Application of bifidobacterium animalis subsp. Lactis BLa80 in preparation of preparation for relieving depression or anxiety

Abstract

The invention relates to application of bifidobacterium animalis subsp. Lactis BLa80 in preparation of a preparation for relieving depression or anxiety. The bifidobacterium animalis subsp. Lactis BLa80 is a strain of the bifidobacterium animalis subsp. Lactis BLa80 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.22547, and the strain of the bifidobacterium animalis subsp. Lactis BLa80 is a strain of the bifidobacterium animalis subsp. Lactis BLa80 with the preservation number of CGMCC No.22547. The invention develops a brand new application of the bifidobacterium animalis subsp. Lactis BLa80 strain, namely, the bifidobacterium animalis subsp. Lactis BLa80 strain is used for relieving depression or anxiety symptoms, specifically, the bifidobacterium animalis subsp. Lactis BLa80 strain can stimulate RIN14B cells to secrete 5-HT, can relieve depression and anxiety-like behaviors of mice suffering from depression and anxiety, can relieve hyperfunction of hypothalamic-pituitary-adrenal axis (HPA axis), and can relieve depression and anxiety-like behaviors of mice suffering from depression and anxiety. The inflammatory response of mice with depression and anxiety is relieved, the expression level of brain-derived neurotrophic factors in hippocampus of the mice with depression and anxiety is improved, the abundance of intestinal beneficial flora of the mice with depression and anxiety is improved, and the diversity of intestinal flora is increased.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, and relates to the application of Bifidobacterium animalis subsp. lactis BLa80 in the preparation of preparations to relieve depression or anxiety. Background Technology

[0002] Depression, also known as major depressive disorder, is often accompanied by feelings of guilt and hopelessness, as well as changes in appetite and sleep, leading to impaired daily functioning. Its core symptoms include low mood, loss of joy, low energy, and fatigue, and may also cause sleep and psychomotor disturbances, feelings of guilt, low self-esteem, and autonomic and gastrointestinal dysfunction. The bidirectional communication network between the gut and the central nervous system, known as the gut-brain axis, is closely related to complex communication between the autonomic nervous system, neuroendocrine system, and immune system.

[0003] Probiotics, as a class of beneficial microorganisms, have attracted attention for their role in maintaining the ecological balance of the human gut. Further research has revealed a close connection between the gut and the brain, the so-called "gut-brain axis," a discovery that provides a theoretical basis for probiotics to actively intervene in depression and anxiety by modulating the gut microbiota.

[0004] The effects of probiotics on anxiety and depression symptoms include endocrine effects related to the hypothalamus-pituitary-adrenal axis, regulation of immune responses, enhancement of epithelial barrier function, neural effects through vagus nerve stimulation, and regulation of neurochemicals such as serotonin, dopamine, gamma-aminobutyric acid, and brain-derived neurotrophic factor.

[0005] Therefore, the potential role of probiotics in improving depression and anxiety not only demonstrates their possibility as a novel treatment method but also provides new scientific evidence for mental health interventions. However, there are relatively few existing probiotic products that can effectively improve anxiety and depression, making the development of more probiotics that can effectively improve these conditions highly significant. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a novel application of Bifidobacterium animalis subsp. lactis BLa80, namely, the application of Bifidobacterium animalis subsp. lactis BLa80 in the preparation of preparations to relieve depression or anxiety.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of Bifidobacterium lactis subsp. BLa80 in the preparation of formulations to relieve depression or anxiety;

[0009] The Bifidobacterium animalis subsp. lactis strain BLa80 is the Bifidobacterium animalis subsp. lactis strain BLa80 with the preservation number CGMCC No. 22547.

[0010] This invention develops a novel use for the animal Bifidobacterium lactis subsp. BLa80 strain, namely, to alleviate symptoms of depression or anxiety. Specifically, the BLa80 strain can stimulate RIN14B cells to secrete 5-HT, reduce depressive and anxiety-like behaviors in mice with depression and anxiety, alleviate hyperfunction of the hypothalamus-pituitary-adrenal axis (HPA axis), alleviate inflammatory responses in mice with depression and anxiety, increase the expression level of brain-derived neurotrophic factor in the hippocampus of mice with depression and anxiety, increase the abundance of beneficial gut bacteria and increase gut microbiota diversity in mice with depression and anxiety.

[0011] Preferably, the number of viable bacteria in the preparation is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc.

[0012] Preferably, the dosage form of the preparation includes solution, powder, tablet, capsule or granule.

[0013] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0014] In this invention, the formulation alleviates HPA axis hyperfunction.

[0015] In this invention, the formulation alleviates the inflammatory response.

[0016] In this invention, the formulation enhances the expression level of brain-derived neurotrophic factor in the hippocampus.

[0017] In this invention, the formulation increases the abundance of beneficial intestinal flora and enhances the diversity of intestinal flora.

[0018] More preferably, the strain in the preparation further includes Weizmannia coagulans strain BC179; the Weizmannia coagulans strain BC179 is the Weizmannia coagulans strain BC179 with accession number CGMCC No. 33768.

[0019] The aforementioned BLa80 strain can not only be used alone to relieve symptoms of depression or anxiety, but can also be used in combination with the Weizmann's coagulans BC179 strain, and the two strains have a synergistic effect in relieving depression or anxiety.

[0020] Preferably, the ratio of viable bacteria of the BLa80 strain to the BC179 strain is 1:3 to 3:1, such as 1:3, 1:2, 1:1, 2:1, 3:1, etc.

[0021] Based on the potential synergistic relationship between the two strains, when the specific usage ratio mentioned above is met, they are more effective in relieving depression or anxiety.

[0022] Secondly, the present invention provides a probiotic composition for relieving depression or anxiety, wherein the strains in the probiotic composition include Bifidobacterium animalis subsp. lactis BLa80 strain with accession number CGMCC No. 22547 and Weizmannia coagulans BC179 strain with accession number CGMCC No. 33768.

[0023] This invention creatively combines *Bifidobacterium animalis* subsp. *lactospirum* BLa80 and *Weizmannii coagulans* strain BC179 to obtain a strain combination product. Studies have found that this combination is more effective than either *Bifidobacterium animalis* subsp. *lactospirum* BLa80 or *Weizmannii coagulans* BC179 alone in improving depression and anxiety, exhibiting a synergistic effect. This invention provides an effective combination strategy for improving depression or anxiety, and is of significant importance.

[0024] Preferably, the number of live bacteria in the probiotic composition is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc.

[0025] Preferably, the ratio of viable bacteria of the BLa80 strain to the BC179 strain is 1:3 to 3:1, such as 1:3, 1:2, 1:1, 2:1, 3:1, etc.

[0026] All other point values ​​not specifically listed in the numerical ranges involved in this invention are within the protection scope of this invention. Considering the length and brevity of the explanation, they will not be described in detail here.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention develops a novel use for the animal Bifidobacterium lactis subsp. BLa80 strain, namely, to alleviate symptoms of depression or anxiety. Specifically, the BLa80 strain can stimulate RIN14B cells to secrete 5-HT, reduce depressive and anxiety-like behaviors in mice with depression and anxiety, alleviate hyperfunction of the hypothalamus-pituitary-adrenal axis (HPA axis), alleviate inflammatory responses in mice with depression and anxiety, increase the expression level of brain-derived neurotrophic factor in the hippocampus of mice with depression and anxiety, increase the abundance of beneficial gut bacteria and increase gut microbiota diversity in mice with depression and anxiety.

[0029] The BLa80 strain involved in this invention is classified as *Bifidobacterium animalis* subsp. *lactis*, deposited on May 17, 2021, with accession number CGMCC No. 22547, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] The BC179 strain involved in this invention is classified and named *Weizmannia coagulans*, deposited on March 10, 2025, with accession number CGMCC No. 33768, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0031] Figure 1 This is a graph showing the evaluation results of 5-hydroxytryptamine secretion in RIN-14B cells stimulated by BLa80 or BC179 strains;

[0032] Figure 2This is a graph showing the statistical results of the percentage of time (%) of mice in each group spent in the central region during the open field test;

[0033] Figure 3 This is a graph showing the statistical results of the percentage of time (%) spent in the open arm for each group of mice in the elevated cross maze experiment;

[0034] Figure 4 This is a graph showing the statistical results of the immobility time of mice in each group during the tail suspension test;

[0035] Figure 5 This is a graph showing the statistical results of the sugar water preference values ​​(%) of mice in each group during the sugar water preference experiment;

[0036] Figure 6 This is a graph showing the statistical results of serum adrenocorticotropic hormone (ACTH) levels in each group of mice;

[0037] Figure 7 This is a graph showing the statistical results of serum corticosterone levels in each group of mice;

[0038] Figure 8 This is a graph showing the statistical results of the serum levels of the pro-inflammatory factor TNF-α in each group of mice;

[0039] Figure 9 This is a graph showing the statistical results of the serum levels of the anti-inflammatory factor IL-10 in each group of mice;

[0040] Figure 10 This is a graph showing the statistical results of BDNF levels in the hippocampus of mice in each group;

[0041] Figure 11 This is a graph showing the statistical results of the Simpson diversity index of the gut microbiota in each group of mice;

[0042] Figure 12 This is a graph showing the NMDS analysis results of the gut microbiota of each group of mice;

[0043] Figure 13 This is a graph showing the statistical results of the relative abundance of Bifidobacterium in the gut microbiota of each group of mice;

[0044] Figure 14 This is a graph showing the statistical results of the relative abundance of unnamed genera of the Prevotaceae family in the gut microbiota of mice in each group. Detailed Implementation

[0045] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0046] The following are the methods for preparing bacterial suspensions involved in the experiments:

[0047] Preparation of BLa80 bacterial suspension: The required strain was inoculated into L-MRS liquid medium and activated by culturing at 37℃ for 16-18 hours. The activation was repeated twice to obtain the activated solution. The activated solution was inoculated into L-MRS liquid medium at an inoculation rate of 3% (v / v) and cultured at 37℃ for 24 hours to obtain the bacterial suspension.

[0048] Preparation of BC179 bacterial suspension: The desired strain was inoculated into 30 mL of MRS liquid medium for primary activation and cultured on a shaker at 37℃ and 250 r / min for 12-15 h. The primary culture was then inoculated into a 250 mL Erlenmeyer flask containing 30 mL of MRS medium at a 5% inoculation rate and cultured on a shaker at 37℃ and 250 r / min for 18-24 h to obtain the desired bacterial suspension.

[0049] Centrifuge the bacterial culture at 4000 rpm for 5 min at 4°C, filter to obtain bacterial cells, resuspend the bacterial cells in pure water (Example 2) or HBSS buffer (Example 1) and dilute as needed to obtain the bacterial suspension of the corresponding strain.

[0050] L-MRS medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween-80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0051] MRS medium: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, diammonium citrate 2 g / L, glucose 20 g / L, Tween-80 1 mL / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L.

[0052] The rat insulinoma cells RIN-14B used in the following experiments were purchased from the China Center for Type Culture Collection; the C57BL / 6 male mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0053] Example 1

[0054] Evaluation of the ability of the strain to stimulate RIN-14B cells to secrete 5-hydroxytryptamine:

[0055] The occurrence of depression is related to the imbalance of monoamine neurotransmitters in the brain, mainly manifested as a decrease in the concentration of 5-HT in the synaptic cleft of brain neurons. RIN14B is derived from a rat islet tumor cell line and is commonly used as a cell for studying depression models because it highly expresses key molecular markers of endocrine cells and enterochromaffin cells.

[0056] (1) Cell culture: RIN-14B cells were cultured in a high-glucose DMEM medium containing 5% newborn fetal bovine serum and 100 μg / mL streptomycin / Penicillium at 37°C and 5% CO2. The culture medium was changed every 2 days. After the cells entered the logarithmic phase, cells with good growth were selected for passage.

[0057] (2) Cell induction: In 96-well plates, 4 × 10⁶ cells were inoculated at a concentration of 4 × 10⁶ cells / well. 5 RIN-14B cells were seeded at a density of 1 cell / mL and cultured at 37°C for 48 hours. The culture medium was then discarded. Cells were washed with 1 mL of HBSS buffer containing 0.1% BSA and 2 μM fluoxetine hydrochloride, followed by the addition of 1 mL of HBSS buffer containing 1 × 10⁻⁶ cells / mL. 9 CFU / mL live bacteria HBSS suspension (BLa80 bacterial suspension, BC179 bacterial suspension), control group only added HBSS buffer, incubated at 37℃ for 30 min, supernatant collected, centrifuged at 4℃, 10000×g for 5 min to remove precipitate, supernatant collected, 5-hydroxytryptamine (5-HT) content detected using ELISA kit.

[0058] (3) The results are as follows Figure 1 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05), it can be seen from the figure that both BLa80 strain and BC179 strain can significantly stimulate RIN14B cells to secrete 5-HT, indicating that BLa80 strain or BC179 strain has the potential to alleviate depression.

[0059] Example 2

[0060] Evaluation of the ability of bacterial agents to improve depression and anxiety in mice:

[0061] (1) Experimental animals: Male C57BL / 6 mice aged 6-8 weeks were raised in a controlled environment with a room temperature maintained at 22±1°C and a humidity of 55%±10%, following a 12-hour light / dark cycle, and were allowed to eat and drink freely. All methods used in this animal experiment were reviewed and approved by the Ethics Committee of Hubei Center for Disease Control and Prevention (Ethics Assessment Center Animal (Fujian) No. 202510304).

[0062] (2) Animal grouping: After 1 week of adaptive feeding, 90 mice were randomly divided into 9 groups (10 mice in each group): blank group (NC), model group (MC), fluoxetine hydrochloride drug group (YAO), animal bifidobacterium lactis subsp. BLa80 intervention group (BLa80), Weizmann's coagulans BC179 intervention group (BC179), commercial animal bifidobacterium lactis subsp. ATCC700541 intervention group (ATCC700541), compound group 1 (BLa80+BC179(1), live bacteria ratio of 3:1), compound group 2 (BLa80+BC179(2), live bacteria ratio of 1:3), compound group 3 (ATCC700541+BC179, live bacteria ratio of 3:1).

[0063] (3) Animal modeling and intervention methods:

[0064] The model group, drug intervention group, and various probiotic intervention groups were used to establish a mouse model of depression and anxiety by receiving chronic unpredictable mild stimulation (CUMS) for 7 weeks. CUMS included the following 10 different stimuli: forced swimming (mice were placed in a 22cm deep glass jar filled with water at 23±2°C for 10 minutes, carefully covered with a heavy lid to prevent escape, and then returned to a clean, dry cage with fresh bedding to prevent chilling and colds); restraint (mice were placed in a 50mL plastic tube, secured with plastic tape to prevent movement, with a hole at the distal end for breathing); water scarcity (water bottles were removed from the cage within 24 hours and returned after the time limit); and isolation (mice were placed alone in a new cage for 24 hours, and then transported with their companions). The following stimuli were used to create different cages: * **Return to Cage:** Food deprivation (removing food from the cage for 24 hours, then returning it); * **Wet Bedding:** placing mice alone in a new cage with 200 ml of water per 100 g of bedding for 24 hours, then returning them to their cages with their companions; * **No Bedding:** placing mice alone in a new cage without bedding for 24 hours, then returning them to their cages with their companions; * **Tilted Cage:** tilting the cage at a 45° angle for 24 hours, then returning it to its normal position; * **Tail Clamping:** clamping the tail 2 cm from the end of the tail with plastic clamps for 3 minutes at a time, 3 times a day, then returning the animal to its cage with its companions; * **Crowded Space:** placing a plastic partition in the cage, reducing the activity area by half, then removing the partition after 24 hours. To avoid animal adaptation to specific stimuli, which could affect the accuracy of the experimental results, two stimuli were randomly applied daily, and six days a week. Furthermore, to ensure the unpredictability of the experiment, each stimulus was used no more than three times throughout the entire experimental period. This method aims to create a complex and variable stress environment to induce behavioral and physiological responses in mice similar to human depression and anxiety.

[0065] Throughout the experiment, each probiotic intervention group received the same volume of probiotic solution (1×10⁻⁶) via gavage daily while maintaining routine feeding. 10 The drug group received the same volume of fluoxetine hydrochloride solution (10 mg / kg) daily via gavage while maintaining standard feeding. The control and model groups received the same volume of physiological saline daily via gavage while maintaining standard feeding. Evaluation of all mice began at week eight.

[0066] (4) Behavioral evaluation:

[0067] Depressive-like behaviors in mice were evaluated using the tail suspension test and the sucrose preference test. The tail suspension test was used to assess behavioral despair, while the sucrose preference test was used to assess anhedonia. Utilizing the contradictory psychological conflict between rodents' exploratory nature towards novel environments and their aversion to bright light and open spaces, the open field test and the elevated cruciate maze test were used to evaluate their anxiety-like behaviors.

[0068] (4.1) Open field test: The open field test can be used to evaluate depressive and anxious behaviors in animals. The specific procedure is as follows: The open field consists of a square arena (50cm × 50cm) enclosed by a continuous opaque wall made of plexiglass. Mice are placed in the center of the area facing the wall, and their movement is monitored for 10 minutes. The percentage of time the mice spend in the central area is measured. The results are as follows: Figure 2 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0069] (4.2) Elevated Cross Maze Test: The elevated cross maze test can be used to evaluate anxiety behavior in animals. The specific procedure is as follows: Mice are placed in the central area of ​​the maze, facing an open arm, and allowed to move freely for 6 minutes. The percentage of time spent in the open arm for each group is recorded. The results are as follows: Figure 3 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0070] (4.3) Tail Suspension Test: The tip of the mouse's tail (2 cm from the tail tip) was attached to a suspension bar (30 cm above the ground) with adhesive tape. The mouse was then allowed to touch the suspension. Initially, the mouse instinctively struggled violently to escape the predicament. However, if it could not escape, it would become immobile in a state of despair, exhibiting depressive-like behavior. The longer the immobility, the more depressed the mouse became. Movement was monitored using a video tracking system for 6 minutes, and the immobility time for each animal during the test was calculated (immobility was defined as movement without voluntary or escapist intent). The results are as follows: Figure 4 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0071] (4.4) Sugar Water Preference Experiment: Before the test, mice were first trained to drink sucrose for 3 days, i.e., two bottles of 1% sugar water and regular drinking water were placed in the cage, and the positions of the two bottles were changed at irregular intervals. After 3 days, the mice were fasted and deprived of water for 24 hours, and then a 1-hour sugar water test was started: two bottles of regular drinking water and 1% sucrose water of equal volume (50 mL) were placed in the mouse cage. After 1 hour, the experiment was ended, and the amount of drinking water and sucrose water consumed (g) of each group of mice was recorded, and the sugar water preference value was calculated as: (mass of sugar water consumed / total mass of liquid consumed) × 100%.

[0072] The results are as follows Figure 5As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0073] Depend on Figures 2-5 The results showed that 7 weeks of chronic stress could induce significant depressive-like behavior in mice, reflected in an increased time spent refusing to struggle and remaining still during tail suspension, as well as a reduced preference for sucrose. Compared with the model group, mice that took probiotics showed varying degrees of increased activity and sucrose preference. Among them, BLa80 had no significant difference in alleviating depressive-like behavior compared with fluoxetine hydrochloride, and the effect of BLa80 combined with BC179 was further enhanced in alleviating depressive-like behavior.

[0074] Furthermore, the model group mice spent significantly less time in the open field central region and the open arms of the cruciate maze compared to normal mice; while each probiotic group significantly restored the activity of mice in the open field central region and the open arms of the cruciate maze, and fluoxetine hydrochloride had no significant effect on anxiety-like behavior. Among them, the combination of BLa80 and BC179 showed the best effect in alleviating anxiety-like behavior.

[0075] (5) Effects on the hypothalamic-pituitary-adrenal (HPA) axis:

[0076] The hypothalamic-pituitary-adrenal (HPA) axis is a crucial component of the neuroendocrine system, involved in regulating stress responses. Environmental stress is sensed by the cerebral cortex and transmitted to the hypothalamus, which releases corticotropin-releasing factor (CRF), subsequently inducing the pituitary gland to secrete adrenocorticotropic hormone (ACTH). ACTH then circulates through the bloodstream to the adrenal glands, stimulating the secretion of glucocorticoids. Glucocorticoids can act on the hypothalamus and pituitary gland through feedback, forming a feedback regulatory loop that inhibits the synthesis and secretion of CRF and ACTH.

[0077] Blood was collected from each group of mice between 10 and 11 a.m. After centrifugation at 1200×g for 10 min, approximately 100 μL of serum was extracted and stored at -80°C for later use.

[0078] Serum adrenocorticotropic hormone (ACTH) and serum corticosterone concentrations were quantitatively determined and analyzed using an enzyme-linked immunosorbent assay kit, following the manufacturer's instructions.

[0079] The results are as follows Figure 6 and Figure 7 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0080] Depend on Figures 6-7The results showed that 7 weeks of chronic stress led to significant HPA axis hyperactivity. Adrenocorticotropic hormone (ACTH) secretion from the pituitary gland in mice was significantly elevated, resulting in a significant increase in serum corticosterone levels. BLa80 significantly alleviated HPA axis hyperactivity, inhibited excessive secretion of pituitary ACTH, and reduced serum corticosterone levels. Furthermore, the combined use of BLa80 and BC179 further enhanced the effects of inhibiting excessive pituitary ACTH secretion and reducing serum corticosterone levels.

[0081] (6) Effects on the immune status of mice:

[0082] Abnormal expression of glucocorticoid receptors in the brain impairs the negative feedback loop of the HPA axis, which in turn exacerbates the upregulation of adrenocorticotropic hormone (ACTH) and corticosterone levels. This phenomenon is known as "glucocorticoid resistance" and has been demonstrated by numerous clinical studies. Glucocorticoid resistance leads to decreased sensitivity of the body's immune cells to glucocorticoids, resulting in a compensatory increase in the level of inflammatory response.

[0083] Blood was collected from each group of mice between 10 and 11 a.m. After centrifugation at 1200×g for 10 min, approximately 100 μL of serum was extracted and stored at -80°C for later use.

[0084] The levels of the pro-inflammatory factor TNF-α and the anti-inflammatory factor IL-10 in serum were quantitatively determined using an enzyme-linked immunosorbent assay kit, following the manufacturer's instructions.

[0085] The results are as follows Figure 8 and Figure 9 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0086] Depend on Figures 8-9 The results showed that the serum levels of the pro-inflammatory factor TNF-α were significantly elevated in depressed mice, and this phenomenon could be reversed after administration of BLa80, with the combined group 1 and combined group 2 showing better effects. Furthermore, the serum levels of the anti-inflammatory factor IL-10 were significantly decreased in depressed mice, and the levels were increased to varying degrees after administration of each group of bacterial agents, with the BLa80 group, combined group 1, and combined group 2 showing the best effects. This indicates that BLa80 alleviates the inflammatory response in depressed mice by inhibiting the levels of pro-inflammatory factors and increasing the levels of anti-inflammatory factors, and the above effects were further enhanced when BLa80 was used in combination with BC179.

[0087] (7) Effects on the brain's BDNF pathway:

[0088] Brain-derived neurotrophic factor (BDNF) is a key protein regulated by the PKA-CREB pathway and widely distributed in the limbic nervous system. BDNF deficiency is highly correlated with the risk of depression, leading to a series of physiological abnormalities such as cognitive deficits, helplessness, and anhedonia. Numerous studies have shown that serum BDNF levels are significantly reduced in patients with depression, and some patients also exhibit symptoms such as hippocampal atrophy. Therefore, BDNF has become a key target for alleviating and treating depression.

[0089] Mice that underwent ocular blood collection were euthanized by dislocation after carbon dioxide treatment. Brain tissue was rapidly dissected, and intact hippocampal tissue was extracted. After tissue processing, pre-cooled PBS was added for homogenization. The homogenate was collected and centrifuged at 10000×g for 10 min at 4°C. The supernatant was collected and stored at -80°C for later use.

[0090] The level of BDNF in hippocampal tissue was determined using an enzyme-linked immunosorbent assay kit, following the manufacturer's instructions.

[0091] The results are as follows Figure 10 As shown in the figure (different letters in the figure represent significant differences between groups, p < 0.05).

[0092] Depend on Figure 10 The results showed that, compared with the NC group, the level of BDNF in the hippocampus of depressed mice was lower than that of healthy mice. BLa80 intervention increased BDNF expression, and its effect was similar to that of the drug group fluoxetine hydrochloride (YAO). Furthermore, the combination of BLa80 and BC179 further increased BDNF expression.

[0093] (8) Effects on the diversity of gut microbiota in mice:

[0094] Mice were euthanized, and after abdominal disinfection and dissection, cecal contents were collected from mice in the control group (NC), model group (MC), fluoxetine hydrochloride drug group (YAO), and Bifidobacterium lactis subsp. Lactans intervention group (BLa80) for microbial diversity analysis. The 16S rDNA V3-V4 hypervariable regions of all bacteria in the samples were sequenced on the MiSeq Illumina sequencing platform to determine the characteristics of the gut microbiota.

[0095] Alpha diversity reflects the richness and diversity of species in a sample. The Simpson diversity index is a commonly used indicator for assessing the abundance and diversity of gut microbiota. The results are as follows: Figure 11As shown, the results indicate that, compared with the NC group, the MC group mice exhibited a higher Simpson diversity index in their gut microbiota, indicating a significant decrease in intraspecific microbiota richness. Compared with the MC group, the BLa80 group mice showed a significantly increased Simpson diversity index in their gut microbiota, approaching that of the NC group. This result suggests that BLa80 intervention can reverse the decrease in microbiota richness in depressed mice.

[0096] β-diversity is used to compare the similarity of different samples in terms of species diversity. NMDS is a common analytical method that reflects the differences and distances between samples. When Stress is less than 0.2, it indicates that NMDS analysis has a certain degree of reliability; samples that are closer together on the coordinate graph have higher similarity. Results for each group are as follows: Figure 12 As shown, BLa80 intervention effectively improved the gut microbiota of depressed mice, bringing them closer to the healthy group.

[0097] To further investigate the effects of BLa80 intervention on the beneficial gut microbiota of depressed mice, the abundance of beneficial species in different treatment groups was statistically analyzed. The relative abundance of *Bifidobacterium* and unnamed genera of *Prevotella* in the gut of mice in each group is shown below. Figure 13 and Figure 14 As shown, the abundance of beneficial bacteria genus Bifidobacterium in the intestines of mice in the BLa80 group was significantly increased, while the abundance of conditionally pathogenic bacteria genus norank-Prevotellaceae was significantly decreased. This indicates that the animal Bifidobacterium lactis subsp. BLa80 strain can effectively colonize the mouse intestines and promote the increase of other beneficial bacteria in the intestines, thus regulating the body's health.

[0098] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0099] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0100] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of Bifidobacterium animalis subsp. lactis BLa80 in the preparation of formulations to relieve depression or anxiety; The Bifidobacterium animalis subsp. lactis strain BLa80 is the Bifidobacterium animalis subsp. lactis strain BLa80 with the preservation number CGMCC No. 22547.

2. The application according to claim 1, characterized in that, The viable bacteria count in the preparation is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

3. The application according to claim 1, characterized in that, The dosage forms of the preparation include solutions, powders, tablets, capsules, or granules.

4. The application according to claim 1, characterized in that, The formulation also contains excipients; the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent or buffer.

5. The application according to claim 1, characterized in that, The formulation alleviates HPA axis hyperfunction, relieves inflammatory response, increases the expression level of brain-derived neurotrophic factor in the hippocampus, increases the abundance of beneficial intestinal flora, and increases intestinal flora diversity.

6. The application according to claim 1, characterized in that, The strain in the preparation also includes Weizmannia coagulans strain BC179; the Weizmannia coagulans strain BC179 is the Weizmannia coagulans strain with accession number CGMCC No. 33768.

7. The application according to claim 6, characterized in that, The ratio of viable cells of the BLa80 strain to the BC179 strain is 1:3 to 3:

1.

8. A probiotic composition for relieving depression or anxiety, characterized in that, The probiotic composition contains strains including Bifidobacterium animalis subsp. lactis BLa80 strain with accession number CGMCC No. 22547 and Weizmannia coagulans BC179 strain with accession number CGMCC No. 33768.

9. The probiotic composition according to claim 8, characterized in that, The probiotic composition contains at least 1×10⁻⁶ live bacteria. 9 CFU / mL or 1×10 9 CFU / g.

10. The probiotic composition according to claim 8, characterized in that, The ratio of viable cells of the BLa80 strain to the BC179 strain is 1:3 to 3:1.

Images