Lactobacillus plantarum for regulating mood health and resisting depression and application of lactobacillus plantarum

Abstract

The invention provides lactobacillus plantarum for regulating mood health and resisting depression and application thereof, and the strain is separated from traditional pickles and is identified as lactobacillus plantarum. Experiments show that KY013 can significantly improve behavioral performance of depression model mice, and the effect of KY013 is equivalent to that of fluoxetine. The action mechanism comprises the following steps: up-regulating the levels of neurotransmitters such as 5-HT, DA, NE and GABA in hypothalamus, regulating the balance of proinflammatory factors (IL-6 and IFN-alpha) and anti-inflammatory factors (IL-10), and playing an anti-depression role through'neurotransmitter-immunity 'double ways. The KY013 disclosed by the invention can be used for developing antidepressant drugs or functional foods and the like, has the advantages of multi-target coordination, high safety and the like, and provides a new microecological preparation choice for treating depression.

Description

Technical Field

[0001] The present invention belongs to the fields of microbial technology and pharmaceutical food applications, and particularly relates to a Lactobacillus plantarum strain with the functions of regulating emotional health and antidepressant effects. Background Art

[0002] Depression is a common mental disorder with a continuously increasing global incidence. Its pathogenesis is complex, involving multiple factors such as neurotransmitter imbalance (such as 5-HT, DA, NE, GABA, etc.), inflammatory response, and gut microbiota dysbiosis. Currently, clinically, selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are mainly used for treatment, but there are problems such as slow onset, large side effects (such as gastrointestinal discomfort, sexual dysfunction), and significant individual differences. In recent years, research on the gut-brain axis has revealed that probiotics can improve depressive symptoms by regulating neurotransmitters, immune inflammation, and other pathways. However, the existing probiotic strains still have the following limitations in terms of antidepressant effects, action mechanisms, and clinical transformation: Insufficient strain specificity: Most studies focus on the genus Lactobacillus, but the neuromodulatory abilities of different strains vary significantly, and there is a lack of efficient strain screening criteria for the core symptoms of depression (such as anhedonia, behavioral despair). Imperfect mechanism research: Existing technologies mostly focus on single neurotransmitters or inflammatory factors, and have not systematically verified the synergistic regulatory effects of strains on multiple neurotransmitters (5-HT, DA, NE, GABA) and immune balance (such as IL-6 / IL-10).

[0003] Although there are also some reports on the use of Lactobacillus plantarum for the treatment of depression in the prior art, there are still certain differences in functions from the present invention. For example, CN117752698A involves strain MA2. First, its antidepressant effect was preliminarily demonstrated through animal behavior tests, proving that the strain has the potential to improve anxiety or depression caused by stress, and verifying that MA2 can improve the anti-stress interference ability and relieve depressive symptoms by affecting the microbial flora diversity and richness in the intestines of mice, promoting intestinal short-chain fatty acid metabolism, and reducing inflammation caused by CUMS. However, in this literature, the principle of the antidepressant effect of strain MA2 is mainly to improve the antidepressant effect by affecting the intestinal flora abundance and then promoting metabolism. CN117243981A involves strain JYLP-326, which detected the effects on emotion-related nerve cells, proteins, and inflammatory factors in the hippocampus of mice, but did not investigate the changes in neurotransmitters in the hypothalamus, a key regulatory brain region for depression. CN115838653A involves strain GM11. Although the levels of 5-HT and BDNF were detected, it was limited to the hippocampus and did not involve the synergistic regulation of other key neurotransmitters such as dopamine (DA), norepinephrine (NE), and gamma-aminobutyric acid (GABA).

[0004] Based on the above problems, it is urgent to obtain a plant lactobacillus that can change the neurotransmitters in the hypothalamus, a key regulatory brain area for depression, and can achieve the synergistic regulation of key neurotransmitters. Summary of the invention

[0005] In response to the above problems, the present invention isolated and screened a strain of Lactobacillus plantarum KY013 with anti-depressant potential from traditional kimchi, verified its behavioral improvement effect through the CUMS depression model system, and clarified its dual mechanism of action by regulating neurotransmitter levels and immune-inflammatory balance, providing a new strategy for the microecological treatment of depression.

[0006] strains A Lactobacillus plantarum with a deposit number of CGMCC 33479, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and deposited on January 20, 2025. Classification name: Lactiplantibacillus argentoratensis

[0007] Its 16S rRNA comprises the RNA sequence shown in SEQ ID NO: 3; The above-mentioned Lactobacillus plantarum is a Gram-positive bacterium and is in the shape of a short rod.

[0008] Pharmaceutical composition comprising bacterial strains The present invention also relates to a composition, comprising the aforementioned Lactobacillus plantarum and pharmaceutically acceptable excipients, carriers, auxiliary materials and vehicles.

[0009] The dosage of Lactobacillus plantarum is 1×10 6 CFU to 1×10 11 CFU / time, preferably 1×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU / time.

[0010] The medication cycle should be based on the ability to achieve the therapeutic effect, for example, three times a day for 1 week; the medication cycle can be appropriately adjusted according to different physical conditions, such as lasting for 2 weeks, one month, etc.

[0011] Examples of excipients, carriers, adjuvants, and vehicles include, but are not limited to, anti-adhesives, binders, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (diluents), film formers or coatings, flavorants, fragrances, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients, carriers, adjuvants, and vehicles include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monobasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0012] The pharmaceutical composition can be prepared in the form of an injection preparation or an oral preparation. The injection preparation includes, according to the physical state, liquid injection, powder for injection, and tablet for injection; according to the injection site, it includes intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and intraspinal injection; preferably, the solvent of the injection preparation includes water for injection or physiological saline.

[0013] Preparations for oral use include tablets containing an active ingredient admixed with a non-toxic pharmaceutically acceptable excipient. These excipients can be, for example, inert diluents or fillers (such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (such as cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid); binders (such as sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adhesives (such as magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil or talc). Preparations for oral use can also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is admixed with an inert solid diluent (such as potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is admixed with a water or oil medium (such as peanut oil, liquid paraffin or olive oil). Powders, granules and pills can be prepared in a conventional manner using the ingredients mentioned above under tablets or capsules, for example using mixers, fluidized bed equipment or spray drying equipment.

[0014] Other pharmaceutically acceptable excipients for oral preparations include, but are not limited to, colorants, flavorants, plasticizers, humectants and buffering agents. Preparations for oral use can also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is admixed with an inert solid diluent (such as potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is admixed with a water or oil medium (such as peanut oil, liquid paraffin or olive oil). Powders, granules and pills can be prepared in a conventional manner using the ingredients mentioned above under tablets or capsules, for example using mixers, fluidized bed equipment or spray drying equipment.

[0015] In some embodiments, the administration includes intramuscular, intravenous (e.g., in the form of a sterile solution and in a solvent system suitable for intravenous use), intradermal, intra-arterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, transdermal, intratumoral, transperitoneal, subcutaneous, subconjunctival, intracapsular, transmucosal, intrapericardial, intraumbilical, intraocular, oral (e.g., tablets, capsules, cachets, caplets or syrups), transdermal (e.g., in the form of creams, gels, lotions or ointments), topical, by inhalation, by injection or by infusion (e.g., continuous infusion in the form of creams or lipid compositions, local perfusion directly soaking the target cells, catheterization, lavage) of the compositions described herein.

[0016] Food and health product compositions containing strains The present invention provides a functional bacterium agent containing Lactobacillus plantarum, specifically it can be a food or health product composition, and the composition contains an effective dose of Lactobacillus plantarum and excipients acceptable in food or health product science.

[0017] The usage amount of the Lactobacillus plantarum is 1×10 6 CFU to 1×10 11 CFU / composition per time, preferably 1×10 8 CFU / g, 1×10 9 CFU, 1×10 10 CFU / composition per time.

[0018] The usage dose of health products and foods is preferably once a day, lasting for 1 - 6 months, 2 - 6 months, 3 - 6 months, etc., and can be adjusted according to different constitutions.

[0019] The food or health product composition may contain one or more of the following excipients: Carriers or fillers: maltodextrin, microcrystalline cellulose, starches (such as corn starch, potato starch), lactose, sucrose, mannitol, sorbitol, etc.; Protectants: skim milk powder, trehalose, glycerol, ascorbic acid, sodium glutamate, etc.; Embedding or sustained-release materials: gum arabic, gelatin, pectin, chitosan, β-cyclodextrin, liposomes, etc.; Flavoring agents: sweeteners (such as aspartame, sucralose, steviol glycosides), souring agents (such as citric acid, malic acid), flavors (such as fruit flavors, milk flavors), etc.; Nutritional fortifiers: vitamins (such as vitamin B group, vitamin C, vitamin D), minerals (such as calcium, magnesium, zinc), dietary fiber (such as fructooligosaccharide, inulin), etc.; Stabilizers or emulsifiers: xanthan gum, carrageenan, gum arabic, lecithin, glycerol monostearate, etc.; Preservatives: potassium sorbate, sodium benzoate, natamycin, etc.; Prebiotics: galactooligosaccharides, fructooligosaccharides, xylooligosaccharides, inulin, etc.

[0020] The dosage forms of the food or health product composition include but are not limited to: solid beverages, capsules, tablets, granules, powders, chewable tablets, gummies, yogurt, fermented dairy products or other functional food forms.

[0021] Uses of strains and compositions containing the strains Strains, compositions containing them, and functional bacterial agents containing Lactobacillus plantarum are used for correcting the immune imbalance state of the body, relieving depressive mood, treating and / or preventing depression; for preparing drugs for treating depression; for preparing food compositions; and for preparing health product compositions. Among them, correcting the immune imbalance state of the body is achieved by regulating the levels of cytokines in the body.

[0022] Specifically Correcting the immune imbalance state of the body means that the strains and their compositions can improve the immune imbalance state of the body by regulating the levels of pro-inflammatory cytokines (such as IL-6, IFN-α) and anti-inflammatory cytokines (such as IL-10), thereby relieving depressive-like behaviors caused by chronic inflammation.

[0023] For relieving depressive mood and treating / preventing depression, the strains and their compositions exert antidepressant effects through the following mechanisms: (1) Up-regulating the levels of monoamine neurotransmitters (including 5-HT, DA, NE) and GABA in the hypothalamus and central nervous system; (2) Regulating the function of the "gut-brain axis", improving the composition of the gut microbiota, and enhancing the integrity of the gut barrier; (3) Inhibiting the over-activation of the hypothalamic-pituitary-adrenal (HPA) axis and reducing the stress response.

[0024] The above uses are applicable to the prevention or adjuvant treatment of mood disorders such as depression and anxiety caused by stress, neuroinflammation or gut microbiota dysregulation.

[0025] Fermenting agents, functional bacterial agents A fermented composition, fermenting agent or functional bacterial agent containing the aforementioned Lactobacillus plantarum, including the bacterial liquid prepared from the bacterial liquid of the aforementioned Lactobacillus plantarum, or the powder or granule obtained by further processing; one or more non-antagonistic microbial bacterial agents can be further contained in the fermenting agent, and a compound bacterial agent is prepared from one or more selected from Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus.

[0026] The effective concentration of the single-use bacterial agent and the viable bacteria count are 1×10 6 CFU to 1×10 11 CFU, preferably 1×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU.

[0027] The starter or functional bacterial agent can also be used as a functional food or nutritional product.

[0028] The symbiotic bacteria refer to those foods containing a mixture of prebiotics and probiotics. They generally contain prebiotic components that are beneficial for growth and / or metabolic activity, and overall probiotic effects in combination with, such as but not limited to, Lactobacillus plantarum and fructooligosaccharides or galactooligosaccharides.

[0029] Beneficial effects The Lactobacillus plantarum KY013 (CGMCC 33479) isolated and screened in the present invention exhibits significant antidepressant potential through systematic verification. Its innovation lies in the multi-dimensional synergistic action mechanism: this strain not only specifically improves the core symptoms of depression (such as anhedonia, behavioral despair), but also exerts its effects through a dual regulatory pathway - on the one hand, comprehensively regulating the multi-neurotransmitter system in key brain regions of the hypothalamus (simultaneously increasing the levels of 5-HT, DA, NE, and GABA), and on the other hand, precisely balancing the immune-inflammatory response (regulating the ratio of IL-6 / IL-10), thus breaking through the limitations of the single-target action of existing probiotics. Compared with the prior art, the KY013 strain has a more specific brain region targeting (hypothalamus) and a more systematic neurotransmitter network regulation ability. The prepared drug or functional food composition can improve the function of the HPA axis and enhance the intestinal barrier through the gut-brain axis within the dose range of 1×10^8 - 10^10 CFU, providing a new microecological intervention strategy for the treatment of depression with rapid onset and low side effects, and at the same time expanding the high-value application approach of traditional fermented food resources. Brief description of the drawings

[0030] Figure 1 Gram staining microscope results of KY013 bacteria.

[0031] Figure 2 Sucrose preference rate of mice in the sucrose preference experiment.

[0032] Figure 3 Immobile time of mice in the forced swimming experiment.

[0033] Figure 4 Results of the open field experiment of mice: The left figure shows the number of rearing times and the number of grid crossings of mice; the right figure shows the residence time of mice in the central area.

[0034] Figure 5Results of the elevated plus-maze test in mice: The left figure shows the number of entries into the open arms of the mice, and the right figure shows the residence time of the mice.

[0035] Figure 6 Immobility time of mice in the tail suspension test.

[0036] Figure 7 Content of 5-HT in the hypothalamus of mice.

[0037] Figure 8 Content of DA in the hypothalamus of mice.

[0038] Figure 9 Content of NE in the hypothalamus of mice.

[0039] Figure 10 Content of GABA in the hypothalamus of mice.

[0040] Figure 11 Content of the cellular inflammatory factor IFN-α in the serum of mice.

[0041] Figure 12 Content of the cellular inflammatory factor IL-6 in the serum of mice.

[0042] Figure 13 Content of the cellular inflammatory factor IL-10 in the serum of mice. Specific implementation mode

[0043] Example 1 Isolation, screening and identification of Lactobacillus plantarum 1. Strain isolation and screening Sample source: Homemade pickles from farmers. The sample was evenly mixed with PBS and serially diluted 10 -2 ~10 -6 , in 5 gradients. 100 μL was taken and evenly spread on MRS medium, and anaerobically cultured at 37 °C for 24 - 48 h. When colonies formed, the morphological characteristics of the colonies were observed, and single colonies conforming to the morphological characteristics of Lactobacillus plantarum were picked out.

[0044] 2. Molecular identification Universal primer sequences for 16S rRNA amplification: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1) 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2) Table 1

[0045] Prepare 1% agarose gel (1 g agarose plus 100 mL deionized water), heat to boiling 2 - 3 times, and when cooled to about 55 °C, add 10 μL of 4S Green buffer and mix well, then pour into the gel casting tray. After solidification, mix 1 μL of Loading buffer and 5 μL of PCR product mixture and add them into the gel wells, and perform gel electrophoresis at 220 V for 30 min. If a band appears at the target band (1500 bp), send the sample to Shanghai Branch of Beijing Liuhe Huada Gene Technology Co., Ltd. for 16S rRNA sequencing. Compare the sequencing results on the ezbiocloud website.

[0046] The 16S rRNA sequence was obtained by sequencing the PCR product: Sequence alignment was performed through the ezbiocloud website to identify the strain as Lactiplantibacillus argentoratensis 3. Morphological identification: The KY013 strain was stained using the Gram staining method and its morphology was observed under a 100× oil immersion microscope. The specific operation steps are as follows. Inoculate the KY013 strain into MRS solid medium and culture it anaerobically at 37 °C for 48 h. Take a small amount of single colony of Lactiplantibacillus, dip it in 2 μL of physiological saline, and smear it clockwise into a uniform thin circle of about 1 cm2, and place it in a normal temperature environment until the bacterial solution dries. Pass the slide through the flame of an alcohol lamp 1-2 times for fixation, taking care not to overheat, and it is appropriate that the slide is not hot to the touch. Drop crystal violet staining solution on the bacteria and stain for 1 min, then wash with water. Drop iodine solution and stain for 1 min, then wash with water. Drop decolorizing solution, shake the slide, decolorize for 30 s, wash with water, and absorb the water. Drop safranin staining solution and stain for 1 min, then wash with water. Blot off the excess water with filter paper and perform microscopy under a 100× oil immersion microscope.

[0047] The results of the Gram staining microscope showed that it was a Gram-positive bacterium (see Figure 1 ), showing a short rod-shaped morphology.

[0048] Example 2 Behavioral tests Behavioral tests were conducted from 9:00 am to 4:00 pm in the light cycle. All behavioral tests started on the 8th day, and the mice were allowed to acclimatize to the test environment for 2 hours before the test. Different batches of mice were used for different behavioral tests. After each animal experiment, urine and feces were cleaned, and to eliminate the influence of animal odor on the next experiment, the open field was cleaned with 75% ethanol solution.

[0049] In this study, a mouse model of chronic unpredictable stress (CUMS) was selected. Before model establishment, all mice were weighed and randomly divided into 4 groups of 10 mice each. Lactobacillus plantarum KY013 and fluoxetine were suspended or dissolved in normal saline, and the bacterial suspension and fluoxetine solution for gavage were prepared freshly before use. The administration frequency was once a day. This model used male C57BL / 6J mice at 5 weeks of age, weighing 18 ± 2 g. The mice were acclimated for 7 days, and then the CUMS model was established for a total of 28 days. The stressors selected for establishing the CUMS mouse model included: (1) water deprivation for 24 h, (2) restraint in a restraint cage for 2 h, (3) tail suspension for 1 min, (4) cage shaking for 10 min, (5) cage tilting at 45˚ for 24 h, (6) food deprivation for 24 h, and (7) keeping the cage wet overnight (200 mL / cage). When selecting stressors, randomness should be maintained, and the interval between the same stressor should be greater than 7 days. The average body weight of the mice in the blank control group (NC) was 26.1 g on the 28th day, and the average body weight of the CUMS model mice (MS) was 22.5 g on the 28th day. The average body weight of the mice in the MS group was significantly lower than that of the mice in the NC group, which was in line with the characteristics of the CUMS mouse model.

[0050] Control group (NC): Normal mice were gavaged with 0.2 mL of 0.85% normal saline. CUMS model group (MS): Mice were induced with CUMS and gavaged with 0.2 mL of 0.85% normal saline. Lactobacillus plantarum KYOO13 intervention group of CUMS model (KY-13): Mice were induced with CUMS and gavaged with Lactobacillus plantarum KY013 at a dose of 10 9 CFU / mouse / day. Fluoxetine intervention group of CUMS model (FLU): Mice were induced with CUMS and treated with fluoxetine as a positive drug, and the gavage dose was 2.5 mg / kg body weight.

[0051] (1) Sucrose preference test By replacing the water bottle with a bottle containing sucrose solution (1%), mice were accustomed to sucrose during 3 d. The test included water deprivation for 23 h, and then the animals were allowed to freely use two bottles, one containing tap water and the other containing 1% sucrose solution. After 1 h, the weights of the bottles were measured and the liquid consumption was calculated. The sucrose preference was determined as follows: Sucrose preference (%) = sucrose intake / (sucrose intake + water intake) × 100. The sucrose preference was evaluated for 2 d. The positions of the sucrose and the water bottle were alternated daily to avoid false effects caused by side bias.

[0052] The results were as Figure 2As shown, compared with the NC group, the sucrose preference index of mice in the MS group was significantly decreased, indicating that the mice had depressive behaviors; the sucrose preference indices of mice in the FLU group and the KY-13 group were significantly higher than those in the MS group. It was shown that fluoxetine and probiotic interventions could alleviate the depressive behaviors of animals.

[0053] (2)Forced swimming test (FST) The mice were placed in a glass cylinder (25 cm high, 10 cm in diameter), and the cylinder was filled with water (22 °C) to a height of 18 cm. The test time was 6 minutes, and the percentage of immobile time was recorded. Immobility was defined as only slightly moving the tail or front paws to keep the head floating on the water surface, and the front paws were usually placed on both sides of the body. Swimming was defined as actively using the front paws to move forward, and the body was usually parallel to the cylinder wall. Climbing was defined as actively using the front paws to grasp the cylinder wall, and the body was perpendicular to the cylinder wall. Start video recording before putting the animal into the water tank, and slowly put the mouse into the water by holding its tail. Once the mouse is in the water, slowly release the tail. Using this method can prevent the animal's head from being immersed in the water and affecting the results. When all the mice enter the water tank, start the countdown on the stopwatch. The usual test length for mice is 6 min.

[0054] The results are as Figure 3 shown. In the forced swimming experiment, compared with the NC group, the immobile time of mice in the MS group was significantly higher than that in the NC group; the immobile times of mice in the FLU group and the KY-13 group were significantly lower than those in the MS group. It was shown that fluoxetine and probiotic interventions could significantly improve the survival desire of mice.

[0055] (3)Open field test The open field was a 40 cm × 40 cm area, with a 20 cm× 20 cm center and 35 cm high walls around. During the test, the mice were placed in the center of the open field, and their behaviors were recorded for 10 minutes. A video tracking system (Smart) was used to record the movement distance and the time spent in the central and peripheral areas. Each mouse was placed separately in the center of the open box and allowed to freely explore for 2 minutes before the test. Record the behaviors of each mouse in the next 4 min, record the number of grids crossed by the animal's front limbs, the number of times of hind limb standing, and the time spent moving in the designated central area (20×20 cm) of the box.

[0056] The results are as Figure 4 shown. The number of times of rearing and grid crossing of mice in the MS group throughout the open field and the time spent in the central area were significantly lower than those in the NC group; the number of times of rearing and grid crossing of mice in the FLU group and the KY-13 group in the open field and the time spent in the central area were significantly higher than those in the MS group. It was shown that fluoxetine and probiotic interventions could significantly improve the autonomous exploration ability and locomotor activity of mice.

[0057] (4)Elevated plus maze The maze is made of black stainless steel and consists of four arms (30 cm long and 5 cm wide): two enclosed arms (with black walls 30 cm high) and two open arms (with a 0.5 cm high edge). The four arms are connected by a central platform (5 cm × 5 cm), and the maze is 50 cm high. Each mouse starts a 5-minute test from the central platform facing the open arms. The video tracking system records the time spent in the open arms and the number of entries into the open arms and the enclosed arms (an entry into an arm is defined as all four paws entering the arm).

[0058] The results are as Figure 5 shown. The number of entries into the open arms and the time spent in the open arms of the mice in the MS group were significantly lower than those in the NC group in the elevated plus maze; the number of entries into the open arms and the time spent in the open arms of the mice in the FLU group and the KY-13 group were significantly higher than those in the MS group. It indicates that fluoxetine and probiotic intervention can significantly improve the spontaneous exploration ability and locomotor activity of mice.

[0059] (5) Tail suspension test The dimensions of the tail suspension test box are 55 cm high * 60 cm wide * 11.5 cm deep. There is a baffle in the middle of the test box to separate the experimental animals. Medical tape is used to stick 1 cm away from the tip of the tail so that the animal's tail can be suspended from the hanging rod of the device. The height between the tip of the animal's tail and the ground is about 30 cm, making the mouse in a head-down position. The experiment needs to record a total of 6 minutes of video, and the video records the behavior of the animal during the experiment, but records the immobile time of the animal in the last 4 minutes. The experimental animal is gently taken out of the breeding cage, and the mouse's tail is fixed with tape at 2 cm from the tail tip, about 7 cm long; in order to prevent the mouse from climbing its tail, a 1 cm long straw is used to cover it. The experimental animal is quickly hung at about 20 cm from the bottom of the test box and left immediately; it is filmed with a camera to record the whole process of the animal and record the immobile time of the mouse for 6 minutes; the definition of immobile time is the process where the mouse's front legs move but the hind legs do not participate. The movement caused by inertia at the beginning is also considered immobile time.

[0060] The results are as Figure 6 shown. In the forced swimming test, compared with the NC group, the immobile time of the mice in the MS group was significantly higher than that in the NC group; the immobile time of the mice in the FLU group and the KY-13 group was significantly lower than that in the MS group. It indicates that fluoxetine and probiotic intervention can significantly improve the survival desire of mice.

[0061] Example 3 Detection of hypothalamic neurotransmitters Collection of serum and tissue samples: After the behavioral tests, blood was quickly drawn from the eyeballs, and then the mice were sacrificed. The brain was placed in a petri dish on ice, and then the hippocampus and hypothalamus were quickly removed. Mouse feces were collected from the large intestine and stored in an -80°C refrigerator for subsequent experiments. The hypothalamus was placed in sterile physiological saline, homogenized thoroughly with a homogenizer, then placed in a centrifuge, set at 6000 r / min and 4°C, centrifuged for 10 min, the supernatant was taken, the supernatant was aliquoted, the sample to be tested was retained, and the rest was frozen at -80°C for standby; when using serum, it needs to be transferred from -80°C to a 4°C refrigerator 12 hours in advance and then used normally.

[0062] Determination of the contents of 5-HT, DA, NE and GABA in the hypothalamus (1)5-HT Serotonin The ELISA kit was used to detect the content of 5-HT in the hypothalamus, and the results are as Figure 7 shown. Compared with the NC group, the concentration of 5-HT in the hypothalamus of mice in the MS group was significantly decreased, which indicated that stress had a negative effect on the brain regions of mice in the MS group, making them show the characteristics of depression; while the concentration of 5-HT in the hypothalamus of mice in the FLU group and KY-13 group was significantly increased. This indicated that the intervention of probiotics and fluoxetine could have a positive effect on the content of 5-HT in the body and relieve depression.

[0063] (2)DA Dopamine The ELISA kit was used to detect the content of DA in the hypothalamus, and the results are as Figure 8 shown. Compared with the NC group, the concentration of DA in the hypothalamus of mice in the MS group was significantly decreased; while the concentration of DA in the hypothalamus of mice in the FLU group and KY-13 group was significantly increased. This indicated that the intervention of probiotics and fluoxetine could have a positive effect on the content of DA in the body and relieve depression.

[0064] (3)NE Norepinephrine The ELISA kit was used to detect the content of DA in the hypothalamus, and the results are as Figure 9 shown. Compared with the NC group, the concentration of NE in the hypothalamus of mice in the MS group was significantly decreased; while the concentration of NE in the hypothalamus of mice in the FLU group and KY-13 group was significantly increased. This indicated that the intervention of probiotics and fluoxetine could have a positive effect on the content of NE in the body and relieve depression.

[0065] (4)GABA γ-Aminobutyric acid The ELISA kit was used to detect the content of GABA in the hypothalamus, and the results are as Figure 10 shown. Compared with the NC group, the concentration of GABA in the hypothalamus of mice in the MS group was significantly decreased; while the concentration of GABA in the hypothalamus of mice in the FLU group and KY-13 group was significantly increased. This indicated that the intervention of probiotics and fluoxetine could have a positive effect on the content of GABA in the body.

[0066] Many neurotransmitters such as 5-HT, DA, NE, and GABA are secreted by cells and are positively related to emotions such as positive, optimistic, and happy. The levels and functions of these neurotransmitters are closely related to mental and neurological dysfunctions such as depression, anxiety, and autism. Among them, the reduced availability of 5-HT in the brain is a key feature in the pathogenesis of depression. In the experimental results of the present invention, the levels of neurotransmitters 5-HT, DA, NE, and GABA in the hypothalamus of mice in the NC group were significantly reduced, while the levels of these several neurotransmitters in the hypothalamus of mice in the experimental groups FLU and KY-13 were significantly increased, indicating that probiotic and fluoxetine interventions can restore the 5-HT deficiency in the brain regions caused by stress in model mice, restore the 5-HT level to the normal level, and have a positive impact on the other three neurotransmitters, thereby improving the depressive state of mice.

[0067] Example 4 Determination of Serum Indexes After the behavioral tests, blood was collected from the retro-orbital venous plexus of mice to measure serum biochemical indexes.

[0068] (1)IFN-α The IFN-α in mouse serum was measured using a kit, and the results are as Figure 11 shown. Compared with the NC group, the concentration of the inflammatory factor IFN-α in the serum of mice in the MS group was significantly increased, while the concentration of the inflammatory factor IFN-α in the serum of mice in the FLU group and the KY-13 group was significantly decreased.

[0069] (2)IL-6 The IL-6 in mouse serum was measured using a kit, and the results are as Figure 12 shown. Compared with the NC group, the concentration of the inflammatory factor IL-6 in the serum of mice in the MS group was significantly increased, while the concentration of the inflammatory factor IL-6 in the serum of mice in the FLU group and the KY-13 group was significantly decreased.

[0070] (3)IL-10 The IL-10 in mouse serum was measured using a kit, and the results are as Figure 13 shown. Compared with the NC group, the concentration of the inflammatory factor IL-10 in the serum of mice in the MS group was significantly increased, while the concentration of the inflammatory factor IL-10 in the serum of mice in the FLU group and the KY-13 group was significantly decreased.

[0071] When continuous inflammation occurs in the body, it will lead to immune imbalance in the body, which may then trigger depression. The content of serum inflammatory factors in each group of mice is as Figures 11 - 13As shown, the levels of pro-inflammatory cytokines IL-6 and IFN-α in the sera of mice in the MS group were significantly higher than those in the NC group, and the level of the anti-inflammatory cytokine IL-10 was significantly lower than that in the NC group, indicating that the immune system of the MS model mice was in a state of imbalance; after intervention with fluoxetine and probiotic KY-13, the levels of pro-inflammatory cytokines IL-6 and IFN-α in the model mice were significantly decreased, and the level of the anti-inflammatory cytokine IL-10 was significantly increased. This indicates that probiotic KY-13 can correct the immune imbalance state of the body by regulating the levels of cytokines in the body, thereby improving depression.

Claims

1. A Lactobacillus argentoratensis, characterized in that: The deposit number is CGMCC 33479, and it is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit date is January 20, 2025.

2. The Lactobacillus plantarum according to claim 1, wherein the 16S rRNA is shown as SEQ ID NO:

3.

3. A pharmaceutical composition comprising the Lactobacillus plantarum according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutically acceptable carrier is one or more carriers selected from fillers, binders, wetting agents, disintegrants, lubricants or flavoring agents commonly used in pharmacy.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is in the form of granules, capsules, tablets, pills and oral liquid.

6. Use of the Lactobacillus plantarum according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of a medicament for correcting an immune imbalance in an organism or relieving depression or for treating, relieving or preventing depression.

7. A food composition or a health product composition, comprising the plant lactobacillus according to any one of claims 1-2.

8. A starter, functional bacterial agent or nutritional composition comprising the Lactobacillus plantarum according to claim 1, 2 or 3.

9. The plant lactobacillus fermentation agent, functional bacterial agent or nutritional composition according to claim 6, wherein the nutritional composition is a food, a nutrient, a supplement, a probiotic or a symbiotic bacteria.

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