Probiotic composition for improving mood as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to a probiotic composition capable of improving emotion and a preparation method and application of the probiotic composition capable of improving emotion. The composition comprises bifidobacterium pseudolongum, Adellomyces aldelii, microbacter cecum, streptococcus thermophilus, Ackermann mucophilus and intestinal pasteurella, and the probiotic composition capable of improving emotion and the preparation method and application of the probiotic composition capable of improving emotion and the preparation method and application of the probiotic composition capable of improving emotion and the probiotic composition capable of improving emotion and the probiotic composition capable of improving emotion and the probiotic composition capable of improving emotion. The obtained probiotic composition has a remarkable improvement effect on animal depression and anxiety behaviors caused by chronic social contusion stress, and the technical problem that in the prior art, depression drugs cannot target intestinal flora metabolite regulation and accurate intervention of neurotransmitter balance at the same time is solved. The probiotic composition can quickly and enduringly improve depression and anxiety behaviors caused by chronic stress, has better safety, and can be used for preventing and treating depression and related mental diseases thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and more specifically, relates to a probiotic composition for improving mood, and a preparation method and application thereof. Background Art

[0002] Depression is a global mental health problem, affecting approximately 3.8% of the world's population and projected to become the leading cause of disability worldwide by 2030. Major depressive disorder (MDD) is characterized by persistent low mood, loss of interest or pleasure in previously pleasurable activities, recurrent thoughts of death, and physical and cognitive symptoms, which severely impair patients' physical and mental health and quality of life.

[0003] MDD poses a serious threat to human health, yet our understanding of its pathogenesis remains limited. However, with the rapid advancement of diagnostic technologies, a large number of studies in recent years have revealed a crucial role for the gut-brain axis (GBA) in the development and progression of MDD. Neuroscience research suggests that intestinal microbes, or the gut microbiota, can influence and regulate nervous system function through their metabolites. However, current microbial therapies for depression (including probiotics and metabolite interventions) have been less than ideal. For example, mixed preparations of Lactobacillus and Bifidobacterium can partially alleviate depressive symptoms, but these lack strain specificity and are associated with inconsistent efficacy. Furthermore, while metabolites such as short-chain fatty acids (SCFAs) can modulate neuroinflammation, their poor ability to penetrate the blood-brain barrier significantly limits their effects on the central nervous system. Consequently, there is a lack of precise interventions that simultaneously target gut microbiota regulation and neurotransmitter homeostasis. Summary of the Invention

[0004] In response to the above-mentioned defects or improvement needs of the prior art, the present invention provides a probiotic composition for improving mood, a preparation method and application thereof, thereby solving the technical problem in the prior art that depression drugs cannot simultaneously target the precise intervention of intestinal flora metabolite regulation and neurotransmitter balance.

[0005] To achieve the above objectives, according to one aspect of the present invention, a mood-improving probiotic composition is provided, comprising Bifidobacterium pseudolongum, Adlercreutzia equolifaciens, Parvibacter caecicola, Streptococcus thermophilus, Akkermansia muciniphila, and Barnesiella intestinihominis.

[0006] Preferably, in each milliliter of the probiotic composition, the viable counts of Bifidobacterium pseudolongum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica are all 1×10 9 CFU~1×10 12 CFU.

[0007] Preferably, the weight ratio of Bifidobacterium pseudolongum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica is (1.5-2.5): (0.5-1.5): (0.5-1.5): 1:1: (0.5-1.5).

[0008] Preferably, the probiotic composition further comprises a prebiotic excipient; preferably, the prebiotic excipient is one or more of galacto-oligosaccharide, fructo-oligosaccharide and inulin.

[0009] Preferably, the probiotic composition further comprises at least one of a carrier, an excipient, a diluent or a vehicle.

[0010] According to another aspect of the present invention, a method for preparing the mood-enhancing probiotic composition is provided, comprising resuspending Bifidobacterium pseudolongum, Kreutzeria adlerae, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica in sterile phosphate buffer and mixing the suspensions to obtain the probiotic composition.

[0011] According to another aspect of the present invention, there is provided a use of the mood-improving probiotic composition in the preparation of a medicine or functional food for preventing or treating depression.

[0012] Preferably, the drug or functional food is administered orally or by gavage.

[0013] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0014] (1) The probiotic composition provided by the present invention is prepared from Bifidobacterium pseudolongum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica in a specific ratio. Animal experiments have shown that the resulting probiotic composition significantly improves depression and anxiety behaviors in animals induced by chronic social defeat stress.

[0015] (2) The present invention has been experimentally verified to find that the prepared probiotic composition can effectively improve the depressive and anxious behaviors of mice caused by chronic social frustration stress, and the formula ratio of the probiotic composition has been determined. The present invention also explored the mechanism of action of the probiotic composition in improving the depressive and anxious behaviors of mice caused by chronic social frustration stress through animal experiments. The results showed that the probiotic composition can promote the production of intestinal flora metabolite vitamin K2, which enters the brain through the blood circulation and increases the levels of neurotransmitters 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) through the gut-brain axis, quickly and persistently improving the depressive and anxious behaviors caused by chronic stress. It has good safety and can be used for the prevention and treatment of depression and related mental illnesses.

[0016] The typical mechanism of action of vitamin K is through vitamin K-dependent proteins (VKDPs). VKDPs rely on vitamin K for γ-glutamyl carboxylation, a post-translational modification of proteins necessary for their biological activity. Although traditionally believed to play a role primarily in blood coagulation, vitamin K2 is crucial for maintaining brain health and cognitive function. Endogenous vitamin K2 is primarily produced by intestinal flora. Therefore, the present invention improves depression and anxiety symptoms by increasing vitamin K2 content to affect neurotransmitter levels in the brain.

[0017] (3) The raw materials of the probiotic composition provided by the present invention are safe and healthy, without toxic side effects. By regulating the metabolites of intestinal flora, it acts on the gut-brain axis to improve behavioral disorders such as stress-related depression, thereby solving the problems existing in the prevention and treatment of depression in the existing technology, providing theoretical and technical support for clinical prevention and treatment applications, and suggesting the wide application of probiotic compositions in the preparation of neuroprotective drugs, pharmaceutical compositions or functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This figure shows the test results of the effect of the probiotic composition on the social behavior and sugar water preference behavior of mice subjected to chronic social frustration stress, wherein A: social contact time of the social contact experiment; B: social contact ratio of the social contact experiment; C: sugar water consumption of the sugar water preference experiment; D: sugar water preference rate of the sugar water preference experiment.

[0019] Figure 2The graph shows the test results of the effect of the probiotic composition on the despair behavior of mice induced by chronic social defeat stress, wherein A: immobility time in the tail suspension test; B: immobility time in the forced swimming test.

[0020] Figure 3 This figure shows the test results of the effect of the probiotic composition on the anxiety behavior of mice induced by chronic social frustration stress, wherein: A: open arm time in the elevated plus maze test; B: number of entries into the open arm in the elevated plus maze test; C: central zone time in the open field test; D: total movement distance in the open field test.

[0021] Figure 4 This figure shows the test results of the effect of the probiotic composition on the intestinal flora metabolite vitamin K2 and brain neurotransmitters in mice with chronic social frustration stress, where A: the content of vitamin K2 in the intestinal contents of mice; B: the content of serotonin (5-HT) in the brain tissue of mice; C: the content of glutamate (Glu) in the brain tissue of mice; D: the content of γ-aminobutyric acid (GABA) in the brain tissue of mice; E: the content of dopamine (DA) in the brain tissue of mice; F: the content of norepinephrine (NE) in the brain tissue of mice. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0023] In the following examples, standard strains of Bifidobacterium pseudolongum, Adlercreutzia equolifaciens, Parvibacter caecicola, Streptococcus thermophilus, Akkermansia muciniphila, and Barnesiella intestinihominis were all provided by Ningbo Mingzhou Biotechnology Co., Ltd. Unless otherwise specified, other reagents, instruments, and equipment used in the present invention, for which the manufacturer is not indicated, can be purchased through regular channels or can be prepared by existing methods.

[0024] In the following examples, all data are expressed as mean ± standard error (SEM). GraphPad Prism 8 software was used to analyze the experimental data. Comparisons of multiple group means were performed using one-way analysis of variance, followed by Bonferroni's post hoc analysis. P < 0.05 indicates a statistically significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0025] Example 1

[0026] The present invention provides a probiotic composition for improving mood and a preparation method thereof.

[0027] The probiotic strains Bifidobacterium pseudolongum, Kreutzeria adlerii, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica were cultured in Lactobacillus delbrueckii (MRS) broth (Ningbo Mingzhou Biotechnology Co., Ltd.) at 37°C and shaken at 1000 rpm for 48 h. Bacillus cecum was cultured in chocolate agar medium (Ningbo Mingzhou Biotechnology Co., Ltd.) at 37°C and shaken at 1000 rpm for 48 h. All strains were subcultured three times before the experiment. The bacteria were collected by centrifugation at 8000 rpm for 2 min, washed three times with sterile phosphate buffer, and centrifuged at 8000 rpm for 2 min to remove the supernatant, and finally 10 per ml. 9 The colony-forming unit concentration was resuspended in sterile phosphate buffered saline. The bacterial suspensions of Bifidobacterium pseudolongum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica were added with 1% trehalose, 0.5% sodium glutamate, 1% skim milk, 0.25% ascorbic acid, and 0.25% dextran at a weight ratio of 2:1:1:1:1:1 / 100 mL distilled water, mixed well, and set aside.

[0028] Example 2

[0029] This example provides a construction of a chronic social defeat stress (CSDS) depression animal model and a treatment experimental protocol

[0030] 1. Experimental Animals

[0031] Male C57BL / 6J mice, 7–8 weeks old, weighing 20–22 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.

[0032] 2. Rearing conditions

[0033] All mice were housed in a standard laboratory animal environment (temperature: 22 ± 2 ° C; humidity: 50% ± 10%; light cycle: 12h / 12h) with free access to food and water. Mice were fed a standard diet containing 13.8% of calories from fat, 22.8% of calories from protein, and 63.4% of calories from carbohydrates (laboratory mouse maintenance diet from Jiangsu Xietong Company, China). Before establishing the model, mice were housed for 2 weeks to acclimate to the environment. The use of animals and experimental protocols involved in the experiment complied with the animal use standards of the International Committee for Assessment and Review of Laboratory Animal Care.

[0034] 3. Animal grouping

[0035] C57BL / 6J male mice were randomly divided into 3 groups: a control group, a model group, and a model group treated with a probiotic composition (treatment group), with 10 mice in each group. All mice were housed in single cages, and all other housing conditions were the same.

[0036] 4. Construction of the Chronic Social Frustration Stress Model

[0037] The chronic social defeat stress (CSDS) model in mice is one of the most commonly used preclinical models of stress-induced depression. CSDS induces depressive-like behaviors in rodents, including social avoidance, loss of preference for sucrose (i.e., anhedonia), and increased reward threshold. The specific experimental procedures are as follows:

[0038] (1) Screening of CD1 mice with appropriate aggressiveness: CD1 mice, 8-10 months old, weighing 25-32 g, were purchased from Beijing Weitonglihua Company. The criteria for "appropriate aggressiveness" were that the latency period of the first attack was less than 1 min and the number of attacks was greater than 3.

[0039] (2) Modeling phase: C57BL / 6J mice were subjected to social defeat by being challenged by different CD1 mice every day for 10 consecutive days (5-10 min). The resident mice were CD1 mice, and the C57BL / 6J mice, as intruder mice, were placed in a new CD1 compartment every day to be challenged. At the end of the social defeat phase, the resident CD1 mice and the intruder C57BL / 6J mice were placed on opposite sides of the cage, separated by a perforated transparent plastic plate, to allow visual, olfactory, and auditory contact for the remainder of the 24 h.

[0040] (3) All C57BL / 6J mice were housed in single cages 24 h after the last social defeat and underwent subsequent behavioral testing.

[0041] 5. Treatment with probiotic compositions

[0042] Bifidobacterium pseudolongum, Kreutzeria adlerii, Bacillus cecum, Streptococcus thermophilus, Akkermansia muciniphila and Pasteurella enterica were prepared at a weight ratio of 2:1:1:1:1:1 to a concentration of 1×10 9 A probiotic composition solution containing 100 CFU / ml of probiotic composition was prepared using sterile phosphate buffered saline as a solvent, aliquoted and refrigerated at 4°C for oral gavage of mice. After the CSDS model was completed, the mice were gavaged with 200 μL of the probiotic composition solution for 10 consecutive days according to the above grouping.

[0043] Example 3

[0044] This example investigates changes in social behavior and sucrose preference in CSDS model mice following administration of the probiotic composition obtained in Example 2. Behavioral changes in social contact and sucrose preference tests were tested in CSDS model mice gavaged with the probiotic composition to assess the efficacy of the probiotic composition on the social and hedonic behaviors of CSDS model mice.

[0045] 1. Behavioral Detection Method in This Example

[0046] (1) Social contact experiment:

[0047] CSDS can induce persistent social avoidance behavior in mice exposed to unfamiliar environments. The social contact test apparatus consists of a white plastic open box (40 cm long, wide, and high) and a transparent rectangular Plexiglas basket (10 cm long, 6 cm wide, and 40 cm high). The bottom of the Plexiglas basket is sealed with wire mesh and has dense ventilation holes, ensuring that C57BL / 6J mice are protected from attack by CD1 mice while allowing them to interact with and sniff the CD1 mice. The social contact test consists of two phases. The first phase is the CD1-free phase: an empty Plexiglas basket is placed at one end of the open field. The second phase is the CD1-free phase: except for the placement of a CD1 in the Plexiglas basket, all other conditions are identical to the first phase, and the test duration is 150 seconds. The social contact ratio is calculated as the social contact time (CD1-free phase) divided by the social contact time (CD1-free phase). A social contact ratio less than 1 indicates that the mice exhibit depressive-like behaviors associated with social avoidance.

[0048] (2) Sugar water preference experiment:

[0049] Twenty-four hours after the mice were exposed to stress, a sugar water preference test was used to observe their sugar water preference-like behavior. Sugar water acclimation training was conducted 72 hours prior to the experiment. Two water bottles were placed in each cage: one containing a 1% sucrose solution and the other containing tap water. The positions of the two water bottles were rotated every 12 hours. After acclimation to the sugar water, the mice were deprived of water for 12 hours. Following the sugar water preference test, each cage was given one bottle containing 1% sugar water and one bottle containing tap water, with free access to water. After 12 hours, sugar water and tap water consumption were measured, and the sugar water preference rate was calculated using the formula: Sugar water preference rate = sugar water consumption / (sugar water consumption + tap water consumption) × 100%. A sugar water preference rate of less than 75% was considered to indicate that the mice exhibited anhedonic, depressive-like behavior.

[0050] 2. Experimental results

[0051] The results are as follows Figure 1 As shown in Figure A, the results of the social contact experiment showed that compared with the control group mice, the CSDS model group mice spent significantly less time socializing with the unfamiliar CD1 mice in the social contact experiment, with a very significant difference between the groups (P < 0.01). However, after oral administration of the probiotic composition of the present invention, the CSDS model group mice (i.e., the treatment group) spent significantly more time socializing with the CD1 mice (P < 0.01). Figure 1 As shown in Figure B, the social contact ratio of the CSDS model group mice was also significantly lower than that of the control group mice, and this ratio increased after treatment with the probiotic composition. Figure 1 As can be seen from Figure C, the results of the sugar water preference experiment showed that compared with the control group mice, the sugar water consumption of the CSDS model group mice was significantly reduced, and there was a significant difference between the groups (P < 0.01). After oral administration of the probiotic composition of the present invention, the sugar water consumption of the CSDS model group mice (i.e., the treatment group) was significantly increased (P < 0.01); at the same time, Figure 1 As can be seen in Figure D, the CSDS model group mice also had a significantly lower sugar water preference rate than the control group mice, while the sugar water preference rate of mice treated with the probiotic composition of the present invention increased. This indicates that the probiotic composition of the present invention can improve the social avoidance behavior and anhedonia behavior of CSDS model mice.

[0052] Example 4

[0053] Example 4 investigated the effect of the probiotic composition on despair-related behavioral changes in CSDS model mice. Behavioral changes in the tail suspension test and forced swim test were tested in CSDS model mice gavaged with the probiotic composition to evaluate the intervention effect of the probiotic composition on despair-related behaviors in CSDS model mice.

[0054] 1. Behavioral Detection Method in This Example

[0055] (1) Tail suspension test:

[0056] The tail suspension test is one of the most commonly used assays for studying depressive-like behaviors in rodents and evaluating the efficacy of antidepressants. In this test, mice are placed under mild, unavoidable stress, and the duration of their immobility reflects their level of despair. Before the experiment begins, mice are placed in a behavioral chamber to acclimate to the testing environment for 1 hour. The mice are suspended with their noses 20 cm above the ground. The animals are then recorded for 6 minutes using the ANY-maze animal behavior analysis system. The mice are then removed and returned to their original cages. The duration of immobility during the 6-minute period is calculated as the immobility time in the tail suspension test.

[0057] (2) Forced swimming test:

[0058] The forced swim test, similar to the tail suspension test, is used to assess desperation behavior in mice. Before the experiment begins, mice are placed in a behavioral chamber to acclimate to the testing environment for 1 hour. The test is conducted in a transparent glass cylinder filled with water. A timer begins when the mouse is gently placed in the cylinder, and the video is recorded for 6 minutes using the ANY-maze animal behavior analysis system. At the end of the test, the mouse is removed and returned to its home cage. The immobility time during the final 4 minutes is calculated as the immobility time for the forced swim test.

[0059] 2. Experimental results

[0060] The results are as follows Figure 2 As shown, from Figure 2 As shown in Figure A (tail suspension behavior), the immobility time of the CSDS model group mice in the tail suspension test was significantly longer than that of the control group mice, and the immobility time of the CSDS model group mice (i.e., the treatment group) after oral administration of the probiotic composition in the tail suspension test was significantly reduced (P < 0.01); Figure 2 As can be seen from Figure B (forced swimming behavior), the immobility time of the CSDS model group mice in the forced swimming test was significantly increased compared with the mice in the control group. The immobility time of the CSDS model group mice (i.e., the treatment group) after treatment with the probiotic composition was significantly reduced (P < 0.05), indicating that stress promotes the despair behavior of mice, and the probiotic composition product of the present invention can improve this depressive behavior to a certain extent.

[0061] Example 5

[0062] Example 5 investigates the effect of the probiotic composition on anxiety behavior changes in CSDS model mice.

[0063] The behavioral changes of CSDS model mice gavaged with the probiotic composition in the elevated plus maze test and the open field test were tested to evaluate the intervention effect of the probiotic composition on the anxiety behavior and motor ability of CSDS model mice.

[0064] 1. Behavioral Detection Method in This Example

[0065] (1) Elevated plus maze test:

[0066] The elevated plus maze test is a method for assessing anxiety in rodents. It exploits the animals' exploratory nature toward novel environments and their fear of suspended, open arms, creating a conflict and assessing their anxiety. The apparatus consists of a cross-shaped area with two arms (open and closed), each measuring 30 cm × 5 cm, and a blank area (10 cm × 10 cm) between them. The closed arm is 15 cm high, and the experimental area is maintained at 60 cm above the ground. A camera is positioned directly above the apparatus to observe the movements of the mice. Each mouse is placed in the same position on the central platform upon entry. The mouse is allowed to move freely within the maze for 5 minutes. An entry into either an open or closed arm is considered complete when all four limbs are within the arms. Video recording is performed using the ANY-maze animal behavior analysis system. The mice are then removed and returned to their original cages. The number and duration of entries into the open arms within 5 minutes are recorded.

[0067] (2) Open field test:

[0068] The open field test can reflect indicators such as mice's anxiety level and motor ability. Mice were placed in a white plastic open box (45 cm long, wide, and high). The experiment was conducted using Anymaze video monitoring and recording software. The room was kept dimly lit. The time the freely moving mice spent in the center of the open box (22.5 cm long and 22.5 cm wide) was recorded over a 6-minute period. The distance the mice traveled and the time they spent in the central area were also recorded. After each experiment, the mice's excrement was promptly cleaned and the box was wiped with 75% alcohol.

[0069] 2. Experimental results

[0070] The results are as follows Figure 3 As shown, from Figure 3 As can be seen from Figure A, the open arm time of the CSDS model group mice in the elevated plus maze experiment was significantly reduced compared with the control group mice. The open arm time of the CSDS model group mice (i.e., the treatment group) after oral administration of the probiotic composition in the elevated plus maze was significantly increased (P < 0.05). Figure 3 As can be seen in Figure B, the CSDS model group mice entered the open arms significantly less frequently than the control group mice in the elevated plus maze test. After treatment with the probiotic composition, the CSDS model group mice (i.e., the treatment group) entered the open arms significantly more frequently (P < 0.01). Figure 3As shown in Figure C, in the open field experiment, the movement time of the model group mice in the central area of ​​the open field was shorter than that of the control group mice. After treatment with the probiotic composition, the movement time of the mice in the central area was increased (treatment group); Figure 3 As can be seen from Figure D, there was no significant difference in the movement distance of mice in the open field test between different groups, indicating that chronic stress can cause anxiety behavior in mice, and the probiotic composition product of the present invention can improve this anxiety behavior without affecting the movement ability of mice.

[0071] Example 6

[0072] Example 6 investigates the effect of the probiotic composition on the changes in vitamin K2 levels in the intestinal contents of CSDS model mice.

[0073] Because intestinal microorganisms can affect immune function, neuroendocrine regulation and other processes through microbial metabolites, and then affect neural development and function through the gut-brain axis, ultimately leading to depression-anxiety-like behaviors. The intestinal flora in normal organisms is in a steady state. When stimulated by stress, the structural composition of the intestinal microbiome will change, resulting in an imbalance in intestinal flora metabolites, which in turn induces depression-anxiety behaviors through the gut-brain axis. We first discovered that the intestinal flora that can synthesize vitamin K2 in the intestines of CSDS model mice is reduced compared to the control group mice. Therefore, we next analyzed the vitamin K2 in the intestinal contents of CSDS model mice to determine whether the probiotic composition intervenes in depression-anxiety-like behaviors by affecting intestinal flora metabolites. The specific experimental methods are as follows:

[0074] 1. Intestinal content sample collection: After mice were anesthetized with intraperitoneal injection of chloral hydrate, the entire intestine was removed. The intestinal surface was cleaned in a clean bench, and the colon was excised using a sterile scalpel. Next, intestinal content samples were collected using a sterile scalpel and placed in a sterile centrifuge tube. The tubes were quickly frozen in liquid nitrogen and stored in a -80°C freezer.

[0075] 2. Enzyme-linked immunosorbent assay (ELISA) to detect vitamin K2 levels in intestinal contents

[0076] (1) Sample pretreatment: The colon and intestinal contents of each group of mice were accurately weighed, added to pre-cooled cell culture buffer at a mass-to-volume ratio of 1:9, fully ground, and centrifuged to aliquot the supernatant.

[0077] (2) Prepare antibodies, washing buffer, and standards according to the instructions of the vitamin K2 ELISA kit;

[0078] (3) Sample addition: Add 50 μL of sample to the blank wells, standard wells, and sample wells according to their arrangement order. Incubate at 37°C for 1 h. Wash the plate with 300 μL of washing solution and pat dry. Repeat this cycle 5 times.

[0079] (4) Adding antibodies: dilute the antibodies with antibody diluent, add 100 μL to each well, incubate at 4°C for 1 h, add 300 μL of washing solution, wash the plate 5 times, pat dry, and repeat 5 times;

[0080] (5) Color development: Add 60 μL of color development solution to each well and incubate at 37°C for 30 min;

[0081] (6) Stop reaction: Add stop solution to each well and read the result using a microplate reader.

[0082] 3. Experimental results

[0083] The results are as follows Figure 4 As shown, from Figure 4 As can be seen from Figure A, the level of vitamin K2 in the intestinal contents of the CSDS model group mice was significantly lower than that of the control group mice. The level of vitamin K2 in the intestinal contents of the CSDS model group mice (treatment group) after oral gavage with the probiotic composition was significantly increased (P < 0.01), indicating that stress can cause changes in the vitamin K2 metabolites produced by intestinal microorganisms in mice, and the probiotic composition of the present invention can reverse this change and may play a role in improving depression and anxiety through the gut-brain axis.

[0084] Example 7

[0085] Example 7 investigates the effects of the probiotic composition on changes in brain neurotransmitter levels in CSDS model mice.

[0086] Existing literature confirms that intestinal flora metabolites can affect the levels of neurotransmitters in the animal brain, thereby leading to the occurrence and development of depression. Among them, reports have found that vitamin K2 may be related to the synthesis or metabolism of monoamine neurotransmitters, and that glutamine synthetase in astrocytes in the central nervous system can convert ammonia into glutamine, which is a precursor for the synthesis of neurotransmitters glutamate and GABA in the brain. Therefore, the neurotransmitters in the brain tissue of CSDS model mice were analyzed to determine whether the probiotic composition can affect neurotransmitter levels through intestinal flora metabolites and thus intervene in depressive and anxiety-like behaviors. The specific experimental methods are as follows:

[0087] 1. Experimental Methods: ELISA kits were used according to the instructions to detect the levels of serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), dopamine (DA), and norepinephrine (NE) in mouse brain tissue samples. The ELISA kits used used a double antibody sandwich method. The specific operation method is as follows:

[0088] (1) Sample addition: Set up a blank control group, a standard group, and a sample group. Add 50 μL of the standard at different concentrations to the standard wells. First, add 40 μL of the sample diluent to the sample wells, followed by 10 μL of the sample to be tested. No sample or enzyme-labeled reagent is required to the blank control wells.

[0089] (2) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank well. Seal the plate with sealing film and incubate at 37°C for 60 min.

[0090] (3) Washing: Remove the sealing film, discard the liquid, and spin dry. Then, fill each well with washing solution, let it stand for 30 seconds, and then discard it. Repeat this step 5 times and pat dry.

[0091] (4) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, and gently shake to mix. Incubate at 37°C in the dark for 15 min.

[0092] (5) Measurement: Add 50 μL of stop solution to each well to terminate the reaction. The blue color immediately turns yellow. Measure the absorbance at 450 nm.

[0093] 2. Experimental results

[0094] The results are as follows Figure 4 As shown, from Figure 4 As can be seen from Figure B, the 5-HT in the brains of the model group mice was significantly reduced compared with the control group mice, and the 5-HT in the brains of the mice treated with the probiotic composition (treatment group) was significantly increased compared with the model group mice (P < 0.01); Figure 4 As can be seen from Figure C, the probiotic composition treatment did not affect the level of glutamate in the mouse brain tissue; Figure 4 As can be seen from Figure D, the GABA in the brains of the model group mice was significantly reduced compared with the control group mice, and the GABA in the brains of the mice treated with the probiotic composition (treatment group) was significantly increased compared with the model group mice (P < 0.001); Figure 4 Zhong E and Figure 4 As can be seen from Figure F, the probiotic composition treatment did not affect the levels of DA and NE in mice; the above test results indicate that chronic stress can cause a decrease in 5-HT and GABA in the mouse brain, and the probiotic composition of the present invention can improve depression and anxiety behaviors by increasing 5-HT and GABA.

[0095] In summary, the probiotic composition can promote the expression of vitamin K2 and ammonia in intestinal metabolites, increase the levels of neurotransmitters 5-HT and GABA in the brain, and have a certain therapeutic effect on depressive symptoms caused by chronic stress.

[0096] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0097] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A probiotic composition for improving mood, characterized in that: The composition comprises Bifidobacterium pseudolongum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila and Pasteurella enterica.

2. A mood-enhancing probiotic composition according to claim 1, characterized in that: In each milliliter of the probiotic composition, the number of viable bacteria of Bifidobacterium pseudolongum, Kreutzeria adlerii, Bacillus cecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica is 1×10 9 CFU~1×10 12 CFU.

3. The mood-enhancing probiotic composition according to claim 1, characterized in that: The weight ratio of the pseudo-Bifidobacterium longum, Kreutzeria adlerii, Microbacterium caecum, Streptococcus thermophilus, Akkermansia muciniphila and Pasteurella intestinalis is (1.5-2.5): (0.5-1.5): (0.5-1.5): 1:1: (0.5-1.5).

4. The mood-enhancing probiotic composition according to claim 1, wherein: The probiotic composition further comprises a prebiotic excipient; preferably, the prebiotic excipient is one or more of galacto-oligosaccharide, fructo-oligosaccharide and inulin.

5. A mood-enhancing probiotic composition according to claim 4, characterized in that: The probiotic composition further comprises at least one of a carrier, an excipient, a diluent or a vehicle.

6. A method for preparing the mood-enhancing probiotic composition according to any one of claims 1 to 5, characterized in that: Bifidobacterium pseudolongum, Kreutzeria adlerii, Bacillus cecum, Streptococcus thermophilus, Akkermansia muciniphila, and Pasteurella enterica are resuspended in sterile phosphate buffer and mixed to obtain a probiotic composition.

7. Use of the mood-enhancing probiotic composition according to any one of claims 1 to 5 in the preparation of a medicine or functional food for preventing or treating depression.

8. Use of the probiotic composition product according to claim 7 in the preparation of a medicine or functional food for preventing or treating depression, characterized in that: The medicine or functional food is administered orally or by gavage.

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