Bifidobacterium longum subsp. infantis capable of alleviating autism spectrum disorder and uses thereof

Abstract

The present application relates to long bifidobacterium infantis capable of relieving autism spectrum disorder and application thereof, and belongs to the technical field of biological medicine.The present application provides a long bifidobacterium infantis XA-9111, the preservation number of the long bifidobacterium infantis XA-9111 is CGMCC No.26615;experimental demonstration shows that the long bifidobacterium infantis XA-9111 can significantly improve the anxiety behavior, repetitive stereotyped behavior and social impairment of male offspring of MIA model mice, and the long bifidobacterium infantis XA-9111 can significantly improve the hyperactivity, anxiety behavior, repetitive stereotyped behavior and social impairment of BTBR model mice, so the long bifidobacterium infantis XA-9111 can significantly improve the core symptoms and concurrent symptoms of autism spectrum disorder.

Description

Technical Field

[0001] This invention relates to Bifidobacterium longum infantis subspecies, which can alleviate autism spectrum disorder, and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder in which individuals with ASD face varying degrees of difficulty and developmental differences in social communication, cognition, emotion, and behavior. In addition to affecting interpersonal interactions, ASD is characterized by repetitive and rigid behaviors, as well as abnormal responses to specific interests and senses. ASD accompanies a child's development and has a profound impact on parent-child relationships, peer relationships, school life, and community adaptation. Besides social and communication impairments, it may also involve intellectual, language, and other developmental issues.

[0003] Currently, there is no specific drug that can completely cure ASD. Treatment for ASD is typically comprehensive, including behavioral therapy, educational interventions and support, as well as medication to manage related symptoms. To date, the U.S. Food and Drug Administration (FDA) has approved only two drugs for the treatment of ASD: risperidone (brand name Risperdal) and aripiprazole (brand name Abilify). Risperidone is an antipsychotic drug approved for treating stereotyped and aggressive behaviors in children and adolescents aged 5 to 16 with ASD; however, it may cause adverse reactions such as weight gain, drowsiness, and tremors. Aripiprazole is also an antipsychotic drug approved for treating stereotyped behaviors, aggressive behaviors, and bipolar disorder in patients aged 5 to 17 with ASD; however, it can cause side effects such as constipation, indigestion, nausea, vomiting, headache, fatigue, anxiety, insomnia, drowsiness, blurred vision, and orthostatic hypotension. Therefore, there is an urgent need to develop drugs with fewer side effects that can effectively treat ASD in order to overcome the shortcomings of existing ASD treatments.

[0004] Bifidobacterium longum subsp. infantis is a probiotic belonging to the genus Bifidobacterium. It is an important member of the infant gut microbiota, possessing various beneficial functions and effects. For example, Bifidobacterium longum subsp. infantis aids in the digestion and absorption of nutrients (it breaks down and ferments complex polysaccharides in the intestine, helping to digest food and producing nutrients such as short-chain fatty acids, which are beneficial for intestinal cells to absorb and utilize nutrients), maintains intestinal health (it can reduce the number of harmful bacteria in the gut, maintain the normal balance of the gut flora, inhibit the growth of harmful bacteria, thereby reducing adverse effects on the host and maintaining a healthy gut), and supports the immune system (Bifidobacterium longum...). Bifidobacterium longum subsp. infantis helps enhance an infant's immunity and resistance to disease by stimulating the development and function of the immune system; alleviates intestinal discomfort and bloating (Bifidobacterium longum subsp. infantis helps reduce discomfort symptoms such as bloating and diarrhea, improving intestinal comfort and making the infant more comfortable and calm); synthesizes vitamins and enzymes (Bifidobacterium longum subsp. infantis can synthesize vitamins such as B vitamins and vitamin K, as well as some enzymes, which play an important role in promoting intestinal health and overall growth and development); and improves lactose digestion (Bifidobacterium longum subsp. infantis produces lactase, which helps infants digest lactose better, thereby reducing symptoms of lactose intolerance). Finding a Bifidobacterium longum subsp. infantis that can effectively treat autism spectrum disorder is very helpful in developing drugs with fewer side effects that can effectively treat autism spectrum disorder. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a strain of Bifidobacterium longum subsp. infantis XA-9111, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26615 and deposit date of February 21, 2023.

[0006] The *Bifidobacterium longum* subspecies *XA-9111* was derived from a fecal sample of a healthy female infant (5 months old, exclusively breastfed) in Shenzhen. Sequencing analysis of this strain revealed its 16S rDNA sequence as shown in SEQ ID NO.1. The sequenced sequence was compared with the nucleic acid sequence in NCBI's Blasten program, and the results showed that the strain was *Bifidobacterium longum* subspecies *XA-9111*.

[0007] This invention also provides the application of the above-mentioned *Bifidobacterium longum* subsp. *infantii* XA-9111 in the preparation of a drug, said drug having any of the following functions:

[0008] (a) Treatment of autism spectrum disorder (ASD);

[0009] (b) Inhibit inflammation.

[0010] In one embodiment of the present invention, the treatment of autism spectrum disorder includes improving the core symptoms and / or complications of autism spectrum disorder.

[0011] In one embodiment of the present invention, the core symptoms of autism spectrum disorder include repetitive and stereotyped behaviors and / or social impairments; the concurrent symptoms of autism spectrum disorder include hyperactive behaviors and / or anxious behaviors.

[0012] In one embodiment of the present invention, the improvement of core symptoms of autism spectrum disorder includes improvement of repetitive and stereotyped behaviors and / or improvement of social impairment; the improvement of concurrent symptoms of autism spectrum disorder includes improvement of hyperactive behaviors and / or improvement of anxious behaviors.

[0013] In one embodiment of the present invention, the drug contains live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111.

[0014] In one embodiment of the present invention, the viable bacterial content of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111 in the drug is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0015] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0016] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes surfactants, excipients, stabilizers, suspending agents, isotonic agents, preservatives, fillers, disintegrants, antioxidants, emulsifiers, coating agents, binders, lubricants, and / or flavoring agents.

[0017] In one embodiment of the present invention, the dosage form of the drug is a powder, tablet, granule, capsule, or oral liquid; the oral liquid is a solution, syrup, emulsion, or suspension.

[0018] The present invention also provides a medicament for treating autism spectrum disorder, wherein the medicament comprises live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111.

[0019] In one embodiment of the present invention, the treatment of autism spectrum disorder includes improving the core symptoms and / or complications of autism spectrum disorder.

[0020] In one embodiment of the present invention, the core symptoms of autism spectrum disorder include repetitive and stereotyped behaviors and / or social impairments; the concurrent symptoms of autism spectrum disorder include hyperactive behaviors and / or anxious behaviors.

[0021] In one embodiment of the present invention, the improvement of core symptoms of autism spectrum disorder includes improvement of repetitive and stereotyped behaviors and / or improvement of social impairment; the improvement of concurrent symptoms of autism spectrum disorder includes improvement of hyperactive behaviors and / or improvement of anxious behaviors.

[0022] In one embodiment of the present invention, the viable bacterial content of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111 in the drug is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0023] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0024] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes surfactants, excipients, stabilizers, suspending agents, isotonic agents, preservatives, fillers, disintegrants, antioxidants, emulsifiers, coating agents, binders, lubricants, and / or flavoring agents.

[0025] In one embodiment of the present invention, the dosage form of the drug is a powder, tablet, granule, capsule, or oral liquid; the oral liquid is a solution, syrup, emulsion, or suspension.

[0026] The present invention also provides a drug for inhibiting inflammation, wherein the drug comprises live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111.

[0027] In one embodiment of the present invention, the viable bacterial content of the above-mentioned Bifidobacterium longum subsp. infantis XA-9111 in the drug is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

[0028] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0029] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes surfactants, excipients, stabilizers, suspending agents, isotonic agents, preservatives, fillers, disintegrants, antioxidants, emulsifiers, coating agents, binders, lubricants, and / or flavoring agents.

[0030] In one embodiment of the present invention, the dosage form of the drug is a powder, tablet, granule, capsule, or oral liquid; the oral liquid is a solution, syrup, emulsion, or suspension.

[0031] The technical solution of this invention has the following advantages:

[0032] This invention provides a strain of *Bifidobacterium longum subsp. infantis* XA-9111, with the accession number CGMCC. No. 26615; Experiments have shown that this *Bifidobacterium longum* infant subspecies XA-9111 can significantly improve anxiety, repetitive and stereotyped behaviors, and social impairment in male offspring of MIA model mice. Furthermore, *Bifidobacterium longum* infant subspecies XA-9111 can significantly improve hyperactivity, anxiety, repetitive and stereotyped behaviors, and social impairment in BTBR model mice. This indicates that *Bifidobacterium longum* infant subspecies XA-9111 can significantly improve the core symptoms and complications of autism spectrum disorder, showing great promise in the preparation of therapeutic drugs for autism spectrum disorder. Simultaneously, experiments have shown that *Bifidobacterium longum* infant subspecies XA-9111 can effectively reduce the levels of cytokines such as IL-6 and TNF-α in the plasma of male offspring of MIA model mice, and significantly increase the levels of chemokines such as BLC and RANTES in the plasma of male offspring of MIA model mice. This indicates that *Bifidobacterium longum* infant subspecies XA-9111 can significantly inhibit inflammation induced by the MIA model, showing great promise in the preparation of anti-inflammatory drugs.

[0033] Preservation of biological materials

[0034] A strain of *Bifidobacterium longum* subsp. *infantis* XA-9111, taxonomically named *Bifidobacterium longum* subsp. *infantis*, was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26615. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0035] Figure 1 Phylogenetic tree of Bifidobacterium longum infantis subspecies XA-9111.

[0036] Figure 2 : Behavioral results of open field tests (MIA model) in different groups of mice.

[0037] Figure 3Behavioral results of elevated cross maze test (MIA model) in different groups of mice.

[0038] Figure 4 : Behavioral results of grooming tests in different groups of mice (MIA model).

[0039] Figure 5 Behavioral results of bead embedding test in different groups of mice (MIA model).

[0040] Figure 6 Behavioral results of the three-box social test (MIA model) in different groups of mice.

[0041] Figure 7 Plasma multifactor detection results of different groups of mice (MIA model). Figure 7 In the table, a represents the results of IL-6 cytokine detection; b represents the results of TNF-α cytokine detection; c represents the results of BLC chemokine detection; and d represents the results of RANTES chemokine detection.

[0042] Figure 8 : Behavioral results of open field tests in different groups of mice (BTBR model). Figure 8 In the figure, a represents the total distance the mouse travels in the open field box; b represents the percentage of time the mouse spends moving in the central area of ​​the open field box.

[0043] Figure 9 : Behavioral results of grooming tests in different groups of mice (BTBR model).

[0044] Figure 10 Behavioral results of bead embedding test in different groups of mice (BTBR model).

[0045] Figure 11 Behavioral results of the three-box social test (BTBR model) in different groups of mice.

[0046] Figure 12 Microscopic images of cFOS neurons in the striatum of different groups of mice (BTBR model).

[0047] Figure 13 Statistical analysis of the number of activated cFOS neurons in the striatum of different groups of mice (BTBR model). Detailed Implementation

[0048] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0049] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0050] The method for detecting the number of viable bacteria in the following examples is as follows: the national standard GB 4789.35-2016, "National Food Safety Standard for Microbiological Testing of Food - Lactic Acid Bacteria Detection", is adopted.

[0051] Experimental Example 1: Acquisition and Identification of Bifidobacterium longum subsp. infantileum XA-9111

[0052] The specific steps are as follows:

[0053] Using fecal samples from healthy female infants (5 months old, exclusively breastfed) in Shenzhen as the isolation source, 1.0 g of fresh fecal sample was added to 9 mL of sterile anaerobic phosphate buffer and vortexed to obtain a homogenized sample; 0.5 mL of the homogenized sample was then added to 4.5 mL of sterile anaerobic phosphate buffer to obtain 10 -1 Dilute the solution, then take 0.5 mL of 10 -1 The diluent was diluted in 4.5 mL of sterile anaerobic phosphate buffer to obtain 10 -2 Diluent, follow these steps to obtain 10 -3 10 -4 10 -5 10 -6 10 -7 Diluent; 100 μL of the serially diluted solution was spread onto MRS solid medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) containing 1% (w / v, g / 100 mL) L-cysteine ​​hydrochloride. -4 10 -5 10 -6 One plate was used for each gradient, and the culture was anaerobic at 37°C for 72 hours to obtain colonies. Colonies exhibiting typical characteristics of *Bifidobacterium longum* subsp. *infantii* were selected from MRS solid medium based on their shape, size, edge, and transparency. These colonies were then streaked onto MRS solid medium using an inoculation loop and anaerobically cultured at 37°C for 72 hours to obtain purified single colonies. These purified single colonies were then inoculated into 5 mL of MRS liquid medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) and anaerobically cultured at 37°C for 24 hours to obtain bacterial suspensions. Each bacterial suspension was numbered, and 16S rDNA sequencing was performed for identification and analysis. Strains exhibiting typical characteristics of *Bifidobacterium longum* subsp. *infantii* were selected, resulting in strain XA-9111.

[0054] The strain identification process is as follows:

[0055] 10 μL of bacterial culture of strain XA-9111 was taken, and DNA was extracted using a bacterial genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.). A lysate was obtained. Using the universal bacterial primers 27F and 1492R (27F: AGAGTTTGATCMTGGCTCAG, 1492R: TGTACGGYTACCTTGTTACGACTT, where M and Y are degenerate bases, M = A or C, Y = C or T), as shown in SEQ ID NO.2 and SEQ ID NO.3 respectively, the lysate was used as a template for amplification to obtain the 16S rRNA of strain XA-9111 (the 16S rDNA sequence of strain XA-9111 is shown in SEQ ID NO.1). The 16S rDNA of strain XA-9111 was sequenced using the NCBI Blastn program (the constructed phylogenetic tree is shown in [link to phylogenetic tree]). Figure 1 The results showed that this strain was *Bifidobacterium longum* subsp. *infantis*, and it was named *Bifidobacterium longum* subsp. *infantis* XA-9111. The bacterial culture of *Bifidobacterium longum* subsp. *infantis* XA-9111 was placed in a 20% (v / v) glycerol aqueous solution (glycerol aqueous solution to bacterial culture volume ratio 1:1) and deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 26615.

[0056] Experimental Example 2: Effects of Bifidobacterium longum infant subspecies XA-9111 on ASD-like behavior in male offspring of MIA mice

[0057] This experimental example demonstrates the effect of Bifidobacterium longum infant subspecies XA-9111 on ASD-like behavior in male offspring of MIA mice. The experimental procedure is as follows:

[0058] 1. Preparation of bacterial suspension test samples

[0059] 1 mL of the preserved bacterial culture of *Bifidobacterium longum* subsp. *infantii* XA-9111 was inoculated into 100 mL of MRS medium (purchased from Solarbio) and anaerobically cultured at 37°C for 24 h to obtain the activated solution. The activated solution was then inoculated into MRS medium at a rate of 5% (v / v) and anaerobically cultured at 37°C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000g for 10 min at room temperature (25°C) to collect the bacterial cells. 1 / 10 volume of D-PBS buffer (pH 10) was added to the bacterial cells. 7.3 (purchased from Servicebio) After washing the bacterial cells, centrifuge again at 8000g for 10 min at room temperature (25℃) and collect the washed bacterial cells; add 1 / 10 of the fermentation broth volume of an aqueous solution containing 5% (w / w) trehalose, 5% (w / w) levodopa, 10% (w / w) maltodextrin and 0.5% (w / w) arginine to the washed bacterial cells to obtain a bacterial suspension; dispense the bacterial suspension into 2mL centrifuge tubes, 1mL per tube, and store at -80℃. Before administration by gavage, the bacterial suspension was removed from the -80°C freezer, thawed and revived by incubation at 37°C for 5 min, and centrifuged at 8000g for 5 min at room temperature (25°C) to collect the revived bacterial cells. An equal volume of PBS buffer (pH 7.3, purchased from Servicebio) containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride was added to the revived bacterial suspension for washing. The cells were then centrifuged again at 8000g for 10 min at room temperature (25°C) to collect the washed revived bacterial cells. The washed revived bacterial cells were resuspended in PBS buffer containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride to a bacterial concentration of 2×10⁻⁶. 10 CFU / mL was used to obtain the bacterial solution for gavage.

[0060] 2. Construction of the Maternal Immune Activation (MIA) Model

[0061] Maternal overreaction during pregnancy (maternal immune activation, MIA) is a commonly used animal model in preclinical research in the field of autism spectrum disorder (see references: "Elaine Y. Hsiao, et al., Cell. 2013; Gloria B. Choi, et al., Science, 2016; Sangdoo Kim et al., Nature. 2017; Yeong shin Yim, et al., Nature. 2017."). Male offspring of this model exhibit significant core symptoms of ASD, namely repetitive and stereotyped behaviors and social impairments, accompanied by other ASD complications such as anxiety and hyperactivity.

[0062] 2.1 Laboratory Animals

[0063] Twenty male and eighty female SPF-grade 8-12 week old C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. Before mating, the female mice's feces were tested for the presence of segmented filamentous bacteria (SFB); mice without SFB could not be used for MIA model construction. SPF-grade mice were housed in individually ventilated cages at a density of 5 mice per cage, and all mice were ear-tagged and numbered. Five days before breeding, male mice were housed individually in each cage. The ambient temperature and humidity were maintained at 23±1℃ and 50±10%, respectively, with an automatic light-dark cycle of 12 hours. Unless otherwise specified in the experimental methods below, the mice had free access to food and water.

[0064] 2.2 MIA Model

[0065] F0 generation male and female mice were mated at a ratio of 2:1. The vaginal plug was examined the day after mating. If a plug was found, pregnant mice were injected intraperitoneally with Poly(I:C) (20 mg / kg, Sigma-Aldrich, CAS No. 42424-50-0) on day 12.5 of embryonic development (E12.5). Control group pregnant mice were injected with saline. Pregnant mice were housed individually after the intraperitoneal injection, with toys and nesting materials provided in their cages. Care was taken to avoid startling the pregnant mice during this stage to prevent the females from cannibalizing their offspring after birth. The mother mice nursed the pups for 21–28 days after birth. Then, the male offspring were separated into individual cages, with a maximum density of 5 mice per cage, and each cage was tagged with an ear tag.

[0066] 3. Bowel cleansing and drug administration

[0067] 3.1 Antibiotic bowel cleansing in mice

[0068] All male offspring mice in all groups underwent bowel cleansing with antibiotics.

[0069] The specific steps for antibiotic bowel cleansing are as follows: Vancomycin (50 mg / kg by gavage, Sigma Aldrich, CAS No. 1404-93-9), neomycin (100 mg / kg by gavage, Sigma Aldrich, CAS No. 1405-10-3), and metronidazole (100 mg / kg by gavage, Sigma Aldrich, CAS No. 443-48-1) were dissolved in sterile water and administered orally to male offspring mice. The volume of solution administered was 0.1 mL per 10 g body weight. The solution was freshly prepared daily and administered by gavage every 12 hours for 7 consecutive days.

[0070] During the antibiotic bowel cleansing period in mice (7 days), ampicillin was prepared into a 1 mg / mL ampicillin solution using sterile drinking water and given to the mice to drink, with the solution being changed every three days.

[0071] During the antibiotic bowel cleansing period for mice (7 days), all procedures must be performed in a biosafety cabinet, including bedding changes, drinking water changes, food additions, and gavage. It is forbidden to open the cage lid outside the biosafety cabinet or expose the mice to the space outside the biosafety cabinet.

[0072] 3.2 Grouping of mice and administration of drugs by gavage

[0073] Twenty male offspring of MIA mice were randomly divided into two groups using a block design: a model group (Poly(I:C) + Vehicle group) and an experimental group (Poly(I:C) + B. infantis group), with 10 mice in each group. Ten male offspring of pregnant female mice injected with saline were used as the control group (Saline + Vehicle group). The total body weight of the control group mice was compared with the total body weight of the male offspring mice in the other two groups, with a difference within ±2g. After a 7-day antibiotic bowel cleansing period, all male offspring mice were immediately administered the test sample by gavage at a volume of 0.1 mL per 10g of body weight. Mice in the Saline + Vehicle and Poly(I:C) + Vehicle groups were administered PBS buffer (Gibco) containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride (Sigma-Aldrich, CAS No. 52-89-1) by gavage. TM (Catalog No. 10010023), a bacterial suspension of Bifidobacterium longum infantis XA-9111 was administered orally to mice containing Poly(I:C)+B. infantis via gavage (each 1 mL of the suspension contained 2 × 10⁻⁶ bacteria). 10 (individual bacterial cells). Gavage was administered once daily at a fixed time for 21 consecutive days before the behavioral test, and continued after the start of the behavioral test until the day before the end of the experiment.

[0074] 4. Behavioral tests

[0075] Before the experiment, mice needed to be undisturbed for 60 minutes in the behavioral testing room. After each test, the behavioral equipment needed to be wiped with 75% (v / v) alcohol, and the alcohol odor was allowed to dissipate before the next mouse was tested. NoldusEthoVision XT software (Noldus Information Technology; Leesburg, VA, USA) was used to record video and track the mice's movements, identifying three areas: the mouse's nose, center of body, and tail. The behavioral experimenters were unaware of the mice's group assignments.

[0076] 4.1 Open Field Test

[0077] Spontaneous mouse behavior was recorded in an open field box (41cm×41cm×38cm) using a camera positioned directly above the box. The central area of ​​the open field was defined as a virtual region of 20cm×20cm. Each mouse was recorded in the open field for 10 minutes, and the total distance traveled (Total Distance, cm) and the percentage of time spent in the central area (Time in Center, %) were recorded and analyzed.

[0078] 4.2 Hair trimming test

[0079] After the open field test, the researchers analyzed the spontaneous grooming behavior of the mice in the open field box over 10 minutes using video analysis, and calculated the total cumulative grooming time.

[0080] 4.3 Elevated Cross Maze Test

[0081] Mice were placed in a cross-shaped maze for testing. The closed arms of the maze measured 15cm in height, 30cm in length, and 5cm in width, while the open arms measured 1cm in height, 30cm in length, and 5cm in width. The maze was gray, made of plexiglass, and 50cm above the ground. Each mouse was placed in the center of the maze, and its movement time in the open arms was recorded.

[0082] 4.4 Bead embedding test

[0083] The mice were placed in a test box (40cm×20cm×30cm, with a bedding depth of 5cm) and a 30-minute video recording was performed. The box contained 20 glass beads (arranged in 4 rows of 5 beads each, with a consistent spacing of 4cm between the beads). At the end of the 30-minute mark, the mice were carefully removed from the box. Beads that were buried more than 2 / 3 of their volume were recorded as buried beads, and the number of buried beads was counted.

[0084] 4.5 Three-box social test

[0085] The three-chamber socialization test was conducted in a 60cm×40cm×22cm blue acrylic box. The box was divided into three 20cm×40cm×22cm areas (left, center, and right) by two partitions (transparent acrylic glass with a 5cm×8cm sliding door in the central area). Each of the left and right chambers had a circular cage (10cm in diameter, 18cm high, 3mm in diameter grid, 7mm grid spacing) in the center. A camera was positioned directly above the central chamber to record video and track the mouse's movements. First, the mouse was placed in the box from the central chamber and allowed to move freely within the three chambers, becoming familiar with the environment of the three-chamber setup with circular cages in each chamber (without any objects inside). This was recorded for 10 minutes. Then, a mouse (strain, sex, and age identical to the experimental mouse) was placed in a cage on one side of the box, not in the same cage as the experimental mouse. This mouse was placed in the box from the central chamber. The cage on the other side was empty, and the mouse was placed in the box from the central chamber and allowed to explore freely. This was recorded for 10 minutes.

[0086] 5. Analysis of behavioral results

[0087] Open field testing results showed that, compared with the Saline+Vehicle group (central area movement time percentage 13.66±0.34%), the Poly(I:C)+Vehicle group mice had significantly reduced movement time in the central area of ​​the open field (reduced to 7.89±0.82%), while the Poly(I:C)+B. infantis group mice administered Bifidobacterium infantis subsp. XA-9111 by gavage had increased activity time in this area (increased to 13.32±1.01%). Figure 2 One-way ANOVA, F (2,27) =15.69, P<0.0001; Dunnett post-hoc test: P Saline+VehicleVSPoly(I:C)+Vehicle <0.0001, P Poly(I:C)+Vehicle vs Poly(I:C)+B.infantis =0.0001). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve anxiety behavior in male offspring of MIA model mice.

[0088] The elevated cross maze test results showed that, compared with the Saline+Vehicle group mice (open arm region movement time 71.12±8.81s), the Poly(I:C)+Vehicle group mice had a significantly reduced movement time in the open arm region of the cross maze (reduced to 30.08±4.41s), while the Poly(I:C)+B. infantis group mice administered Bifidobacterium infantis XA-9111 by gavage had a significantly increased movement time in this region compared with the model group mice (increased to 64.78±13.03s). Figure 3 One-way ANOVA, F (2,27) =5.557, P=0.0095; Dunnett post-hoc test: PSaline+Vehicle VS Poly(I:C)+Vehicle =0.0086, P Poly(I:C)+Vehicle vs Poly(I:C)+B.infantis =0.0286). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve anxiety behavior in male offspring of MIA model mice.

[0089] The results of the grooming test showed that, compared with the Saline+Vehicle group (grooming time 61.80±2.32s), the Poly(I:C)+Vehicle group mice had a significantly increased cumulative grooming time in the open field test (increased to 111.70±4.84s), while the grooming time of the Poly(I:C)+B. infantis group mice administered Bifidobacterium infantis XA-9111 by gavage was significantly reduced compared with the model group mice (reduced to 80.40±5.02s). Figure 4 One-way ANOVA, F (2,27) =35.34, P<0.0001; Dunnett post-hoc test: P Saline+Vehicle VS Poly(I:C)+Vehicle <0.0001, P Poly(I:C)+Vehicle vs Poly(I:C)+B.infantis <0.0001). This indicates that *Bifidobacterium longum* infant subspecies XA-9111 can improve repetitive and stereotyped behaviors in male offspring of the MIA model mouse.

[0090] The bead embedding test results showed that, compared with the Saline+Vehicle group mice (1.60±0.22 beads), the number of beads embedded in the Poly(I:C)+Vehicle group mice was significantly increased (to 4.70±0.72 beads), while the number of beads embedded in the Poly(I:C)+B. infantis group mice administered Bifidobacterium infantis XA-9111 via gavage was significantly decreased compared with the model group mice (to 0.80±0.55 beads). Figure 5 One-way ANOVA, F (2,27) =14.67, P<0.0001; Dunnett post-hoc test: P Saline+Vehicle VS Poly(I:C)+Vehicle =0.0007, P Poly(I:C)+Vehicle vs Poly(I:C)+B.infantis <0.0001). This indicates that *Bifidobacterium longum* infant subspecies XA-9111 can improve repetitive and stereotyped behaviors in male offspring of the MIA model mouse.

[0091] The results of the three-box socialization test showed that, during the socialization test phase, compared with the Saline+Vehicle group mice (exploration time in the unfamiliar cage area 140.80±3.59s), the Poly(I:C)+Vehicle group mice showed no significant preference for exploring unfamiliar mice (decreased to 93.28±3.89s), while the Poly(I:C)+B. infantis group mice administered Bifidobacterium infantis subsp. XA-9111 via gavage showed a significantly increased exploration time for unfamiliar mice compared with the model group (increased to 146.76±9.06s). Figure 6 Two-way ANOVA, F (2,52) =20.27, P<0.0001; Tukey post-hoc test: P Saline+Vehicle VS Poly(I:C)+Vehicle =0.0002, P Poly(I:C)+Vehicle vs Poly(I:C)+B.infantis <0.0001). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve social impairment in male offspring of MIA model mice.

[0092] In summary, Bifidobacterium longum infant subspecies XA-9111 can improve autism-like behaviors in male offspring of MIA model mice, including anxiety behaviors, repetitive stereotyped behaviors, and social impairments.

[0093] Experimental Example 3: Anti-inflammatory effect of Bifidobacterium longum infantis subsp. XA-9111 on male offspring of MIA model mice

[0094] This experimental example demonstrates the anti-inflammatory effect of Bifidobacterium longum infant subspecies XA-9111 on male offspring of MIA model mice. The experimental procedure is as follows:

[0095] The LEGENplex™ MU Th Cytokine Panel (12-plex) w / VbP cytokine assay used a multifactor kit (Biolegend, catalog number 741044), and the Mouse Proinflammatory Chemokine Panel (13-plex) w / VbP chemokine assay used a multifactor kit (Biolegend, catalog number 740451). After restoring to room temperature (25°C), the relevant working solutions and cytokine standards at different concentrations were prepared according to the kit instructions. Based on Experiment 2, after euthanizing the mice at the experimental endpoint, blood was collected from all groups of mice, and plasma was separated and stored at -80°C for later use. For cytokine and chemokine detection, according to the kit instructions, remove mouse plasma samples from the -80℃ freezer, thaw on ice, and add 25 μL / sample plasma to a V-bottom detection plate. First, add 25 μL of 1× kit buffer, then add 25 μL of well-mixed capture microspheres. After sealing, incubate at 800 rpm at room temperature (25℃) in the dark for 2 hours. After incubation, centrifuge to remove the supernatant, and add 200 μL of washing buffer to each well for washing, retaining the precipitate (centrifugation conditions during washing: room temperature, 250 g, 5 min). After washing, add 25 μL of detection antibody to each well, seal, and react at 800 rpm at room temperature (25℃) in the dark for 1 hour. After the reaction is complete, add 25 μL of detection antibody to each well. SA-PE was sealed and incubated at 800 rpm at room temperature (25°C) in the dark for 30 min. After incubation, the supernatant was removed by centrifugation, and 200 μL of washing buffer was added to each well for washing, retaining the precipitate (centrifugation conditions during washing: room temperature, 250 g, 5 min). After washing, 150 μL of 1× kit buffer was added to each well to obtain the test sample. Flow cytometry was used to detect particle size, APC, and PE fluorescence in the test sample, and cytokine standard curves and sample concentration analysis were performed according to the kit instructions and analysis system prompts.

[0096] The results are as follows Figure 7As shown, compared with the Poly(I:C)+Vehicle group (IL-6: 4.28±1.57 ng / mL; TNF-α: 5.38±1.86 ng / mL; BLC: 93.13±3.48 ng / mL; RANTES: 16.84±0.12 ng / mL), the levels of cytokines such as IL-6 (decreased to 0.72±3.45 ng / mL) and TNF-α (decreased to 1.08±0.45 ng / mL) in the plasma of mice in the Poly(I:C)+B. infantis group were significantly reduced. Simultaneously, the levels of chemokines such as BLC (increased to 129.20±14.73 ng / mL) and RANTES (increased to 25.74±2.47 ng / mL) were significantly increased. This indicates that *Bifidobacterium longum* subsp. infantis XA-9111 has an inhibitory effect on inflammation induced by the MIA model.

[0097] Experimental Example 3: Effects of Bifidobacterium longum infant subspecies XA-9111 on ASD-like behavior in BTBR mice

[0098] This experimental example demonstrates the effect of Bifidobacterium longum infant subspecies XA-9111 on ASD-like behavior in BTBR mice. The experimental procedure is as follows:

[0099] 1. Preparation of bacterial suspension test samples

[0100] 1 mL of the preserved bacterial culture of *Bifidobacterium longum* subsp. *infantii* XA-9111 was inoculated into 100 mL of MRS medium (purchased from Solarbio) and anaerobically cultured at 37°C for 24 h to obtain the activated solution. The activated solution was then inoculated into MRS medium at a rate of 5% (v / v) and anaerobically cultured at 37°C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000g for 10 min at room temperature (25°C) to collect the bacterial cells. 1 / 10 volume of D-PBS buffer (pH 10) was added to the bacterial cells. 7.3 (purchased from Servicebio) After washing the bacterial cells, centrifuge again at 8000g for 10 min at room temperature (25℃) and collect the washed bacterial cells; add 1 / 10 of the fermentation broth volume of an aqueous solution containing 5% (w / w) trehalose, 5% (w / w) levodopa, 10% (w / w) maltodextrin and 0.5% (w / w) arginine to the washed bacterial cells to obtain a bacterial suspension; dispense the bacterial suspension into 2mL centrifuge tubes, 1mL per tube, and store at -80℃. Before administration by gavage, the bacterial suspension was removed from the -80°C freezer, thawed and revived by incubation at 37°C for 5 min, and centrifuged at 8000g for 5 min at room temperature (25°C) to collect the revived bacterial cells. An equal volume of PBS buffer (pH 7.3, purchased from Servicebio) containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride was added to the revived bacterial suspension for washing. The cells were then centrifuged again at 8000g for 10 min at room temperature (25°C) to collect the washed revived bacterial cells. The washed revived bacterial cells were resuspended in PBS buffer containing 1% (w / v, g / 100mL) L-cysteine ​​hydrochloride to a bacterial concentration of 2×10⁻⁶. 10 CFU / mL was used to obtain the bacterial solution for gavage.

[0101] 2. Laboratory animals

[0102] SPF grade 4-week-old male BTBR T + Itpr3 tf Twenty male C57BL / 6J mice (purchased from Jackson Laboratory, USA, strain number 002282) and ten 4-week-old male C57BL / 6J mice (purchased from Jackson Laboratory, USA, strain number 000664) were used. Mice were housed in individually ventilated cages at a density of 5 mice per cage. All mice were ear-tagged and numbered. The ambient temperature and humidity were maintained at 23±1℃ and 50±10%, respectively, with an automatic light-dark cycle of 12 hours. Unless otherwise specified in the experimental procedures below, mice had free access to food and water.

[0103] The BTBR mouse model not only exhibits the core symptoms of ASD (reduced social interaction, fewer ultrasound emissions in social situations, and increased grooming behavior), but also shows similar brain developmental abnormalities and abnormal immunochemical indicators as ASD. Therefore, the BTBR mouse model is an ideal model for studying autism (see references "Gil Sharon, et al. Cell. 2019; Martina Sgritta, et al. Neuron. 2018; Jamshid Faraji, et al. Translational Psychiatry. 2018.").

[0104] 3. Bowel cleansing and drug administration

[0105] 3.1 Antibiotic bowel cleansing in mice

[0106] All mice underwent bowel cleansing with antibiotics.

[0107] The specific steps for antibiotic bowel cleansing are as follows: Vancomycin (50 mg / kg by gavage, Sigma Aldrich, CAS No. 1404-93-9), neomycin (100 mg / kg by gavage, Sigma Aldrich, CAS No. 1405-10-3), and metronidazole (100 mg / kg by gavage, Sigma Aldrich, CAS No. 443-48-1) were dissolved in sterile water and administered orally to mice. The volume of solution administered was 0.1 mL per 10 g body weight. The solution was freshly prepared daily and administered by gavage every 12 hours for 7 consecutive days.

[0108] During the antibiotic bowel cleansing period in mice (7 days), ampicillin was prepared into a 1 mg / mL ampicillin solution using sterile drinking water and given to the mice to drink, with the solution being changed every three days.

[0109] During the antibiotic bowel cleansing period for mice (7 days), all procedures must be performed in a biosafety cabinet, including bedding changes, drinking water changes, food additions, and gavage. It is forbidden to open the cage lid outside the biosafety cabinet or expose the mice to the space outside the biosafety cabinet.

[0110] 3.2 Grouping of mice and administration of drugs by gavage

[0111] 20 BTBR T + Itpr3 tf / J mice were randomly divided into two groups using a block design: a model group (BTBR+Vehicle group) and an experimental group (BTBR+B. infantis group), with 10 mice in each group. Ten male C57BL / 6J mice of the same age and weight were used as a control group (C57+Vehicle group). After a 7-day antibiotic bowel cleanse, all mice were immediately administered the test sample via gavage at a volume of 0.1 mL per 10 g body weight. Mice in both the C57+Vehicle and BTBR+Vehicle groups were gavage with PBS buffer (Gibco) containing 1% (w / v, g / 100 mL) L-cysteine ​​hydrochloride (Sigma-Aldrich, CAS No. 52-89-1). TM (Catalog No. 10010023), BTBR+B. infantis mice were given a bacterial suspension of Bifidobacterium longum infantis subsp. XA-9111 via gavage (each 1 mL of bacterial suspension contained 2 × 10⁻⁶ bacteria). 10 (individual bacterial cells). Gavage was administered once daily at a fixed time for 21 consecutive days before the behavioral test, and continued after the start of the behavioral test until the day before the end of the experiment.

[0112] 4. Behavioral tests

[0113] Before the experiment, mice needed to be undisturbed for 60 minutes in the behavioral testing room. After each test, the behavioral equipment needed to be wiped with 75% (v / v) alcohol, and the alcohol odor was allowed to dissipate before the next mouse was tested. NoldusEthoVision XT software (Noldus Information Technology; Leesburg, VA, USA) was used to record video and track the mice's movements, identifying three areas: the mouse's nose, center of body, and tail. The behavioral experimenters were unaware of the mice's group assignments.

[0114] 4.1 Open Field Test

[0115] Spontaneous mouse behavior was recorded in an open field box (41cm×41cm×38cm) using a camera positioned directly above the box. The central area of ​​the open field was defined as a virtual region of 20cm×20cm. Each mouse was recorded in the open field for 10 minutes, and the total distance traveled (Total Distance, cm) and the percentage of time spent in the central area (Time in Center, %) were recorded and analyzed.

[0116] 4.2 Hair trimming test

[0117] After the open field test, the researchers analyzed the spontaneous grooming behavior of the mice in the open field box over 10 minutes using video analysis, and calculated the total cumulative grooming time.

[0118] 4.3 Bead embedding test

[0119] The mice were placed in a test box (40cm×20cm×30cm, with a bedding depth of 5cm) and a 30-minute video recording was performed. The box contained 20 glass beads (arranged in 4 rows of 5 beads each, with a consistent spacing of 4cm between the beads). At the end of the 30-minute mark, the mice were carefully removed from the box. Beads that were buried more than 2 / 3 of their volume were recorded as buried beads, and the number of buried beads was counted.

[0120] 4.4 Three-box social test

[0121] The three-chamber socialization test was conducted in a 60cm×40cm×22cm blue acrylic box. The box was divided into three 20cm×40cm×22cm areas (left, center, and right) by two partitions (transparent acrylic glass with a 5cm×8cm sliding door in the central area). Each of the left and right chambers had a circular cage (10cm in diameter, 18cm high, 3mm in diameter grid, 7mm grid spacing) in the center. A camera was positioned directly above the central chamber to record video and track the mouse's movements. First, the mouse was placed in the box from the central chamber and allowed to move freely within the three chambers, becoming familiar with the environment of the three-chamber setup with circular cages in each chamber (without any objects inside). This was recorded for 10 minutes. Then, a mouse (strain, sex, and age identical to the experimental mouse) was placed in a cage on one side of the box, not in the same cage as the experimental mouse. This mouse was placed in the box from the central chamber. The cage on the other side was empty, and the mouse was placed in the box from the central chamber and allowed to explore freely. This was recorded for 10 minutes.

[0122] 5. Analysis of behavioral results

[0123] Open field tests showed that compared with the C57+Vehicle group (total movement distance 3487.18±70.71cm), the total movement distance of the BTBR+Vehicle group was significantly increased (to 4190.62±139.00cm), while the total movement distance of the BTBR+B. infantis group, which was administered Bifidobacterium infantis XA-9111 via gavage, was significantly decreased (to 3718.87±184.70cm). Figure 8 a) One-way ANOVA, F (2,27) =6.598, P=0.0046; Dunnett post-hoc test: P C57+VehicleVSBTBR+Vehicle =0.0014, P BTBR+VehiclevsBTBR+B.infantis=0.0241); meanwhile, compared with the C57+Vehicle group mice (whose activity time in the open field center area was 14.31±1.20%), the BTBR+Vehicle group mice had a significantly reduced activity time in the open field center area (reduced to 9.61±0.59%), while the BTBR+B. infantis group mice administered Bifidobacterium infantis subsp. XA-9111 by gavage had an increased activity time in this area (increased to 14.62±1.59%). Figure 8 b, One-way ANOVA, F (2,27) =5.517, P=0.0098; Dunnett post-hoc test: P C57+Vehicle VS BTBR+Vehicle =0.0098, P BTBR+Vehicle vs BTBR+B.infantis =0.0063). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve hyperactivity and anxiety behaviors in BTBR model mice.

[0124] The results of the grooming test showed that, compared with the C57+Vehicle group mice (grooming time 70.20±3.81s), the BTBR+Vehicle group mice had a significantly increased cumulative grooming time in the open field test (increased to 120.90±5.22s), while the grooming time of the BTBR+B. infantis group mice administered Bifidobacterium infantis subsp. XA-9111 by gavage was significantly reduced compared with the model group mice (reduced to 71.00±3.09s). Figure 9 One-way ANOVA, F (2,27) =49.33, P<0.0001; Dunnett post-hoc test: P C57+Vehicle VS BTBR+Vehicle <0.0001, P BTBR+Vehicle vs BTBR+B.infantis <0.0001). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve repetitive stereotyped behaviors in BTBR model mice.

[0125] The bead embedding test results showed that compared with the C57+Vehicle group mice (2.30±0.37 beads), the number of beads embedded in the BTBR+Vehicle group mice was significantly increased (to 5.00±0.58 beads), while the number of beads embedded in the BTBR+B. infantis group mice administered Bifidobacterium infantis subsp. XA-9111 via gavage was significantly decreased compared with the model group mice (to 2.30±0.62 beads). Figure 10 One-way ANOVA, F (2,27) =8.610, P=0.0013; Dunnett post-hoc test: P C57+Vehicle VS BTBR+Vehicle =0.0013, P BTBR+Vehicle vs BTBR+B.infantis =0.0013). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve repetitive stereotyped behaviors in BTBR model mice.

[0126] The results of the three-box socialization test showed that, during the socialization test phase, compared with the C57+Vehicle group mice (exploration time in the unfamiliar cage area 126.82±12.81s), the BTBR+Vehicle group mice showed no significant preference for exploring unfamiliar mice (exploration time 72.86±8.98s), while the BTBR+B. infantis group mice administered Bifidobacterium longum subsp. infanti XA-9111 via gavage showed a significantly increased exploration time for unfamiliar mice compared with the model group (increasing to 159.82±18.39s). Figure 11 Two-way ANOVA, F (2,54) =16.01, P<0.0001; Tukey post-hoc test: P C57+Vehicle VS BTBR+Vehicle =0.0057, P BTBR+Vehicle vs BTBR+B.infantis <0.0001). This indicates that Bifidobacterium longum infant subspecies XA-9111 can improve social impairment in BTBR model mice.

[0127] In summary, Bifidobacterium longum infant subspecies XA-9111 can improve autism-like behaviors in BTBR model mice, including hyperactivity, anxiety, repetitive and stereotyped behaviors, and social impairment.

[0128] Experimental Example 5: Effects of Bifidobacterium longum infantis subspecies XA-9111 on neuronal activation in the brains of BTBR mice

[0129] This experimental example demonstrates the effect of Bifidobacterium longum infantis subspecies XA-9111 on neuronal activation in the brains of BTBR mice. The experimental procedure is as follows:

[0130] 1. Preparation and preprocessing of brain slice samples

[0131] Based on Experiment 4, mice in each group were induced to have anesthesia with 3% (v / v) isoflurane (Shenzhen Ruiwode Biotechnology Co., Ltd.), and then maintained in a state of 1.5% (v / v) isoflurane anesthesia. The mice were then fixed with their abdomens facing upwards and their limbs immobilized. The thoracic cavity of the mice was then opened to expose the heart. An intravenous injection needle was inserted parallel to the left ventricle of each group of mice and fixed in place. 40 mL of phosphate buffer and 30 mL of [unspecified solution] were continuously perfused into the heart of the mice using a 10 mL syringe. Tissue fixation was performed using 4% (w / v, g / 100mL) paraformaldehyde (Shanghai Maclean Biotechnology Co., Ltd., CAS No.: 30525-89-4). After tissue fixation, the brains of mice in each group were removed using instruments on the operating table and immersed in 4% (w / v, g / 100mL) paraformaldehyde, taking care to avoid damaging the brain structure during this process. After immersion in 4% (w / v, g / 100mL) paraformaldehyde for two days, the mouse brains were first transferred to 15% (w / v, g / 100mL) sucrose solution for dehydration for one day, and then transferred to 30% (w / v, g / 100mL) paraformaldehyde solution. The brain tissue was dehydrated for one day with a 100mL sucrose solution and then dried. The olfactory bulb of the brain was then immersed in cryoembedding solution (Tissue-Tek, Ref 4583) with the olfactory bulb facing upwards and cryoembedded at -20°C for 1 hour. After embedding, the brain tissue was sliced ​​in a cryostat (Leica, CM1950) at -20°C, starting from the prefrontal cortex. The entire brain was sliced ​​at a thickness of 40μm per slice. The sliced ​​brain slices were stored in cryoprotectant (Tissue-Tek, REF 4583) at -20°C for cryopreservation.

[0132] 2. Staining of brain sections

[0133] After removing the brain slices from the cryoprotectant, they were first washed in phosphate buffer for 10 min, then washed three times with phosphate-Tween buffer for 5 min each time. After washing, they were blocked with 20 μL of goat serum blocking buffer (ABACM, AB7481) at room temperature (25℃) for 1 h, then 20 μL of c-FOS primary antibody (ABCAM, AB190289, diluted 500 times with phosphate buffer) was added and incubated at 4℃ for 16 h. After incubation, the brain slices were first transferred from the primary antibody to phosphate-Tween buffer for 5 min, 15 min, and 30 min respectively, then 20 μL of anti-rabbit 488 secondary antibody (ABCAM, AB150077, diluted 1000 times with phosphate buffer) was added and incubated at room temperature (25℃) in the dark for 4 h. After incubation, they were washed three times with phosphate-Tween buffer in the dark for 10 min each time, then 20 μL of c-FOS primary antibody (ABCAM, AB150077, diluted 1000 times with phosphate buffer) was added. DAPI (abcam, ab228549, diluted 10 times with phosphate buffer before use) was incubated at room temperature (25°C) in the dark for 5 min, followed by washing with phosphate buffer for 10 min. The brain slices were then transferred to a glass slide. After transfer, the brain slices were allowed to air dry thoroughly, and then 10 μL of mounting medium (abcam, ab15104135) was added. Finally, a coverslip was placed on top to seal the slide. After sealing, the slides were allowed to air dry naturally until imaging was achieved.

[0134] 3. Microscopic imaging

[0135] For single brain slices, after previewing the image using the panoramic tissue quantitative analysis system (Leica, Tissue FAXS Plus) at 5x magnification, the focus range was set, and the entire brain slice was progressively stitched together using the 488 green fluorescence channel and the DAPI fluorescence channel at 20x magnification. After imaging, the complete image of the stitched brain slice containing all complete information from the 20x magnification view was exported from both fluorescence channels and subsequently transferred to ImageJ software for data analysis. In ImageJ software, the brain regions that needed to be counted were selected in the brain slice, and then the number of c-FOS positive neurons was counted.

[0136] 4. Results Analysis

[0137] like Figure 12 and Figure 13 As shown, compared to the BTBR+Vehicle group mice (c-FOS) + Neuron number: 148.80 ± 12.08; striatal c-FOS of BTBR mice treated with XA-9111. +The number of neurons decreased significantly (to 102.80 ± 10.33). This indicates that *Bifidobacterium longum* subsp. infantis XA-9111 can reduce repetitive stereotyped behaviors in BTBR mice by inhibiting the overactivation of striatal neurons in the brain.

[0138] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of Bifidobacterium longum infantis ( Bifidobacterium longum subsp. infantis ), characterized in that, The *Bifidobacterium longum* subsp. *infantii* is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26615.

2. The use of *Bifidobacterium longum* subsp. *infantitidis* as described in claim 1 in the preparation of a medicament for treating autism spectrum disorder, characterized in that... The treatment of autism spectrum disorder includes improving the core symptoms and / or complications of autism spectrum disorder; the core symptoms of autism spectrum disorder include repetitive and stereotyped behaviors and / or social impairments; the complications of autism spectrum disorder include hyperactivity and / or anxiety behaviors; The drug contains live bacteria of *Bifidobacterium longum* subsp. infantis as described in claim 1.

3. The application as described in claim 2, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

4. The application as described in claim 3, characterized in that, Pharmaceutically acceptable carriers include surfactants, excipients, stabilizers, suspending agents, isotonic agents, preservatives, fillers, disintegrants, antioxidants, emulsifiers, coating agents, binders, lubricants, and / or flavoring agents.

5. A drug for treating autism spectrum disorder, characterized in that, The drug contains live bacteria of Bifidobacterium longum subsp. infantis as described in claim 1.

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