Bifidobacterium animalis subsp. Lactis CBTL010 and application thereof in products for improving intestinal flora and relieving depression and anxiety

Abstract

The invention relates to bifidobacterium animalis subsp. Lactis CBTL010 and application thereof in products for improving intestinal flora and relieving depression and anxiety, and belongs to the technical field of microorganisms. The bifidobacterium animalis subsp. Lactis CBTL010 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation time is March 20, 2025, the preservation number is CGMCC NO.33906, and the bifidobacterium animalis subsp. Lactis CBTL010 has the effects of improving intestinal flora and relieving depression and anxiety.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology and relates to an animal Bifidobacterium lactis subspecies CBTL010 and its application in products for improving intestinal flora and alleviating depression and anxiety. Background Art

[0002] The intestine is one of the important organs of the body, and intestinal health plays a vital role in the body. If long-term mental and emotional stress causes anxiety and depression, it can easily cause physical discomfort, especially cause a decline in intestinal function, and have a certain impact on the mental and physical health of the human body through the neural pathways of the microbiome-gut-brain axis.

[0003] Currently, the first-line treatments for depression and anxiety mainly include selective serotonin reuptake inhibitors (SSRIs), benzodiazepines Although these drugs have clear clinical efficacy, they have significant disadvantages: such as significant side effects, delayed efficacy, and easy damage to intestinal flora.

[0004] Therefore, there is an urgent need for a probiotic that can improve intestinal flora and relieve depression and anxiety, and to achieve better therapeutic effects by utilizing the natural, safe and microecological regulatory properties of probiotic therapy. Summary of the Invention

[0005] The present invention aims to provide an animal Bifidobacterium lactis subspecies CBTL010 and its application in products for improving intestinal flora and alleviating depression and anxiety.

[0006] On the one hand, the present invention provides an animal Bifidobacterium lactis subsp. CBTL010, which is isolated from fermented food. The animal Bifidobacterium lactis subsp. CBTL010 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a preservation date of March 20, 2025, a preservation number of CGMCCNO.33906, and a classification name of Bifidobacterium animalis subsp. lactis.

[0007] Furthermore, the modified MRS medium components involved in the cultivation process of the animal Bifidobacterium lactis subspecies CBTL010 include: by mass percentage, 2-4% sucrose, 2-4% lactose, 1.5-3% soy peptone, 1-2% yeast peptone, 1.5-3% yeast powder, 0.2-0.4% dipotassium hydrogen phosphate, 0.2-0.4% diammonium hydrogen citrate, 0.5-1% anhydrous sodium acetate, 0.1-0.2% Tween-80, 0.06-0.12% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, 0.1-0.2% L-cysteine ​​hydrochloride, and the rest is pure water.

[0008] On the other hand, a microbial agent is also provided, comprising the animal Bifidobacterium lactis subspecies CBTL010 according to claim 1 and one or more of its fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, freeze-dried powder and cell lysate.

[0009] Furthermore, the microbial agent also includes a protective agent, and the components of the protective agent are glucose 2-5%, trehalose 5-10%, maltodextrin 2.5-5%, sucrose 4.5-9%, and the rest is pure water.

[0010] Furthermore, the bacterial content of animal Bifidobacterium lactis subspecies CBTL010 in the microbial agent is not less than 10 9 cfu / g.

[0011] Furthermore, the microbial agent is in the form of an oral preparation in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or sprays, and a sterile injection solution.

[0012] In another aspect, there is provided a use of the animal Bifidobacterium lactis subsp. CBTL010 according to claim 1 or the microbial agent according to claim 3 in the preparation of a product for improving intestinal flora and relieving depression and anxiety.

[0013] Furthermore, the product is a food, a health product, a fermented product or a medicine.

[0014] Furthermore, the food, health product, and fermented product also include additives, and the additives are selected from any one or more of flavoring agents, colorants, fillers, disintegrants, sweeteners, lubricants, binders, and pH regulators.

[0015] Furthermore, the product is a medicine, and the medicine also includes pharmaceutical excipients, which are selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives and lubricants.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a strain of Bifidobacterium animalis subsp. lactis CBTL010 that simultaneously improves intestinal function and alleviates depression and anxiety. The strain has strong gastrointestinal tolerance, does not contain active nitrate reductase, does not have nitrate-reducing ability, does not interfere with intestinal nitrogen metabolism balance, and has a good inhibitory effect on foodborne pathogens. This strain can promote intestinal health and intestinal flora balance by increasing the number of lactic acid bacteria in the mouse intestine. Therefore, Bifidobacterium animalis subsp. lactis CBTL010 has the effect of improving intestinal flora and relieving depression and anxiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0019] Figure 1 This is the colony morphology of Bifidobacterium animalis subsp. lactis CBTL010. DETAILED DESCRIPTION

[0020] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0021] Example 1

[0022] 1. Strain screening and purification:

[0023] Use sterile saline to perform gradient dilution of the collected samples, spread them on the prepared MRS culture medium, and culture them at 37°C in a conventional incubator and an anaerobic incubator for 24 to 72 hours or until colonies grow. Pick single colonies suspected of being bifidobacteria as target strains for simple culture and multi-well plate screening.

[0024] 2. Sequencing identification:

[0025] The single colony obtained after screening was sent to a third-party CNAS for identification by the Testing Center of General Biotechnology (Anhui) Co., Ltd. The gene sequence of the strain was as follows:

[0026] CGCGCATTCTCGCCGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGCGATCCGCCCCACGTCACCGTTGTCCCACCGGTTGTACCCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCACATGAGTTCCCGGCATCACCCGCTGGCAACATGCGGGCAGGGTTGCGCTCGGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCGGCCCCGAAGGGAACCGTGTCACCACGGCGATCCGGCACATGTCAGCCCAGGTAAGGTTCCTTCCGCTTGCATCGAATTGATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGGACCCGTGGAAAGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGGTCAGTGACGGCCCAGAGACCTGCCTTGGCCATTGGTGTTCCTTCCCGATATCTACAACATTCGACCGTTACACCGGGAATTCCAGTCTCCCCACCGACACCGCACCCGGCGCGACCCGGCGCAGGACCCGTTAGGCGATGGACTTTCACACCGGACGCGACGACCGCCCTACGAGCCTTTACGCCCAGTAAATCCGCGACGCTCGCACCTACGATACGCGTGCTGGGGAGGAGAATTTTTAAGATATAAGAAA。

[0027] 3. Sequence alignment:

[0028] By comparing the strains with a local database, strains closest to Lactobacillus rhamnosus at the species level were selected based on 16S rRNA sequences. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA 6.0 software. Based on the analysis, CBTL010 was confirmed to be Bifidobacterium animalis subsp. lactis, with a similarity of 98.61%.

[0029] Example 2

[0030] Gastrointestinal tolerance test:

[0031] Preparation of simulated artificial gastric fluid: After sterilizing PBS, adjust the pH to 2.5 with 1 mol / L HCl, add 3.0 mg / mL pepsin, dissolve it, and filter it through a 0.22 μm microporous filter membrane to sterilize it to prepare simulated artificial gastric fluid;

[0032] Preparation method of simulated artificial intestinal fluid: After sterilizing PBS, adjust the pH value to 8.0 with 0.1 mol / L NaOH, add 0.1% trypsin and 1.8% ox bile salt, dissolve and filter with 0.22 μm microporous filter membrane for sterilization to prepare artificial simulated intestinal fluid.

[0033] (1) The isolated and purified strain CBTL010 was activated and cultured for two generations, and the bacteria were washed twice by centrifugation. The bacteria were collected and 0.5 mL of the re-screened bacterial suspension was added to 4.5 mL of the prepared simulated gastric juice with a pH of 2.5. The cells were digested at 37 °C for 3 h. At the same time, the number of viable bacteria was determined by the ML solid culture medium pouring method at 0 h and 3 h, and the survival rate of the strain CBTL010 was calculated. Four parallels were made for each sample.

[0034] Wherein, survival rate = [N1 / N0] × 100%, N0 represents the number of viable bacteria at 0 h, and N1 represents the number of viable bacteria after 3 h of simulated gastric juice digestion.

[0035] (2) Take 0.5 mL of the above-mentioned simulated artificial gastric juice containing bacteria after digestion for 3 hours and add it to 4.5 mL of simulated artificial intestinal fluid. Continue to culture in a 37°C water bath. Count the number of viable bacteria using the ML solid culture medium pouring method after 0 hours and 8 hours. Calculate the survival rate of the strain using the following formula. Make four parallels for each sample.

[0036] Among them, survival rate = [N1 / N0] × 100%, N0 represents the number of viable bacteria at 0 h, and N1 represents the number of viable bacteria after 8 h of digestion in simulated artificial intestinal fluid.

[0037] The test results of the obtained animal Bifidobacterium lactis subsp. lactis CBTL010 after treatment with simulated artificial gastric fluid and simulated artificial intestinal fluid are shown in Table 1 below:

[0038] Table 1 Survival of Bifidobacterium animalis subsp. lactis CBTL010 in simulated gastrointestinal digestive fluid

[0039]

[0040]

[0041] As shown in Table 1, animal Bifidobacterium lactis subsp. lactis CBTL010 has good gastric acid resistance and good tolerance characteristics, and the survival rate can reach 87.54±0.61% after being treated with simulated intestinal fluid for 8 hours.

[0042] Example 3

[0043] Nitrate reductase test of Bifidobacterium animalis subsp. lactis CBTL010:

[0044] Animal Bifidobacterium lactis subsp. lactis CBTL010 was cultured to the third generation stable period, and blank nitrate medium was used as a negative control. The culture medium was inoculated at a 1% inoculum into a pre-prepared nitrate liquid medium. An appropriate amount of sterile paraffin oil was added to each inoculation tube, and then the culture tube was placed at 37°C for 48 hours. Then, 10 drops of 5% potassium iodide solution and 10 drops of 5% starch solution were added dropwise in sequence. After vortex mixing, the color changes in each culture tube were observed and recorded. Three replicates were made for each sample.

[0045] The results show that animal Bifidobacterium lactis subspecies CBTL010 did not produce a color reaction in the nitrate reductase activity test. The results prove that animal Bifidobacterium lactis subspecies CBTL010 does not contain active nitrate reductase, and therefore does not have the ability to reduce nitrate, will not interfere with the intestinal nitrogen metabolism balance, and has no negative impact on the intestinal nitrogen cycle.

[0046] Example 4

[0047] Antibacterial effect on foodborne pathogens:

[0048] Using the Oxford cup method, 100 μL of viable bacteria were taken as 10 7 Place Escherichia coli, Staphylococcus aureus, and Shigella sonnei at 500 CFU / mL on a plate, pour in an appropriate amount of heated and melted LB solid medium, shake evenly, wait for it to cool and solidify, then place Oxford cups at appropriate intervals on the plate, then take 100 μL of CBTL010 fermentation broth and add it to the Oxford cup wells. Use MRS liquid medium as a negative control and nisin as a positive control. Repeat the experiment three times.

[0049] After 24 h of static incubation at 37°C, photos were taken and the diameter of the inhibition zone was measured using a vernier caliper using the cross-cross method. The results are shown in Table 2:

[0050] Table 2

[0051]

[0052] According to the test results in Table 2, the diameters of the inhibition zones of CBTL010 against Escherichia coli, Staphylococcus aureus, and Shigella sonnei were all higher than those of the positive control nisin (13.89±0.36) mm, indicating that CBTL010 has a good inhibitory effect on foodborne pathogens.

[0053] Example 5

[0054] Effects of Bifidobacterium animalis subsp. lactis CBTL010 on specific intestinal flora in constipated mice:

[0055] (1) Establishment of a functional constipation mouse model

[0056] Drug modeling was performed using loperamide hydrochloride to inhibit intestinal water secretion and colon peristalsis, thereby delaying fecal evacuation time and intestinal transit. The main manifestations were reduced fecal quantity, weight and water content in animals, resulting in functional constipation symptoms.

[0057] Kunming mice were selected as experimental subjects for drug modeling of functional constipation:

[0058] First, 80 offspring Kunming mice aged 6 to 8 weeks with similar mental states and activity abilities were selected, including 40 female and 40 male mice, and randomly divided into four groups, with 10 female mice and 10 male mice in each group. One group was randomly selected as the blank group, and the other three groups of mice were modeled.

[0059] The modeling method is to administer loperamide hydrochloride by gavage:

[0060] After one week of adaptive feeding, the mice in the four groups, except for the blank group, were gavaged with 0.25 mL of a 1 mg / mL, filter-sterilized loperamide hydrochloride solution at 8:00 PM each day. The blank group mice were gavaged with an equal volume of sterile saline at the same time. Furthermore, given the long-lasting efficacy of loperamide hydrochloride, daily gavages were performed for the duration of the experiment, using the same timing and method as the modeling procedure.

[0061] Feces from each group of mice were collected every 24 hours and placed separately. The number of fecal pellets in each group was counted and their moisture content was measured. Fecal moisture content was measured using a drying method. Mouse feces were placed in an oven at 105°C to a constant weight. The fecal moisture content was then calculated using the following formula:

[0062] Feces moisture content (%) = (feces weight before drying - feces weight after drying) / feces weight before drying × 100%;

[0063] When the number of fecal particles in the other three groups of mice was significantly reduced and the water content of feces was significantly reduced compared with the blank group, it indicated that the functional constipation model of mice was successfully established.

[0064] During the experiment, mice had free access to food and water. Except for necessary treatments, their feeding process was completely in accordance with the requirements of the feeding instructions. The changes in the number of fecal pellets and fecal moisture content during the mouse modeling process are shown in Table 3, where the data of the modeling group are the average of the three groups of mice:

[0065] Table 3 Changes in mouse fecal characteristics during modeling

[0066]

[0067] It can be concluded from Table 3 that as the treatment time prolonged, the defecation volume and fecal moisture content of the mice in the modeling group gradually decreased, and basically began to stabilize on the third day, and were significantly lower than those in the blank group, indicating that the functional constipation model of mice was successfully modeled.

[0068] (2) Effects of Bifidobacterium animalis subsp. lactis CBTL010 on specific intestinal flora in constipated mice:

[0069] 1. Preparation of bacterial suspension of Bifidobacterium animalis subsp. lactis CBTL010

[0070] The animal Bifidobacterium lactis subsp. CBTL010 stored at -80°C was thawed at room temperature and then inoculated into a test tube containing 10 mL of sterile MRS liquid culture medium, cultured at 37°C under anaerobic conditions for 24 h, and then 1 mL was inoculated into a triangular flask containing 100 mL of sterile MRS liquid culture medium. After cultured at 37°C under anaerobic conditions for 36 h, a fermentation broth of animal Bifidobacterium lactis subsp. CBTL010 was obtained. 40 mL of the fermentation broth was then centrifuged at 6000 rpm for 10 min, the supernatant was discarded, the bottom bacterial mud was washed twice with sterile saline and then resuspended with 10 mL of sterile saline to obtain a high-concentration bacterial suspension; 1 mL of the high-concentration bacterial suspension was then diluted 10 times with sterile saline to obtain a low-concentration bacterial suspension. The two bacterial suspensions were sealed and stored at 4°C for later use.

[0071] 2. Experimental Design of Mouse Experiments

[0072] The three groups of mouse constipation models obtained in step (1) were randomly numbered as model group, high-dose group and low-dose group;

[0073] Gavage regimen: In addition to gavage of loperamide hydrochloride at 8 pm every day, each group was gavaged with 0.25 mL of the high-concentration bacterial suspension prepared above at 10 pm every day, the low-dose group was gavage of 0.25 mL of the low-concentration bacterial suspension prepared above at 10 pm every day, the model group was gavage of 0.25 mL of sterile saline at 10 pm, and the blank group mice were gavage of 0.25 mL of sterile saline at 8 pm and 10 pm, respectively.

[0074] All feces of male and female mice in each group were collected from 10 pm on the tenth day after the start of gavage with strain CBTL010 to 7 pm on the eleventh day. After refrigeration, they were sent to the laboratory and immediately counted for lactic acid bacteria. The changes in the lactic acid bacteria content in the mouse feces were measured according to the "GB4789.35-2016 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test". The data are the number of colony forming units (CFU / g) per gram of feces. The experimental results are shown in Table 4:

[0075] Table 4 Effects of strain CBTL010 on intestinal flora in constipated mice

[0076]

[0077] As shown in Table 4, after oral administration of Bifidobacterium animalis subsp. lactis CBTL010, the number of Lactobacillus and Bifidobacterium genera in both the low-dose group and the high-dose group were close to the level of the blank group. At the same time, the number of lactic acid bacteria in the model group was significantly lower than that in the other three groups. It can be seen that the increase in the number of lactic acid bacteria in the mouse intestine will promote intestinal health and the balance of intestinal flora. Therefore, Bifidobacterium animalis subsp. lactis CBTL010 has the effect of improving intestinal flora.

[0078] Example 6

[0079] Experiment on the effect of animal Bifidobacterium lactis subspecies CBTL010 on alleviating depression in mice:

[0080] 1. Establishment of a Depression Mouse Model

[0081] Forty 6- to 8-week-old SPF-grade C57BL / 6J healthy male mice were purchased from The Jackson Laboratory (JAX) and subjected to one-week adaptive feeding. The breeding conditions were: sufficient food and water, temperature of 25°C, 12 hours of light and 12 hours of darkness.

[0082] After one week of adaptive feeding, SPF mice were randomly divided into four groups, with 10 mice in each group, namely model group, CBTL010 group, blank group, and prevention group. Before modeling, the mice in the prevention group were gavage-administered with 6×109 cfu / mL of Bifidobacterium animalis subsp. lactis CBTL010 solution, with a dosage of 5 million cfu / day per mouse. The mice in the other three groups were gavage-administered with the same volume of normal saline for 14 consecutive days.

[0083] After 14 days, the mice in the model group, CBTL010 group, and prevention group were housed at a rate of 5 per cage and subjected to chronic, unpredictable mild stimulation according to the chronic stress stimulation process for 21 days, simulating the chronic low-intensity stress that humans receive in daily life. This prevented the mice from anticipating the occurrence of stimulation, thereby constructing a depression mouse model and simulating the disease state of patients with depression.

[0084] Chronic stress stimulation process:

[0085] On the first day, electric light was kept on at night;

[0086] The next day, mice were placed in a restraint tank and restrained for 2 h;

[0087] On the third day, add 4°C ice water to the forced swimming buckets during the day, place one mouse in each bucket, and swim for 5 minutes. At night, tilt the cage containing the mice 45°.

[0088] On the fourth day, all mice were isolated in single cages for 3 hours during the day. At night, colored lights were placed above the cages and flashed continuously.

[0089] On the fifth day, pepper was sprinkled into the rat cage during the day to provide odor stimulation for 2 hours, and the rats were not allowed to eat at night.

[0090] On the sixth day, shake the cage for 10 minutes during the day (the intensity is such that the mice cannot stand steadily) in an irregular direction. At night, place all mice in a narrow bucket.

[0091] On the seventh day, all mice were isolated in single cages and kept alone for 3 hours during the day. At night, water was sprayed on the cage bedding to keep it moist.

[0092] On the eighth day, the mice were restrained in a restraint tank for 2 hours during the day and continuously illuminated by electric light at night;

[0093] On the ninth day, each mouse received a foot shock at daytime, with a current intensity of 2 mA, a shock of 4 seconds, and an interval of 10 seconds, repeated twice. At night, all mice were isolated in a single cage and kept alone for 3 hours.

[0094] On the tenth day, pepper was sprinkled into the rat cage during the day to provide odor stimulation for 2 hours, and the rats were not allowed to eat at night.

[0095] On the eleventh day, add 4°C ice water to the forced swimming bucket during the day, place one mouse in each bucket, and swim for 5 minutes. At night, tilt the cage containing the mouse 45°.

[0096] On the twelfth day, each mouse received a foot shock at daytime, with a current intensity of 2 mA, a shock of 4 seconds, and an interval of 10 seconds, repeated twice. At night, a colored light was placed above the mouse cage and flashed continuously.

[0097] On the night of the thirteenth day, all the mice were placed in a narrow barrel;

[0098] On the fourteenth day, add 4°C ice water to the forced swimming bucket during the day, place one mouse in each bucket, and let it swim for 5 minutes. At night, place a colored light above the mouse cage and keep it flashing.

[0099] On the fifteenth day, all mice were isolated in single cages during the day and kept alone for 3 hours;

[0100] On the 16th day, each mouse received foot shock at daytime, with a current intensity of 2 mA, 4 seconds for shock, 10 seconds for interval, repeated twice. At night, water was sprayed on the bedding of the cage to make it wet.

[0101] On the 17th day, mice were placed in a restraint tank and restrained for 2 h during the day;

[0102] On the 18th day, shake the cage for 10 minutes during the day (the intensity is such that the mice cannot stand steadily) in an irregular direction. At night, place all mice in a narrow bucket.

[0103] On the 19th day, all mice were isolated in single cages for 3 hours during the day, and the cages containing the mice were tilted 45 degrees at night.

[0104] On the twentieth day, electric light continued to shine at night;

[0105] On the 21st day, pepper was sprinkled into the mouse cages for 2 hours of odor stimulation. At night, all mice were isolated in single cages and kept alone for 3 hours.

[0106] 2. Behavioral assessment

[0107] (1) Sugar water preference test

[0108] Preparation before the experiment: 1% sucrose solution and pure water were respectively packed into 50 mL centrifuge tubes, and mice in the model group, CBTL010 group, prevention group, and blank group were placed from ordinary cages into experimental cages, and each mouse was raised individually.

[0109] Adaptation phase: Before the formal experiment, mice were first given two solutions, 1% (w / w) sucrose solution and pure water, for 72 hours. To prevent the mice from getting used to side bias, the positions of sucrose and pure water were changed every 12 hours during this period. After 72 hours, the mice were deprived of water and food for 6 hours.

[0110] Formal experiment: Each cage of mice was given 1% sucrose solution and pure water. After 12 hours, the total mass of liquid consumed by each water bottle was measured and the sucrose preference index was calculated using the following formula:

[0111] Sucrose preference index = (sucrose solution consumption / total liquid consumption) × 100%. The results are shown in Table 5 below:

[0112] Table 5

[0113] Group Sucrose preference index Model Group 49.3％ CBTL010 group 50.1％ Prevention Group 72.4％ Blank group 70.7％

[0114] The results are shown in Table 5. The sucrose preference index of the model group and the CBTL010 group was significantly lower than that of the blank group, and P < 0.05, indicating that the depressed mouse model was successfully established. At the same time, there was no significant difference in the sucrose preference index between the prevention group and the blank group, indicating that taking CBTL010 has a certain preventive effect on depression in mice.

[0115] (2) Tail suspension test

[0116] Preparation before the experiment: Prepare an open polyvinyl chloride box with a size of 55 cm × 60 cm × 12 cm. Install the camera on the operating table above the open box. Connect the camera to the computer via USB. Before the experiment, spray the box with 75% ethanol and wipe it with a paper towel to ensure that the box is clean and odorless.

[0117] One hour before the test, four groups of mice were placed in the test chamber for acclimatization, and an absorbent paper was placed at the bottom of each compartment to collect the animal's feces or urine.

[0118] Beginning of the experiment: Gently take the experimental mouse out of the breeding cage, fix the mouse's tail 2 cm long with tape, which is about 7 cm long. In order to prevent the mouse from climbing its tail, use a 1 cm long straw to cover the mouse's tail. Quickly hang the experimental mouse at a distance of about 20 cm from the bottom of the experimental box and leave immediately; use a camera to shoot and record the entire process, and record the mouse's 6 minutes of immobility time (the immobility time is defined as the process in which the mouse's front legs move but the hind legs do not participate. The movement caused by inertia at the beginning is also the immobility time). After the experiment, take the experimental mouse out of the box and put it back in the cage. Before proceeding to the next experimental animal, clean the entire box area with 75% ethanol and paper towels, and measure the immobility time of the tail suspension. The results are shown in Table 6 below:

[0119] Table 6

[0120] Group Tail suspension time / s Model Group 115.2 CBTL010 group 114.3 Prevention Group 60.5 Blank group 59.7

[0121] According to Table 6, the tail suspension immobility time in the model group and CBTL010 group was significantly longer than that in the blank group, and P < 0.05, indicating that the depressed mouse model was successfully established. In addition, the tail suspension immobility time in the prevention group was not significantly different from that in the blank group, indicating that taking CBTL010 has a certain preventive effect on depression in mice.

[0122] (3) Novelty suppression of food intake test

[0123] Preparation before the experiment: Use a white opaque resin test box with an open top and a size of 55 cm × 60 cm × 11.5 cm. Cover the bottom with about 2 cm thick bedding and place 6 feed pellets in the center.

[0124] The experiment began with mice in the model, CBTL010, prevention, and control groups fasting for 24 hours. They were then placed in a box from the same corner and timed. The time it took to chew food was used as the standard, and the time taken was recorded, which was defined as the feeding latency. The test lasted for 5 minutes. Mice that did not eat within 5 minutes had a feeding latency of 5 minutes. The test results are shown in Table 7:

[0125] Table 7

[0126] Group Feeding latency / s Model Group 239.8 CBTL010 group 238.5 Prevention Group 126.4 Blank group 128.1

[0127] The results are shown in Table 7. The feeding latency of the model group and the CBTL010 group was significantly longer than that of the blank group, and P < 0.05, indicating that the depression mouse model was successfully established. At the same time, the feeding latency of the prevention group was not significantly different from that of the blank group, indicating that taking CBTL010 has a certain preventive effect on depression in mice.

[0128] Example 7

[0129] Effects of CBTL010 on body weight in depressed mice:

[0130] According to Example 6, the model group mice, CBTL010 group mice and blank group mice were weighed respectively. The CBTL010 group mice were gavage-fed with 6×10 9 cfu / mL of CBTL010 bacterial solution, and the blank group and model group mice were gavaged with the same amount of normal saline for 24 consecutive days. The body weight of each group of mice was weighed every 6 days. The results are shown in Table 8:

[0131] Table 8

[0132]

[0133]

[0134] According to the data in Table 8, on day 0, compared with the blank group, the body weight of mice in the model group and CBTL010 group was significantly reduced, and P < 0.05, while there was no significant difference in body weight between the model group and CBTL010 group.

[0135] The weight of the mice in the CBTL010 group gradually increased with the increase in the number of days of gavage with CBTL010; while the weight of the mice in the model group gradually decreased with the increase in the number of days of gavage with normal saline; starting from the 6th day, there was a significant difference in the body weight between the model group and the CBTL010 group (P < 0.05), and there was no significant difference in the body weight between the mice in the CBTL010 group and the blank group, indicating that CBTL010 can significantly improve the symptoms of weight loss in depressed mice. It can be seen that CBTL010 has a certain alleviating effect on depression in mice.

[0136] Example 8

[0137] Mice anxiety relief experiment:

[0138] (1) Open field test

[0139] The mice in the blank group, model group, and CBTL010 group of Example 6 were placed in a corner of the open field. Timing and video recording were immediately started. Video recording was stopped after 10 minutes of observation. The surface was wiped with 75% alcohol each time the animal was replaced to remove residual information from the previous animal. Real-time video recording and analysis were performed using Smart 3.0 analysis software. The time (s) the mice stayed in the center of the open field was counted. The results are shown in Table 9 below:

[0140] Table 9

[0141]

[0142]

[0143] As shown in Table 9, compared with the blank group, the time the model group mice spent entering the center of the open field was significantly shorter (p < 0.01). Compared with the model group, the time the CBTL010 group mice spent entering the center of the open field was significantly longer (p < 0.05), indicating that the CBTL010 strain can effectively alleviate anxiety-related behaviors in mice.

[0144] (2) Elevated plus maze test

[0145] Mice from the blank, model, and CBTL010 groups in Example 6 were placed into the center of the elevated plus maze (facing the open arms). Timing and video recording began immediately, and video recording was stopped after 5 minutes of observation. When animals were replaced, the surface was wiped with 75% alcohol to remove residual information from the previous animal. Real-time video recording and analysis were performed using Smart 3.0 analysis software. The number of times mice entered the open arms and the percentage of time spent in the open and closed arms were counted. The results are shown in Table 10 below:

[0146] Table 10

[0147]

[0148] The results are shown in Table 10. Compared with the normal group, the model group mice entered the open arms significantly less frequently and spent less time in the open arms, with p < 0.05. Compared with the model group, the CBTL010 group mice entered the open arms significantly more frequently and spent more time in the open arms, with p < 0.0001. The time spent in the closed arms was significantly reduced, with p < 0.0001 compared with the model group. This suggests that the CBTL010 strain can effectively alleviate anxiety-related behaviors in mice associated with depressive-like symptoms.

[0149] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An animal Bifidobacterium lactis subspecies CBTL010, characterized in that The animal Bifidobacterium lactis subspecies CBTL010 is isolated from fermented food. The animal Bifidobacterium lactis subspecies CBTL010 is deposited in the General Microbiology Center of the China Culture Collection Administration on March 20, 2025, with a deposit number of CGMCC NO.33906 and a classification name of Bifidobacterium animalis subsp.lactis.

2. The animal Bifidobacterium lactis subspecies CBTL010 according to claim 1, characterized in that The modified MRS culture medium components involved in the cultivation process of the animal Bifidobacterium lactis subspecies CBTL010 include: by mass percentage, 2-4% sucrose, 2-4% lactose, 1.5-3% soy peptone, 1-2% yeast peptone, 1.5-3% yeast powder, 0.2-0.4% dipotassium hydrogen phosphate, 0.2-0.4% diammonium hydrogen citrate, 0.5-1% anhydrous sodium acetate, 0.1-0.2% Tween-80, 0.06-0.12% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, 0.1-0.2% L-cysteine ​​hydrochloride, and the rest is pure water.

3. A microbial agent, characterized in that: The microbial agent comprises the animal Bifidobacterium lactis subspecies CBTL010 according to claim 1 and one or more of its fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, freeze-dried powder and cell lysate.

4. The microbial agent according to claim 3, characterized in that The microbial agent also includes a protective agent. According to mass percentage, the protective agent comprises 2-5% glucose, 5-10% trehalose, 2.5-5% maltodextrin, 4.5-9% sucrose, and the rest is pure water.

5. The microbial agent according to claim 3, characterized in that The bacterial content of animal Bifidobacterium lactis subspecies CBTL010 in the microbial agent is not less than 10 9 cfu / g.

6. The microbial agent according to claim 3, characterized in that The microbial agent is in the form of an oral preparation in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup or spray, and a sterile injection solution.

7. Use of the animal Bifidobacterium lactis subsp. CBTL010 according to claim 1 or the microbial agent according to claim 3 in the preparation of a product for improving intestinal flora and relieving depression and anxiety.

8. The product according to claim 7, characterized in that The product is food, health product, fermentation product or medicine.

9. The product according to claim 8, characterized in that The food, health care product and fermented product may further comprise additives, which are selected from any one or more of flavoring agents, colorants, fillers, disintegrants, sweeteners, lubricants, binders and pH regulators.

10. The product according to claim 8, characterized in that The product is a medicine, which also includes pharmaceutical excipients, which are selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives and lubricants.

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