, and abnormal blood pressure or weight. Furthermore, the high cost of treatment limits their application, typically limiting their use to patients with severe symptoms and making them unsuitable for those with mild symptoms or for the prevention of anxiety disorder in sensitive populations. Therefore, developing products with fewer side effects, lower costs, and suitability for the prevention of anxiety disorder in sensitive populations is of significant practical importance and clinical necessity. There are some reports on the effects of *Lactobacillus gasseri* in relieving anxiety and depression. CN202311569598.1 discloses a *Lactobacillus gasseri* that can improve sleep problems such as insomnia, night awakenings, and difficulty falling asleep caused by anxiety and stress, promote deep sleep, improve sleep quality, and regulate negative emotions. However, it does not analyze or study the mechanisms by which it regulates negative emotions and improves sleep. CN202510799977.2 discloses a *Lactobacillus gasseri* isolated from kimchi samples that inhibits α-amylase activity, produces GABA, and has antioxidant capabilities. CN202310840349.5 discloses a *Lactobacillus gasseri* that can produce high levels of GABA and alleviate depressive-like behaviors in a mouse model of depression. However, for anxiety disorders, GABA supplementation does not necessarily lead to relief. Exogenous GABA supplementation has low absorption and bioavailability, is easily decomposed in the body, and has difficulty crossing the blood-brain barrier to enter the central nervous system, thus having limited effect on central anxiety relief.

[0004] Dipeptidyl peptidase-4 (DPP4) is a glycoprotein distributed on the cell surface. One of its main functions is to hydrolyze and inactivate incretin-like substances, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). DPP4 specifically cleaves two amino acid residues in the GLP-1 molecule, rendering it biologically inactive and thus limiting GLP-1's physiological efficacy in blood glucose regulation. Besides GLP-1 and GIP, DPP4 can also degrade various other bioactive peptides, such as matrix-derived factor 1 (SDF-1), which have potential functions in regulating insulin secretion, glucose homeostasis, and cell signal transduction. Unlike α-glucosidase inhibitors, DPP4 inhibitors not only inhibit postprandial hyperglycemia but also fasting hyperglycemia, achieving stable blood glucose regulation throughout the day via the incretin pathway. They represent an important class of drugs in the treatment of type II diabetes, combining safety and efficacy.

[0005] In recent years, research on the "microbe-gut-brain" axis has provided new perspectives for the prevention and treatment of mental illnesses. Evidence suggests that gut microbiota can participate in the pathological processes of mental illnesses through immune responses, regulation of neuroendocrine and metabolic pathways. Some probiotics, as active beneficial microorganisms, actively interact with endogenous microbiota and intestinal cells, inhibiting the colonization of harmful bacteria and promoting the proliferation of beneficial bacteria, regulating the secretion and balance of various neurotransmitters, and increasing the host's endogenous serotonin and GABA levels, thereby regulating the host's brain mood and psychological state. Furthermore, probiotics can reduce systemic chronic inflammation and alleviate neuromorphic inflammation. However, the multifunctional *Lactobacillus gasseri*, which controls blood sugar, promotes the secretion of endogenous neurotransmitters, and inhibits neuroinflammation, still requires further investigation. Summary of the Invention

[0006] Through extensive screening and testing, this invention has discovered a strain of Lactobacillus gasseri that possesses multiple functions, including controlling blood sugar, promoting the secretion of endogenous neurotransmitters, and inhibiting neuroinflammation, thus completing this invention.

[0007] The technical solution of the present invention is as follows: In one aspect, the present invention discloses a strain of Lactobacillus gasseri 198-01 ( Lactobacillus gasseri Lactobacillus gasseri 198-01 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, with accession number GDMCC No: 67302 and deposit date November 14, 2025.

[0008] In one aspect, the present invention provides a probiotic preparation containing Lactobacillus gasseri 198-01 or its fermentation product.

[0009] In some embodiments, the fermentation product is a fermentation broth containing bacteria after fermentation of Lactobacillus gasseri 198-01.

[0010] In some embodiments, the fermentation product is the fermentation supernatant obtained after removing the bacterial cells from the fermentation of Lactobacillus gasseri 198-01. Those skilled in the art know that probiotics can produce a large number of metabolites during fermentation and secrete them into the fermentation broth; these metabolites are also an important component of the efficacy of probiotics.

[0011] In some embodiments, the probiotic preparation is a solid powder, which is prepared by freeze-drying the fermentation broth of Lactobacillus gasseri 198-01; optionally, the solid powder also contains one or more excipients, which can maintain the activity of Lactobacillus gasseri 198-01 and adjust the stability, dispersibility, taste, and solubility of the powder.

[0012] In some embodiments, the probiotic preparation is a liquid preparation containing Lactobacillus gasseri 198-01; optionally, the liquid preparation may further contain one or more excipients to maintain the activity of Lactobacillus gasseri 198-01 and to adjust the taste, stability, viscosity, and pH of the liquid preparation.

[0013] As used herein, the term "probiotic" is defined as any non-pathogenic microorganism that, when administered to a host in sufficient quantities in live form, can have a beneficial effect on the host's health.

[0014] In one aspect, the present invention also provides a composition comprising, in addition to Lactobacillus gasseri 198-01, one or more of the following substances: probiotics, prebiotics, dietary fiber, protein, polypeptides, amino acids, lipids, carbohydrates, vitamins, minerals, and plant extracts.

[0015] In some embodiments, the probiotics are selected from bacteria, fungi (e.g., yeast), or any combination thereof.

[0016] In some embodiments, the bacteria are selected from Bifidobacterium, Lactobacillus, Caseobacillus, Myxobacterium, Plantago, Lactobacillus, Lactobacillus spp., Streptococcus, Lactococcus, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Akkermansia, Clostridium, or any combination thereof.

[0017] In some embodiments, the prebiotic is selected from human milk oligosaccharides, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, resistant dextrin, maltodextrin, polydextrose, psyllium husk, spirulina, arthrophyllum, trachomatis polysaccharides, carrot nitrogenous polysaccharides, or any combination thereof.

[0018] In one aspect, the present invention provides the application of Lactobacillus gasseri 198-01 in the preparation of food, dietary supplements, and pharmaceuticals.

[0019] In this invention, the term "food" has a broad meaning, including food or beverages for humans or animals. The food is selected from solid beverages, confectionery, liquid beverages, biscuits, or dairy products. In animals, the food is provided in the form of feed.

[0020] In this invention, the dietary supplement refers to an edible product that can increase the nutritional value or health benefits for consumers, and is used to supplement the amino acids, vitamins, minerals and other nutrients required by the human body.

[0021] In this invention, the term "medicine" refers to a product intended for the prevention or treatment of disease. The medicine is applicable to both humans and animals, particularly animals closely related to human production and daily life, such as poultry, livestock, and pets.

[0022] In some embodiments, the dosage form of the dietary supplements and pharmaceuticals of the present invention is selected from tablets, capsules, gel candies, oral liquid preparations, pills, powders, and drops.

[0023] In some embodiments, the present invention includes foods, dietary supplements, and pharmaceuticals containing Lactobacillus gasseri 198-01, and also contains additives / excipients acceptable to the food, dietary supplement, and pharmaceutical.

[0024] In some embodiments, acceptable excipients in the pharmaceutical product include: fillers, binders, lubricants, flow aids, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

[0025] In some embodiments, the probiotic agent of the present invention contains more than 10 probiotics. 6 CFU / g or greater than 10 6 CFU / mL.

[0026] In some embodiments, the food, dietary supplement, and pharmaceutical containing Lactobacillus gasseri 198-01 of the present invention contain Lactobacillus gasseri 198-01 in quantities greater than 10. 6 The amount of CFU / dose present (e.g., 10) 7 CFU / dosage, 10 8CFU / dosage, 10 9 CFU / dosage, 10 10 CFU / dosage, 10 11 CFU / dosage, 10 12 CFU / dosage). The unit of dosage can be g or mL.

[0027] As used in this article, the term "CFU (Colony-Forming Units)" refers to the total number of microbial communities such as bacteria, fungi, and yeast in a product, and is usually used to calculate the number of viable cells.

[0028] In one aspect, the present invention discloses the use of Lactobacillus gasseri 198-01 in the preparation of compositions for the prevention and treatment of anxiety disorders.

[0029] In this invention, the prevention and treatment include the functions of preventing, treating and alleviating the symptoms.

[0030] In one aspect, the present invention discloses the use of Lactobacillus gasseri 198-01 in the preparation of compositions for controlling blood sugar.

[0031] In some embodiments, the blood glucose control refers to controlling abnormally high fasting and postprandial blood glucose levels.

[0032] In some embodiments, the blood glucose control composition is a pharmaceutical, health food, or dietary supplement; the pharmaceutical is used for the prevention and treatment of type II diabetes.

[0033] In some embodiments, Lactobacillus gasseri 198-01 of the present invention is used to simultaneously control hyperglycemia and alleviate anxiety.

[0034] Beneficial effects This invention discloses a strain of Lactobacillus gasseri with multiple biological functions, which can control blood sugar elevation, stimulate endogenous serotonin and GABA levels, thereby regulating the host's brain mood and psychological state, preventing and relieving anxiety, and inhibiting inflammation of the body and brain's nervous system, and has good application and development value. Attached Figure Description

[0035] Figure 1 : Statistical results of the total movement distance of zebrafish in each group in the open field experiment.

[0036] Figure 2 : Statistical results of the Thigmotaxis (movement distance) ratio of each group of zebrafish in the open field experiment.

[0037] Figure 3The figures show the movement trajectories and distances of mice in each group during the open field experiment. Figure A shows the movement trajectories of mice in each group during the open field experiment, and Figure B shows the statistical results of the total distances of mice in each group during the open field experiment.

[0038] Figure 4 : Statistical results of the distance traveled by mice in the central area of ​​the open field for each group.

[0039] Figure 5 : Statistical results of the time spent by mice in each group moving in the central area of ​​the open field.

[0040] Figure 6 The figures show the movement trajectories and distances of mice in each group during the elevated cross maze experiment. Figure A shows the movement trajectories of mice in each group in the open and closed arms; Figure B shows the statistical results of the total distances of mice in each group during the elevated cross maze experiment.

[0041] Figure 7 : Statistical results of the distance traveled by mice in the open arm of the elevated cross maze in each group.

[0042] Figure 8 : Statistical results of the movement time of mice in each group in the open arm of the elevated cross maze.

[0043] Figure 9 Neurotransmitter detection in the serum of mice in each group: A is a statistical graph showing the results of serum 5-hydroxytryptamine levels; B is a statistical graph showing the results of serum γ-aminobutyric acid levels.

[0044] Figure 10 Neurotransmitter detection in hippocampal tissue of mice in each group: A is a statistical graph of the results of 5-hydroxytryptamine content in hippocampal tissue; B is a statistical graph of the results of γ-aminobutyric acid content in hippocampal tissue.

[0045] Figure 11 The results of detecting pro-inflammatory factors in the serum of mice in each group are shown in Figure A, which is a statistical graph of the results of the serum pro-inflammatory factor TNF-α; Figure B is a statistical graph of the results of the serum pro-inflammatory factor IL-6; and Figure C is a statistical graph of the results of the serum pro-inflammatory factor IL-1β.

[0046] Figure 12 The detection of pro-inflammatory factors in the hippocampus of mice in each group: A is a statistical graph of the results of TNF-α level in hippocampus; B is a statistical graph of the results of IL-6 level in hippocampus; C is a statistical graph of the results of IL-1β level in hippocampus.

[0047] Figure 13 : Statistical graph showing the changes in average body weight of mice in each group during the test.

[0048] Figure 14: Statistical chart showing the changes in average daily food intake of mice in each group during the test.

[0049] Figure 15 : Statistical chart showing the changes in average daily water intake of mice in each group during the test. Detailed Implementation

[0050] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0051] Source of materials: Zebrafish: Wild-type AB strain zebrafish were purchased from the China Zebrafish Resource Center (Wuhan).

[0052] Lactobacillus rhamnosus HN001: Lactobacillus rhamnosus HN001 is a widely studied probiotic that has been shown to effectively alleviate anxiety and depression levels in patients. Therefore, this invention utilizes commercially available Lactobacillus rhamnosus HN001 as a positive strain.

[0053] Experimental mice: 8-week-old C57BL / 6J mice, weighing 20-25g, were purchased from the Experimental Animal Center of Southern Medical University and then housed in the SPF Animal Room of Southern Medical University at (22 ± 1)°C and 50-70% humidity, with a 12h / 12h light-dark cycle. The testing protocol of this invention has been reviewed and approved by the Animal Experiment Ethics Committee of Southern Medical University (D2024084).

[0054] Statistical analysis: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001. ns indicates no statistically significant difference (P value greater than 0.05).

[0055] Example 1: Isolation and Identification of Lactobacillus gasseri The strain of this invention was isolated from stool samples of normal full-term newborns born at West China Hospital of Obstetrics and Gynecology, Sichuan University. Specifically, fresh stool samples from infants aged 1-4 months after birth were collected using sterile stool collection tubes. Immediately after sampling, the samples were temporarily stored at 4°C and then transported to the laboratory by the sampling personnel for dilution and culture. If immediate operation was not possible, the samples were anaerobically stored at 4°C and cultured on the same day.

[0056] Weigh 0.5g of feces and add 4.5mL of fecal diluent (4.5g KH2PO4, 6.0g Na2HPO4, 0.5g L-cysteine ​​hydrochloride, 0.5g Tween-80, 1.0g agar mixed in 1000mL distilled water, autoclaved at 121℃ for 15min). Mix thoroughly by shaking and serially dilute 10-fold. Take 100μL of the appropriately diluted fecal mixture and spread it onto MRS agar plates (selective medium for lactic acid bacteria) using an L-shaped rod. Incubate anaerobically at 37℃ for 48h. Collect suspected lactic acid bacteria colonies from the plates and subculture them onto MRS agar plates (purified), incubating anaerobically at 37℃ for 48h. Then, pick colonies from the plates and inoculate them onto MRS plates and incubate aerobically at 37℃ for 48h, observing the growth of the bacteria under aerobic conditions. Simultaneously, perform Gram staining and observe bacterial morphology under a microscope. More than 50 strains of facultative anaerobic, Gram-positive bacteria, rod-shaped, without flagella or spores, morphologically suspected to be *Lactobacillus*, were obtained. Further DNA extraction, PCR detection, and NCBI sequence alignment were performed on these strains, initially identifying several strains as *Lactobacillus gasseri*. Subsequently, whole-genome sequencing and analysis of these strains were conducted using the PacBio Sequel2 platform, further confirming their *Lactobacillus gasseri* identity. Based on preliminary tests of growth status and strain viability, the two *Lactobacillus gasseri* strains with strong growth status and viability were named *Lactobacillus gasseri* 198-01 and *Lactobacillus gasseri* BH0010, respectively. *Lactobacillus gasseri* 198-01 was deposited at the Guangdong Provincial Microbial Culture Collection Center, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 67302, and deposit date November 14, 2025.

[0057] Example 2: DPP4 inhibition test of Lactobacillus gasseri (1) Preparation of culture medium for the strain: MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 15g / L, lactose 15g / L, yeast extract 5g / L, diammonium citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​0.5g / L.

[0058] (2) Activation of Lactobacillus gasseri and preparation of fermentation supernatant The bacteria were inoculated into MRS liquid medium and cultured at 37°C for 24 hours to activate them. The activated strain was then inoculated into 2% MRS broth and cultured at 37°C for 12 hours. The bacterial culture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant (fermentation supernatant) was collected. The components in the supernatant were the metabolic products of the strain. The pH of the supernatant was adjusted to 7.4.

[0059] (3) DPP4 enzyme activity inhibition rate test The pH-adjusted fermentation supernatant was screened using a DPP4 enzyme inhibitor screening kit (Elabscience, catalog number: E-BC-D007), following the instructions in the kit's manual. The positive control group used sitagliptin, a highly selective DPP4 inhibitor that specifically inhibits DPP4 enzyme activity. The DPP4 enzyme activity inhibition rate (%) was calculated using the following formula: DPP4 enzyme activity inhibition rate (%) = (Fluorescence intensity of control group - Fluorescence intensity of sample group) / (Fluorescence intensity of control group - Fluorescence intensity of blank group) * 100% The quality control group consisted of working solution containing only DPP4 enzyme, while the blank group consisted of detection buffer containing only DPP4 enzyme.

[0060] Results: The positive control drug (concentration 407.2 μg / mL) showed an inhibition rate of 98%-100% on DPP4 enzyme activity in several replicate tests. The average inhibition rate of DPP4 enzyme activity in *Lactobacillus gasseri* 198-01 reached 66.30%, while the inhibition rate of DPP4 enzyme activity in the *Lactobacillus gasseri* BH0010 group was very low. This demonstrates the application potential of *Lactobacillus gasseri* 198-01 as a DPP4 inhibitor. DPP4 inhibitors have become an important class of clinical hypoglycemic drugs. The mechanism of action of these drugs lies in specifically inhibiting the enzyme activity of DPP4, thereby reducing the degradation of endogenous GLP-1, prolonging its half-life, enhancing its promoting effect on insulin secretion and inhibiting glucagon secretion, and thus achieving stable and effective blood glucose control.

[0061] Example 3: Detection of the effect of Lactobacillus gasseri on anxiety-like behavior in zebrafish As a model organism, zebrafish are often used by researchers to induce an anxiety-like state in them using various external stimuli. This state can trigger a series of abnormal behaviors, such as tactile attraction (manifested as a preference for moving along the edges or corners of a container), motion arrest, or ataxia. These behavioral changes can serve as important indicators for assessing the level of anxiety in zebrafish.

[0062] 3.1 Construction and Testing of a Zebrafish Chronic Anxiety Model (1) Test grouping and intervention Zebrafish with a 5dpf growth rate were randomly divided into 5 groups, including a blank control group, an unpredictable stress group (model group), a Lactobacillus gasseri group (198-01 group), a Lactobacillus gasseri group (BH0010 group), and a Lactobacillus rhamnosus HN001 group (positive treatment group).

[0063] Throughout the test, the blank control group was fed normally without any intervention or modeling treatment. The model group was fed normally and, along with the other intervention groups, underwent subsequent unpredictable stress treatment. The 198-01 group, BH0010 group, and positive treatment group had *Lactobacillus gasseri* 198-01, BH0010, and *Lactobacillus rhamnosus* HN001 added to the system water, respectively, ensuring a bacterial concentration of 1×10⁻⁶ in each group's water. 7 CFU / 250mL. To maintain bacterial concentration, the water in the bacterial system was changed every 24 hours. The intervention lasted for 12 days until behavioral testing was conducted.

[0064] (2) Anxiety model: The zebrafish anxiety model is established using unpredictable stress. Five days after the above intervention, i.e. when the zebrafish were raised to 10 dpf, the model group, 198-01 group, BH0010 group and positive treatment group were given two of the five stressors every day for a total of 7 days.

[0065] The pressure sources included: ① Chasing: In a plastic bowl containing 250 mL of system water, rotate the fish clockwise along the bowl wall evenly for 5 minutes using a plastic dropper, maintaining a speed of 90 revolutions / min. ② Hypertonicity: Add 250 mL of 100 μM NaCl solution to a plastic bowl and immerse the fish in it for 40 minutes. ③ Low pH: Add 250 mL of pH=4.0 solution to a plastic bowl and immerse the fish in it for 3 minutes. ④ Turbulence: Place the fish in a plastic bowl containing 250 mL of circulating water, place an air stone in the bowl, aerate for 3 minutes, rest for 1 minute, repeat 3 times, and increase the aeration intensity of the air stone. ⑤ Flashing: Place the fish in a 90 mm culture dish containing 50 mL of circulating water, allow them to acclimatize to darkness for 3 minutes, and then flash for 15 minutes.

[0066] During the modeling period, paramecium feed was given twice a day, morning and evening, and the system water was changed every 24 hours.

[0067] (3) Detection of anxiety-like behavior in zebrafish The spontaneous movement of zebrafish must be completed between 11:00 and 17:00. Zebrafish were placed in six-well plates, one zebrafish per well, and 2 mL of system water was added to each well. The six-well plates were then placed in a Viewpoint Lab behavioral instrument. The instrument was set to a 6-minute light exposure and a 10-minute dark exposure. The test wells were divided into inner and outer concentric circles of equal area. The instrument automatically recorded the zebrafish's movement time per minute, movement distance, and movement time and distance within both inner and outer circles. Statistical analysis software was used to calculate the total movement distance of the zebrafish in each well and the movement distance within the two concentric circles. The Thigmotaxis (movement distance) was calculated using the formula: Thigmotaxis = (Outer circle movement distance / Total distance) × 100%. The Thigmotaxis (movement distance) ratio is an indicator for evaluating anxiety-like behavior in spontaneous movement of zebrafish. The larger the ratio, the greater the distance the zebrafish moves in the outer circle, suggesting that the zebrafish exhibits obvious anxiety-like behavior.

[0068] 3.2 Results See Figures 1 to 2 ,in, Figure 1 The results show the statistical results of the total movement distance of each group of zebrafish in the open field experiment. The data indicates no statistically significant difference in the total movement distance among the groups, suggesting that the various treatments did not significantly affect the zebrafish's locomotion ability. Figure 2 As can be seen, compared with the blank control group, the Thigmotaxis value of the model group was significantly increased (**P < 0.01), indicating that the zebrafish anxiety model induced by unpredictable stress was successfully constructed, and the zebrafish in the model group exhibited obvious anxiety-like behaviors. It is noteworthy that compared with the model group, the Thigmotaxis values ​​of the 198-01 group (*P < 0.05) and the positive treatment group (**P < 0.01) were significantly decreased, while there was no statistically significant difference in the *Lactobacillus gasseri* BH0010 group. The above experimental results indicate that among the *Lactobacillus gasseri* used in this invention, only *Lactobacillus gasseri* 198-01 has a significant alleviating effect on anxiety-like behaviors in zebrafish. Therefore, this invention successfully constructed a zebrafish anxiety model and used this model to detect the anxiety-relieving efficacy of *Lactobacillus gasseri* 198-01.

[0069] Example 4: X 4.1 Animal Grouping Buspirone hydrochloride tablets are a commonly used anti-anxiety and antidepressant drug in clinical practice. Its mechanism of action mainly involves the selective regulation of serotonin receptors to exert its anti-anxiety effect. Therefore, in this invention, the buspirone hydrochloride treatment group was designated as the positive control group.

[0070] After acclimatizing to the diet for one week, 24 mice were randomly divided into four groups: blank control group, chronic restraint stress anxiety group (model group), Lactobacillus gasseri group (198-01 group), and buspirone hydrochloride group (positive drug group). There were 6 mice in each group.

[0071] 4.2 Construction and intervention of mouse anxiety model Four groups of mice were administered the treatment via gavage for a total of 21 days. The blank control group and the model group received 1×PBS via gavage; the 198-01 group received bacterial suspension (1×10⁻⁶). 9 The control group received CFU / 0.1 mL via gavage; the positive control group received buspirone hydrochloride (5 mg / kg, dissolved in 1×PBS) via gavage, 0.1 mL each time.

[0072] Starting from day 12 after intervention, mice in the model group, 198-01 group, and positive treatment group were placed in 50mL centrifuge tubes with puncture holes for 2-4 hours for chronic restraint stimulation, for 10 consecutive days. The blank control group was gently stroked for 5 minutes to avoid stress on the test subjects. After modeling, behavioral (open field test and elevated cruciate maze test) and histological tests were performed on mice in each group.

[0073] 4.3 Detection of anxiety-like behavior in mice (1) Open field test: The open field test chamber was made of plastic (40cm×40cm), with a 20cm×20cm central square marked with color. A single mouse was placed in the center of the chamber. During the entire experiment, the total distance the mouse traveled in the open field within 10 minutes, the time spent in the central area, and the distance it moved were recorded to assess its anxiety state.

[0074] (2) Elevated Cross Maze Test: The maze device consisted of two opposing open arms (35cm×6cm) and two closed arms (35cm×6cm) extending from a central platform (6cm×6cm). The device was raised to 74cm above the floor. During the test, mice were placed on the central platform of the maze, facing one of the open arms, and their behavior was monitored for 5 minutes. The distance they traveled into the open arms and the time they spent in the open arms were monitored and analyzed to assess their anxiety level.

[0075] 4.4 Results The open field test is a method to assess an animal’s autonomous behavior, exploratory behavior, and stress in a new environment. The time a mouse spends in the central area and its movement trajectory can reflect the mouse’s autonomy and exploratory nature in the new environment. Figure 3 Figure A shows a schematic diagram of the movement trajectories of each group of mice in the open field experiment. Figure 3 B is the correct answer. Figure 3The quantitative statistical graph of the results in the middle A group showed that there was no significant statistical difference in the total movement distance of the mice in the open field, indicating that the treatment conditions did not affect the basic motor ability of the mice.

[0076] Figure 4 and Figure 5 This is a statistical graph showing the distance and time spent moving in the central area of ​​the open field by mice in each group. The results showed that the model group mice had significantly less distance (***P < 0.001) and time (****P < 0.0001) spent moving in the central area compared to the control group. These results indicate that chronic restraint successfully induced an anxiety model in mice, resulting in significant anxiety-like behavior. Importantly, the 198-01 group and the positive treatment group had significantly greater distance (**P < 0.01) and total time (***P < 0.001) spent moving in the central area than the model group, indicating that *Lactobacillus gasseri* 198-01 can significantly alleviate anxiety-like behavior in mice, with effects comparable to the positive control drug.

[0077] The elevated cross maze test is also an internationally recognized classic method for measuring anxiety responses. This test is based on animals' exploratory instincts towards novel environments and their conflicting behaviors with the suspended open arms, and is used to evaluate the anxiety state of rodents. Therefore, this invention also measured the total distance traveled by mice in the elevated cross maze, as well as the distance and time taken to enter the open arms. Figure 6 Figures A and B show the schematic diagrams and corresponding statistical graphs of the movement trajectories of mice in each group during the elevated cross maze experiment, respectively. The lack of significant differences in the total movement distance among the groups indicates that none of the treatments had a significant impact on the mice's basic motor abilities. Figure 7 and Figure 8 As shown, compared with the blank control group, the model group mice showed a significant decrease in both the total distance and total time of open arm movement (***P < 0.001), further confirming that chronic restraint stress can successfully induce anxiety in mice. Importantly, compared with the model group, the 198-01 group showed a significant increase in both the distance (*P < 0.05) and time (**P < 0.01) of open arm movement, indicating that Lactobacillus gasseri 198-01 has a significant alleviating effect on anxiety-like behavior in mice.

[0078] This embodiment demonstrates that the present invention successfully constructed a mouse anxiety model and verified in this model that Lactobacillus gasseri 198-01 has an anxiety-relieving effect and has no significant effect on the mouse's motor ability.

[0079] Example 5: Effects of Lactobacillus gasseri on neurotransmitter and inflammatory factor levels in mice 5.1 Mouse Sample Collection and Detection Blood was collected from each group of mice in Example 4 by enucleation. After centrifugation at 3000 rpm for 30 min at 4°C, the supernatant was collected for serum collection. The levels of the neurotransmitters 5-hydroxytryptamine and γ-aminobutyric acid, as well as the levels of inflammatory factors TNFα, IL-6, and IL-1β in the serum were detected using a mouse ELISA kit diluted according to the instructions (in pg / mL).

[0080] After anesthetizing mice, whole brain tissue was extracted, and the hippocampus was homogenized. Protein content of each sample was determined according to the BCA protein assay kit instructions. The levels of neurotransmitters serotonin and gamma-aminobutyric acid (GABA), as well as inflammatory factors TNFα, IL-6, and IL-1β, in the hippocampal tissue homogenate were measured using an ELISA kit diluted according to the instructions. The results were then normalized to the corresponding tissue protein content (unit: pg / mg).

[0081] 5.2 Results See Figure 9 In this study, the levels of the neurotransmitters serotonin (5-HT) and gamma-aminobutyric acid (GABA) in the serum of mice in each group were detected using an ELISA kit. The serum levels of 5-HT (*P<0.05) and GABA (***P<0.001) in the model group were significantly lower than those in the blank control group, indicating a significant reduction in neurotransmitter secretion in anxious mice. Compared with the model group, the serum levels of 5-HT (**P < 0.01) and GABA (***P < 0.001) in the positive treatment group were significantly increased. Treatment with *Lactobacillus gasseri* 198-01 also increased the serum levels of 5-HT and GABA in mice, with GABA showing a statistically significant difference (**P < 0.01).

[0082] The neurotransmitter serotonin (5-hydroxytryptamine) in the hippocampus of mice in each group Figure 10 (A) and γ-aminobutyric acid (GABA) Figure 10 The levels of serotonin (SHT) and gamma-aminobutyric acid (GABA) in the hippocampus of mice in the model group were detected. The results showed that the levels of SHT and GABA in the hippocampus of mice in the model group were significantly lower than those in the blank control group (**P<0.01), indicating a significant reduction in neurotransmitter secretion in the brain tissue of anxious mice. Conversely, the levels of SHT (**P < 0.01) and GABA (*P < 0.05) in the hippocampus of mice in the Lactobacillus gasseri 198-01 group were significantly higher than those in the model group.

[0083] The levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in mouse serum were detected by ELISA. Figure 11The AC analysis showed that the serum levels of pro-inflammatory factors TNF-α (**P < 0.0001), IL-6 (**P < 0.01), and IL-1β (*P < 0.05) in the model group mice were significantly higher than those in the blank control group, indicating a significant inflammatory response in the anxious mice. Compared with the model group, the serum levels of TNF-α (**P < 0.01), IL-6 (*P < 0.05), and IL-1β (*P < 0.05) in the Lactobacillus gasseri 198-01 group mice were significantly lower, indicating that Lactobacillus gasseri 198-01 can significantly reduce the level of inflammation in mice.

[0084] The levels of TNF-α, IL-6, and IL-1β in hippocampal tissue homogenates from mice in each group were detected by ELISA, and normalized using hippocampal tissue protein content. Figure 12 The AC results showed that, compared with the blank control group, the levels of pro-inflammatory factors TNF-α (**P < 0.01), IL-6 (*P < 0.05), and IL-1β (*P < 0.05) in the hippocampus of the model group mice were significantly increased, indicating that the anxious mice exhibited obvious neuroinflammatory phenomena. Furthermore, intervention with Lactobacillus gasseri 198-01 significantly reduced the levels of TNF-α (**P < 0.01), IL-6 (*P < 0.05), and IL-1β (**P < 0.01) in the hippocampus of mice, indicating that Lactobacillus gasseri 198-01 can effectively reduce the level of neuroinflammatory inflammation in anxious mice.

[0085] Research evidence suggests that anxiety disorders are closely associated with elevated levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the body, including serum and brain tissue. This indicates a chronic, low-grade systemic inflammatory state in patients with anxiety disorders. Pro-inflammatory factors can interfere with the metabolism and function of key neurotransmitters, impair neuroplasticity, and activate disordered secretion of the stress hormone cortisol, leading to the generation and persistence of anxiety symptoms. The *Lactobacillus gasseri* 198-01 of this invention can significantly inhibit the levels of related TNF-α, IL-6, and IL-1β factors, demonstrating its efficacy in inhibiting anxiety disorders and neuroinflammation.

[0086] Example 6: Detection of the effects of Lactobacillus gasseri on body weight and food intake in mice. After the experiment began, the general physiological indicators of the mice were tested weekly, including body weight, food intake and water intake, and the differences in general physiological indicators among the groups of mice were compared.

[0087] The results are as follows Figure 13 As shown in Figures 14 and 15, all mice were in good health during the experiment, with glossy and clean fur and a normal diet. Figure 13As can be seen, there was no significant difference in the average weight of mice in each group (P>0.05). Figure 14 and Figure 15 The results showed no statistically significant differences in average daily food intake and average daily water intake among the groups of mice (P>0.05). These results indicate that *Lactobacillus gasseri* 198-01 has no significant effect on the general physiological indicators of mice.

[0088] The results of Examples 1-6 above demonstrate that this invention provides the use of *Lactobacillus gasseri* 198-01 in the preparation of products for controlling blood sugar and / or for anti-anxiety and antidepressant effects. The DPP4 enzyme activity inhibition test shows that *Lactobacillus gasseri* 198-01, as indicated in this invention, can effectively inhibit DPP4 enzyme activity, thus exhibiting blood sugar control effects. Based on a zebrafish anxiety model, this invention preliminarily screened *Lactobacillus gasseri* 198-01 to significantly alleviate anxiety-like behaviors in zebrafish. Further validation using a mouse anxiety model showed that *Lactobacillus gasseri* 198-01 exhibits a clear anti-anxiety effect, including alleviating anxiety-like behaviors in mice, increasing neurotransmitter levels in mouse serum and brain tissue, and reducing neuroinflammatory levels in the body and brain tissue, while also demonstrating biocompatibility. It is a probiotic with multiple biological functions.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of Lactobacillus gasseri, characterized in that, The Lactobacillus gasseri mentioned is Lactobacillus gasseri 198-01 ( Lactobacillus gasseri (198-01), accession number GDMCC No: 67302, accession address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, accession date: November 14, 2025.

2. A probiotic preparation, characterized in that, The formulation contains Lactobacillus gasseri 198-01 or its fermentation product as described in claim 1; preferably, the formulation is a solid or liquid formulation; more preferably, the formulation further contains excipients.

3. A composition, characterized in that, In addition to containing Lactobacillus gasseri 198-01 as described in claim 1, the composition also contains one or more of the following substances: probiotics, prebiotics, dietary fiber, protein, polypeptides, amino acids, lipids, carbohydrates, vitamins, minerals, and plant extracts.

4. The composition according to claim 3, characterized in that, The probiotics are selected from bacteria, fungi (such as yeast), or any combination thereof.

5. The formulation according to claim 2 or the composition according to claim 3, characterized in that, The content of Lactobacillus gasseri 198-01 is greater than 10. 6 CFU / g or greater than 10 6 CFU / mL.

6. The use of Lactobacillus gasseri 198-01 according to claim 1 in the preparation of food, dietary supplements, and pharmaceuticals.

7. The application according to claim 6, characterized in that, The dosage forms of the dietary supplements and medicines are selected from tablets, capsules, gel candies, oral liquid preparations, pills, powders, and drops.

8. The application according to claim 6, characterized in that, The food, dietary supplement, and medicine also contain additives / excipients acceptable to food, dietary supplement, and medicine.

9. The application according to claim 8, characterized in that, The excipients include: fillers, binders, lubricants, flow aids, thickeners, flavoring agents, edible oils, stabilizers, suspending agents, surfactants, or any combination thereof.

10. The use of Lactobacillus gasseri 198-01 according to claim 1 in the preparation of a composition for preventing and treating anxiety and / or controlling blood sugar; preferably, the composition is a pharmaceutical, health food, or dietary supplement.

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