Application of bifidobacterium animalis subsp. Lactis GOLDGUT-BB21 in preparation of products for relieving depression

Abstract

The invention discloses application of animal bifidobacterium subsp. Lactis GOLDGUT-BB21 in preparation of products for relieving depression, and belongs to the technical field of biological medicine. The probiotics disclosed by the invention can be used for relieving depressive symptoms, improving related symptoms, assisting in treating depression and preventing depression, and also can be used as a nutritional supplement to help maintain the balance of intestinal microflora and promote intestinal health.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, and more particularly to application of animal bifidobacterium lactis GOLDDUT-BB21 in preparation of a product for relieving depression. BACKGROUND

[0002] In today's society, depression has become a major public health problem that seriously affects human psychological health and quality of life. Traditional depression treatment methods, such as drug therapy (antidepressants) and psychological therapy, can alleviate symptoms to a certain extent, but have many limitations. For example, drug therapy can cause a series of side effects such as nausea, vomiting, insomnia, sexual dysfunction, etc., and some patients have poor response to drugs, with treatment resistance problems; and psychological therapy requires long-term investment and has high requirements for the professional level of therapists, which is difficult to meet the needs of the majority of patients.

[0003] Probiotics, as a new type of intervention means, have shown great potential in the field of depression treatment. Studies have shown that there is a close relationship between the gut microbiota and the brain, i.e. the "gut-brain axis". The gut microbiota can affect the function and behavior of the brain, including mood regulation, through various pathways such as nerves, endocrine, and immune. Probiotics, as beneficial microorganisms in the gut, can regulate the balance of gut microbiota, improve the intestinal environment, and then have a positive impact on the brain through the "gut-brain axis" to relieve depression symptoms.

[0004] Compared with traditional treatment methods, probiotic intervention in depression has significant advantages. As a natural biological agent, it does not have the side effects of drug therapy, is highly safe, and is suitable for long-term use. At the same time, probiotic intervention can regulate the physiological and psychological state of the human body as a whole, not only relieving depression symptoms, but also improving the intestinal function, immune function and metabolic level of patients, and improving the overall health status of patients. Patients can achieve this through dietary adjustment or supplementation of probiotic preparations, without the need for complex medical equipment and professional medical personnel.

[0005] The pathogenesis of depression involves multiple factors, such as imbalance of neurotransmitters in the brain, decrease of neural plasticity, dysfunction of hypothalamic-pituitary-adrenal axis, chronic inflammation, and gut-brain dysfunction. Probiotics play a role through the microbiota-gut-brain axis, and the mechanisms include increasing brain-derived neurotrophic factor / serotonin levels, reducing microbe-mediated inflammation, and activating the vagus nerve. However, the specific details and interactions of these mechanisms are still not well understood, making it difficult to precisely intervene in different pathogenesis of depression. In addition, different probiotic strains have different characteristics, and their therapeutic effects on depression also differ. The strains that have been studied more are Bifidobacterium or Lactobacillus, but not all strains have significant effects on depression, and the synergistic effects between different strains are also not clear. In addition, the strains effective for specific subtypes of depression have not been determined, which brings uncertainty to the clinical application of probiotics in the treatment of depression.

[0006] Therefore, how to develop a strain and its preparation capable of relieving depression is a problem that those skilled in the art need to solve urgently. SUMMARY

[0007] Therefore, how to develop a strain and its preparation capable of relieving depression is a problem that those skilled in the art need to solve urgently.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application claims the application of animal Bifidobacterium lactis subsp. GOLDGUT-BB21 in the preparation of products for relieving depression, wherein the animal Bifidobacterium lactis subsp. (Bifidobacterium animalis subsp. lactis) GOLDGUT-BB21 is preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 29347, the preservation time being December 18, 2023, and the preservation address being No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1, Postcode 100101.

[0010] Further, the above-mentioned product is a biological product, a food or a drug.

[0011] Further, the above-mentioned biological product is the cell body, metabolite and fermentation liquor of animal Bifidobacterium lactis subsp. GOLDGUT-BB21.

[0012] Further, the above-mentioned food is a general food, a special medical purpose formula food, a functional food and a health food.

[0013] Further, the health food is a solid beverage, a milk beverage, a compressed tablet candy, a soy product, a dairy product, or a fruit and vegetable product.

[0014] Further, the pharmaceutical product further includes a pharmaceutical excipient.

[0015] Further, the pharmaceutical excipient is at least one of a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid, and a release retardant.

[0016] Further, the pharmaceutical excipient is at least one of microcrystalline cellulose, hydroxypropyl methylcellulose, and lecithin.

[0017] Further, the pharmaceutical product is in the form of a granule, a capsule, a tablet, a pill, or an oral liquid.

[0018] Further, the number of viable bacteria of Bifidobacterium animalis lactis GOLDGUT-BB21 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0019] According to the technical solutions described above, compared with the prior art, the present application has the following beneficial effects:

[0020] 1. Alleviating depressive symptoms

[0021] Alleviating anxiety symptoms: Depression patients often have anxiety symptoms, and probiotics also show certain effects in alleviating anxiety. For example, certain probiotic strains can reduce experimentally induced anxiety behavior.

[0022] 2. Improving related symptoms

[0023] Directly improving mood: Probiotics significantly reduce depressive symptoms.

[0024] Regulating neurotransmitters: Probiotics can regulate neurotransmitter levels in the brain by affecting the "gut-brain axis", such as increasing serotonin levels, thereby improving depressive mood.

[0025] 3. Assisting in the treatment of depression

[0026] Combined with drugs: Probiotics can be used as an adjunctive therapy for traditional antidepressants. For example, certain probiotic supplements, when combined with antidepressants, can enhance the efficacy of the drugs and reduce the side effects of the drugs.

[0027] Improved treatment adherence: For some people who do not want to take antidepressant medication or feel that the current therapy is too expensive, probiotic therapy provides a gentle and effective alternative.

[0028] 4. Prevention of depression

[0029] Modulation of intestinal microbial community: By improving the balance of intestinal microbial community, enhancing the intestinal barrier function, and reducing the inflammatory response, the risk of depression is reduced.

[0030] 5. As a nutritional supplement

[0031] Supplementing intestinal beneficial flora: In daily diet, probiotics can be used as a nutritional supplement to help maintain the balance of intestinal microbial community and promote intestinal health. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Schematic diagram of the process of CUMS modeling operation;

[0033] Figure 2 Results of behavioral experiments after BB21 intervention;

[0034] Figure 3 Common flora Wayne diagram and difference flora heat map of Model and BB21 intervention groups;

[0035] Figure 4 Fecal SCFAs detection (butyric acid, isovaleric acid);

[0036] Figure 5 Partial least squares discriminant analysis (PLS-DA) results;

[0037] Figure 6 Top 20 important pathways in KEGG metabolic annotation of BB21 intervention group and Model;

[0038] Figure 7 KEGG enrichment topology diagram of differential metabolites of BB21 intervention group compared with Model group;

[0039] Figure 8 5-HT content in brain tissue and colon tissue (*p<0.05, **p<0.01, ***p<0.001, ****p<0.000);

[0040] Figure 9 Corticosterone content in brain tissue and serum (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0041] Figure 10The expression amount of TRKB-FL / TRKB-T protein in brain tissue;

[0042] Figure 11 The expression amount of 5-HT / SERT protein in colon tissue;

[0043] Figure 12 The HE staining and MLCK / CREB immunofluorescence diagram of brain tissue;

[0044] Figure 13 The HE staining and MLCK / CREB immunofluorescence diagram of colon tissue. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0046] Embodiment 1

[0047] Obtaining, isolating, identifying, culturing and preserving of animal Bifidobacterium lactis subsp. GOLDGUT-BB21

[0048] The present application refers to the patent application with the application number 202410024101.6, the application date 2024.01.05, the applicant Shenzhen Bo Time Health Biotechnology Co., Ltd. and the title A strain of animal Bifidobacterium lactis subsp. GOLDGUT-BB21 and its application.

[0049] Embodiment 2

[0050] Experimental exploration of animal Bifidobacterium lactis subsp. GOLDGUT-BB21 in relieving depression

[0051] The present study aims to explore the intervention effect of animal Bifidobacterium lactis subsp. GOLDGUT-BB21 intervention program on chronic unpredictable mild stress (CUMS) induced depression mouse model. At the same time, the regulation mechanism of the “gut-brain axis”, neurotransmitter system and immune inflammatory response is focused on, and the regulatory role of probiotics in neurotransmitter metabolism and inflammation inhibition is elucidated. By comparing the differences between probiotics and drugs, the potential target and mechanism of probiotics in relieving depression-like behavior are revealed, which provides a basis for precise strain screening.

[0052] 1. Experimental materials

[0053] 1.1 Animal Bifidobacterium lactis subsp.

[0054]

[0055] 1.2 Experimental Mice

[0056] Four-week-old male C57BL / 6J mice (20 ± 2 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. All mice were housed under standard conditions (temperature 25 ± 2℃, humidity 50 ± 10%, 12 h light / dark cycle) with free access to food and water. The experimental protocol was approved by the Laboratory Animal Ethics Committee of Hainan University (HNUAUCC-2024-00298), and all procedures followed laboratory animal welfare and ethical guidelines.

[0057] 2. Experimental Methods

[0058] 2.1 Animal Experiment Grouping

[0059] Table 1 CUMS Modeling Operation Table

[0060]

[0061] Each cycle lasts four weeks, for a total of 12 weeks of modeling.

[0062] A CUMS depression model was established using C57BL / 6J mice. Figure 1 As shown, after 7 days of acclimatization, all mice were first divided into a control group and a model group. They were initially trained with sucrose water, followed by an 8-week CUMS (Cumulative Depression Model) treatment. Specific modeling procedures are shown in Table 1. The first behavioral experiment was conducted in week 9 to assess the effectiveness of the mouse depression model. After successful modeling, the mice were divided into a control group, a model group, a drug group, and an intervention group containing *Bifidobacterium lactis* subsp. *golden* (n = 8) (n = 8 per group). These mice were then treated with fluoxetine (10 mg / kg) and probiotics (10 mg / kg) for 4 weeks. 9 The mice were administered CFU / d via gavage. During this period, CUMS modeling was performed simultaneously. A second behavioral experiment was conducted after four weeks of intervention to determine the antidepressant effect of the intervention group using mouse behavioral data. Finally, the mice were dissected for necessary sampling and subsequent indicator testing.

[0063] 2.2 Mouse behavioral experiments

[0064] Behavioral experiments, including the tail suspension test, forced swimming test, sucrose preference test, elevated maze test, and open field test, were used to determine the degree of depression in mice based on the behavioral results.

[0065] (1) Tail suspension test: The mice were hung upside down on the device with a head distance of 20 cm from the ground, and the tail suspension immobility state of the mice was recorded for 6 min. When the mice showed passive suspension and no limb movement, it was determined to be immobile, and the cumulative immobility time in the last 4 min period was calculated with a stopwatch.

[0066] (2) Forced swimming test: The mice were placed in a high 24.5 cm, diameter 16 cm high borosilicate glass container, the container contained 20 cm high water, and the water temperature was maintained at (23 ± 2) ℃, and the swimming state of the mice was recorded for 6 min. When the mice showed no obvious swimming action in the container, it was considered to be floating immobile, and the cumulative immobility time in the last 4 min period was calculated with a stopwatch.

[0067] (3) Sucrose water preference test: Sucrose drinking test is a detection index to reflect whether the hedonic is missing. Hedonic deficiency refers to the lack of interest in reward stimulation, which is a manifestation of emotional disorders (including depression).

[0068] The sucrose preference test includes two parts: adaptation training part and test part. Training part: On the fourth day of the adaptive feeding stage, two bottles of 1% (w / v) sucrose solution were placed in each cage for 24 h, then one of them was replaced with pure water, and the positions of the sucrose water bottle and the pure water bottle were exchanged at the 12th hour to avoid the interference of the water bottle position on the experiment. Test part: A bottle of 1% (w / v) sucrose solution and a bottle of pure water solution were placed in the mouse cage, and the water consumption of the mice was recorded and calculated after 1 h.

[0069] (4) Elevated plus maze test: The mice were placed in the central area of the maze with their heads facing the open arms, and it was noted that each mouse was placed in the same position thereafter. At the same time, the camera monitor was turned on to record the number of times the mice entered the open arms and closed arms and the time spent in each arm within 5 min. Behavior analysis software VisuTrack was used to analyze the data. During the experiment, the experimenter should be at a distance of 1 m from the maze.

[0070] (5) Open field test: The activity and exploration behavior of mice in a strange open environment were observed, and their activity ability and anxiety level were analyzed. The mice were gently placed in the center of the open field box to adapt to the environment. The behavior of the mice in the open field box was recorded for 6 min, and the parameters such as the time spent in the central area and the activity speed were recorded. The central area is relatively open, and anxious mice tend to avoid this area and spend less time in it; non-anxious mice will spend more time in the central area. It can be observed whether the probiotic intervention improves the activity inhibition and anxiety-like behavior of CUMS-induced depressed mice.

[0071] 3 Sampling and biomarker analysis

[0072] 3.1 Sampling

[0073] After the completion of the second behavioral experiment, mice were fasted for 12 h, and then fresh fecal samples were collected for subsequent microbiome and metabolome analysis. Then the mice were anesthetized with 3% sodium pentobarbital (45 mg / kg, intraperitoneal injection), and whole blood was collected through the orbital venous plexus. The whole blood sample was centrifuged at 3000 rpm at 4°C for 20 min to separate the serum. After the mice were euthanized, the brain and colon tissues were quickly separated. Part of the tissue samples were immediately fixed in 4% paraformaldehyde solution for subsequent histopathological examination; the remaining samples were quickly frozen in liquid nitrogen and transferred to a -80°C ultra-low temperature freezer for long-term storage.

[0074] 3.2 Metagenomic sequencing and bioinformatics analysis

[0075] After the fresh fecal sample was collected, it was immediately treated with liquid nitrogen and transferred to a -80°C ultra-low temperature freezer for storage. Subsequent samples were sent to Meiji Bio Co., Ltd. for metagenomic sequencing analysis. Bioinformatics analysis was based on R language (v4.2.3), RStudio software and Meiji Bio cloud platform.

[0076] 3.3 Short-chain fatty acid (SCFA) analysis

[0077] The contents of SCFAs (butyric acid and isovaleric acid) in the fecal sample were determined by gas chromatography-mass spectrometry (GC-MS). 50 mg of fecal sample was added to 1 mL of extraction solution (containing 0.5% phosphoric acid) and ultrasonically extracted, and the supernatant was collected after centrifugation. Ethyl acetate extraction, organic phase was dried by nitrogen, then derivatized and analyzed by GC-MS.

[0078] 3.4 Non-targeted metabolome analysis

[0079] Fresh fecal samples were collected, quickly transferred to -80°C after liquid nitrogen freezing, and then sent to Meiji Bio Co., Ltd. for subsequent non-targeted metabolome sequencing analysis.

[0080] Sample Chromatography Conditions: 3 μL sample was separated on a HSS T3 column (100 mm x 2.1 mm i.d., 1.8 μm) and then detected by mass spectrometry. Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The positive ion mode separation gradient was as follows: 0 - 3 min, mobile phase B increased from 0% to 20%; 3 - 4.5 min, mobile phase B increased from 20% to 35%; 4.5 - 5 min, mobile phase B increased from 35% to 100%; 5 - 6.3 min, mobile phase B maintained 100%; 6.3 - 6.4 min, mobile phase B decreased from 100% to 0%; 6.4 - 8 min, mobile phase B maintained 0%. The negative ion mode separation gradient was as follows: 0 - 1.5 min, mobile phase B increased from 0 to 5%; 1.5 - 2 min, mobile phase B increased from 5% to 10%; 2 - 4.5 min, mobile phase B increased from 10% to 30%; 4.5 - 5 min, mobile phase B increased from 30% to 100%; 5 - 6.3 min, mobile phase B linearly maintained 100%; 6.3 - 6.4 min, mobile phase B decreased from 100% to 0%; 6.4 - 8 min, mobile phase B linearly maintained 0%. The flow rate was 0.40 mL / min, and the column temperature was 40°C.

[0081] Mass Spectrometry Conditions: The sample mass spectrometry signal was collected in positive and negative ion scanning modes, and the mass scanning range was 70 - 1050 m / z. The sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the auxiliary gas heating temperature was 425°C, the positive mode ion spray voltage was set to 3500 V, the negative mode ion spray voltage was set to -3500 V, the ion transmission tube temperature was 325°C, and the normalized collision energy was 20 - 40 - 60 V cycle collision energy. The first mass spectrometry resolution was 60000, the second mass spectrometry resolution was 7500, and the data was collected in DDA mode.

[0082] 3.5 Neurotransmitter and metabolite determination

[0083] The concentrations of 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), and corticosterone (CORT) in serum and brain tissue were determined by Elisa kits.

[0084] 3.6 Western Blot analysis

[0085] (1) Protein extraction and quantification

[0086] Tissue samples were homogenized in pre-cooled RIPA lysis buffer (containing 1% protease inhibitor) and incubated at 4°C for 30 min.

[0087] 12,000 x g, 4°C for 20 min, and the supernatant was collected; the total protein concentration was determined by BCA method (Pierce™ BCA kit, Thermo Fisher).

[0088] (2) Electrophoresis and membrane transfer

[0089] 30 μg of equal amounts of protein were separated by 10% SDS-PAGE electrophoresis (constant voltage 80 V→120 V); the protein was transferred to a PVDF membrane (0.22 μm, Millipore) by wet transfer method, and the transfer conditions were as follows: constant current 300 mA, 90 min.

[0090] (3) Blocking and antibody incubation

[0091] After membrane transfer, the PVDF membrane was quickly rinsed with TBST (5 min); 5% skim milk powder (prepared with TBST) was used for room temperature blocking for 60 min; primary antibody incubation: anti-TRKB (1:1000), anti-5-HT (1:800), anti-SERT (1:1000), and anti-β-actin (1:5000) were incubated at 4°C on a shaker overnight (16 h); TBST washing: 3 x 10 min; HRP-labeled secondary antibody (1:5000) was incubated at room temperature for 60 min; TBST washing: 3 x 10 min.

[0092] (4) Development and analysis

[0093] ECL chemiluminescence reagent (Advansta) was used for development; ImageJ v 1.53 was used to analyze the gray value of the band; the expression of the target protein was standardized with β-actin as the internal reference.

[0094] 3.7 Histopathology and immunofluorescence analysis

[0095] Paraffin-embedded brain and colon tissue sections (5 μm) were subjected to HE staining to observe the tissue structure, or immunofluorescence staining was performed to detect the expression of MLCK and CREB. After deparaffinization and hydration, the tissue sections were subjected to antigen retrieval, 3% BSA blocking for 1 h, primary antibody (anti-MLCK, 1:100; anti-CREB, 1:200) incubation at 4°C overnight, fluorescence-labeled secondary antibody incubation at room temperature for 1 h, DAPI staining of the nucleus, fluorescence microscope observation, and analysis of the fluorescence intensity using Image J software.

[0096] Data were expressed as mean ± standard error of mean (SEM) and analyzed by GraphPad Prism 8.0 software. One-way ANOVA was used for multiple comparisons followed by Tukey's multiple comparison test. Differences were considered statistically significant at p < 0.05. Correlation analysis between gut microbiota and metabolites was performed by Spearman algorithm and heat map was drawn.

[0097] 4. Results of the study

[0098] 4.1 Results of the behavioral experiment

[0099] After 8 weeks of CUMS modeling operation, the mouse depression model was successfully modeled. As shown in Table 1, after 4 weeks of probiotic gavage intervention, the immobility time of the tail suspension test (TST) and forced swimming test (FST) of the probiotic intervention group was significantly reduced, the sucrose water intake of the sucrose preference test (SPT) was significantly increased, and the number of times of passing through the center area and the time of staying in the open area of the open field test (OFT) were increased, indicating that after probiotic intervention, the despair emotion of the mice was relieved, the pleasure was restored, and the depression was relieved. Figure 2

[0100] 4.2 Analysis of gut microbiota composition and short-chain fatty acid levels

[0101] 4.2.1 Analysis of gut microbiota composition

[0102] As shown in Table 2, metagenomic sequencing was used to analyze the gut microbiota structure of mice in each group, and the set analysis and common difference microbiota heat map were analyzed using a Venn diagram. BB21 produced 41 non-common difference microbiota compared with the model group, indicating that probiotics have a regulatory effect on gut microbiota. At the genus level, the abundance of pathogenic bacteria Klebsiella pneumoniae (Klebsiella pneumoniae) in the BB21 intervention group was significantly reduced, the immune-regulating Oscillibacter_sp. (Oscillibacter_sp.), the intestinal physical barrier-enhancing Eisenbergiella_porci (Eisenbergiella_porci), and the butyric acid-producing Anaerofustis_stercorihominis (Anaerofustis_stercorihominis) were increased, indicating that probiotics can achieve gut-brain axis repair by regulating specific functional microbiota. Figure 3

[0103] 4.2.2 Analysis of short-chain fatty acid levels

[0104] ​​Short-chain fatty acids (SCFAs) are important bioactive molecules in the metabolic products of gut microbiota, and are metabolites of most beneficial bacteria in the gut. SCFAs can effectively inhibit the abundance of pathogenic bacteria in the gut. Isovalerate mainly originates from the fermentation products of branched-chain amino acids in the gut, and elevated isovaleric acid levels usually indicate an imbalance in the gut environment. Figure 4 As shown, the butyrate concentration in the BB21 intervention group increased significantly, while the isovaleric acid content decreased significantly, which may improve depression by balancing the intestinal environment and promoting butyrate production.

[0105] 4.3 Non-targeted metabolomics

[0106] To comprehensively evaluate the impact of probiotic intervention on intestinal metabolism, non-targeted metabolomics analysis was performed on mouse feces. Figure 5 As shown, the levels of metabolites in the BB21 intervention group were significantly different from those in the model group.

[0107] like Figure 6 As shown, to elucidate the metabolic pathway mechanism of BB21's antidepressant effect, KEGG pathway annotation based on gut microbiota metabolites was performed. Related metabolites were annotated in pathways related to depression, including tryptophan metabolism, phenylalanine, tyrosine and tryptophan metabolism, central carbon metabolism, and histidine metabolism.

[0108] like Figure 7As shown, BB21 focuses on riboflavin metabolism, synaptic vesicle circulation, choline metabolism in cance, GABAergic synapses, and alanine, aspartate, and glutamate metabolism pathways. BB21 specifically regulates nucleotide metabolism, phenylalanine-tyrosine-tryptophan biosynthesis, and purine metabolism: tryptophan is a precursor to serotonin, and enhanced biosynthesis can increase serotonin levels in the brain; purine metabolism participates in the energy supply for neurotransmitter release, while nucleotide metabolism maintains nucleic acid synthesis in nerve cells, ensuring neuronal proliferation and repair.

[0109] 4.4 Analysis of neurotransmitter indicators and Western blot results

[0110] The levels of 5-hydroxytryptamine (5-HT) in mouse brain and colon tissues were determined. Figure 8 As shown, after CUMS modeling, the 5-HT content in the Model group decreased significantly, while the 5-HT content increased significantly after BB21 intervention.

[0111] The levels of corticosterone (CORT) in the brain tissue and serum of mice were measured. For example... Figure 9 As shown, after CUMS modeling, the CORT content in the Model group increased significantly and GABA decreased significantly. After BB21 intervention, the CORT content decreased significantly and GABA increased significantly, returning to normal levels.

[0112] like Figure 10 As shown, Western blot analysis further confirmed that the probiotic BB21 intervention group increased the expression of brain-derived neurotrophic factor (BDNF) receptor tyrosine kinase B-FL (TRKB-FL) in brain tissue compared with the model group (p<0.05). However, the TRKB-T content in the BB21 intervention group did not change significantly compared with the model group. The TRKB-FL / TRKB-T ratio can reflect the antidepressant effect, and the higher the value, the greater the effect. The TRKB-FL / TRKB-T ratio in the BB21 intervention group was close to that in the normal group.

[0113] like Figure 11As shown, BB21 intervention increased the 5-HT content in the colon tissue compared with the model group (p<0.01), and reduced the expression of 5-HT transporter (SERT).

[0114] MLCK is a serine / threonine protein kinase, mainly involved in regulating the permeability of intestinal epithelium and blood brain barrier. Studies have shown that the overexpression of MLCK is closely related to the dysfunction of intestinal barrier and blood brain barrier induced by inflammatory factors, and MLCK inhibitors have been proved to improve stress-induced depressive-like behavior and cognitive dysfunction. CREB is a key transcription factor that plays a core role in neural plasticity, synaptic function and memory formation. Many studies have shown that the expression and phosphorylation level of CREB are reduced in patients with depression and animal models, and almost all known antidepressants can increase the activity of CREB, promote the expression of neurotrophic factors such as BDNF, thereby enhancing neural plasticity and neurogenesis, and improving depressive symptoms. For example, Figure 12-13 As shown, BB21 intervention can reduce the expression of MLCK in brain tissue and colon tissue and up-regulate the expression of CREB.

[0115] 5. Conclusion

[0116] Probiotics are one of the effective measures to intervene depression. This study aimed to investigate the improvement effect of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 intervention on depressive-like behavior in mice induced by chronic unpredictable mild stress (CUMS). Through the multidimensional synergistic effect of "gut microbiota-metabolome-intestine-brain axis-neuroimmunity", the depressive-like behavior of CUMS-induced mice was significantly improved. The core mechanism is to regulate the balance of intestinal flora by reducing the abundance of pathogenic bacteria such as Klebsiella pneumoniae and enriching beneficial bacteria such as butyrate-producing bacteria; relying on common pathways such as riboflavin metabolism and strain-specific metabolic pathways (such as bile acid synthesis, tryptophan metabolism, and arginine metabolism) to reprogram intestinal metabolism and increase the levels of SCFAs and beneficial metabolites; then activate the BDNF-TRKB / CREB signaling pathway to repair neural plasticity, regulate the balance of neurotransmitters such as 5-HT, inhibit the expression of MLCK to protect barrier function, and improve neuroimmune inflammatory response. This study provides comprehensive data support for the flora, metabolism, and molecular mechanism of probiotics as an adjunctive intervention for depression, and provides a scientific basis for the precise screening of anti-depression probiotic strains.

[0117] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in the preparation of products for relieving depression, characterized in that, The Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29347 on December 18, 2023.

2. The application according to claim 1, characterized in that, The product is a biological product, food, or medicine.

3. The application according to claim 2, characterized in that, The biological products are the bacterial cells, metabolites, and fermentation broth of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21.

4. The application according to claim 2, characterized in that, The food products mentioned are general food, food for special medical purposes, functional food, and health food.

5. The application according to claim 4, characterized in that, The health food products mentioned are solid beverages, milk beverages, compressed candies, soy products, dairy products, or fruit and vegetable products.

6. The application according to claim 2, characterized in that, The medicine also includes pharmaceutical excipients.

7. The application according to claim 6, characterized in that, The pharmaceutical excipients are at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, and release inhibitors.

8. The application according to claim 7, characterized in that, The pharmaceutical excipient is at least one of microcrystalline cellulose, hydroxypropyl methylcellulose, and lecithin.

9. The application according to claim 2, characterized in that, The dosage form of the medicine is granules, capsules, tablets, pills, or oral liquid.

10. The application according to claim 2, characterized in that, The viable count of Bifidobacterium animalis subsp. lactis GOLDGUT-BB21 in the product is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

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