Application of lactobacillus sanfrancisco to prevention and / or treatment of attention deficit hyperactivity disorder

Abstract

The present invention belongs to the field of microorganisms, and more particularly, the present invention relates to the use of Lactobacillus sanfranciscensis in the prevention, diagnosis and / or treatment of attention deficit hyperactivity disorder, and a microbial preparation for the prevention and / or treatment of attention deficit hyperactivity disorder. Therefore, a further evidence is provided for the correlation between the intestinal microbiota and the ADHD, and a new candidate is also provided for the treatment of the ADHD.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and more specifically, the present invention relates to Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis ) in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder and a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder. Background Art

[0002] Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a high prevalence worldwide. [1] , which is characterized by inattention, hyperactivity, and impulsivity, places a heavy burden on many children and adolescents [2，3] Although the etiology of ADHD is complex, researchers agree that genetic influences and environmental factors play a major role in the development of this disorder. Today, the importance of the gut microbiota in a variety of psychiatric disorders has been demonstrated through the microbiota-gut-brain axis. [4，5] The human intestinal microbiome is composed of more than 1,000 bacterial species, which are involved in metabolism, immune response, synthesis and other functions. [6-8] , plays an important role in maintaining human health. However, when the intestinal microbiota is disturbed, it may cause various diseases. Existing studies have shown that the imbalance of intestinal microbiota is related to a variety of mental illnesses, including autism spectrum disorder, schizophrenia, bipolar depression [4，5，9] Over the past few years, about a dozen studies have explored the potential link between the gut microbiome and ADHD, but there is no consensus among researchers. [10-19] .

[0003] Therefore, there is an urgent need in this field to conduct more in-depth research on the intestinal microbiota of ADHD patients in order to provide a new solution for preventing and / or treating attention deficit hyperactivity disorder through intestinal microbiota. Summary of the invention

[0004] As mentioned above, most existing studies on the gut microbiota of ADHD patients are still focused on identifying differential bacteria at the genus or family level. More in-depth studies on the gut microbiota of ADHD patients are urgently needed to understand the role of the gut microbiota in the pathogenesis of ADHD and provide a new approach to prevent and / or treat ADHD through gut microbiota.

[0005] In view of this, in a first aspect, the present invention provides Lactobacillus sanfrancisco ( Lactobacillus_ sanfranciscensis ) for use in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder.

[0006] In some embodiments, the attention deficit hyperactivity disorder is characterized by a lack of or a lower abundance of Lactobacillus sanfrancisco compared to a healthy individual.

[0007] In some embodiments, the product comprises a functional food or a pharmaceutical.

[0008] In some embodiments, the product is administered gastrointestinally.

[0009] In some embodiments, the dosage forms of the drug include pills, powders, capsules, tablets, suspensions, and granules.

[0010] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0011] In a second aspect, the present invention provides a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder, the microbial preparation comprising Lactobacillus sanfrancisco.

[0012] In some embodiments, the content of Lactobacillus sanfrancisco is at least 1×10 8 CFU / dose.

[0013] In some embodiments, the microbial preparation is administered gastrointestinal tract.

[0014] In a third aspect, the present invention provides a method for preventing and / or treating attention deficit hyperactivity disorder, comprising administering Lactobacillus sanfranciscous or the microbial preparation of the second aspect of the present invention to a subject in need thereof.

[0015] In some embodiments, the attention deficit hyperactivity disorder is characterized by a lack of or a lower abundance of Lactobacillus sanfrancisco compared to a healthy individual.

[0016] In some embodiments, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco deposited in the American Type Culture Collection with the deposit number ATCC27651.

[0017] In some embodiments, the Lactobacillus sanfrancisco is isolated from the intestinal microbiota of a stool sample of a healthy human.

[0018] In some embodiments, the Lactobacillus sanfrancisco is isolated from the intestinal microbiota of an autologous fecal sample of a subject.

[0019] The beneficial effects of the present invention are: by studying the composition of the intestinal microbiota of ADHD patients at the bacterial species level, further evidence is provided for the correlation between the intestinal microbiota and ADHD, thereby proposing the use of probiotics in the preparation of products for the prevention and / or treatment of attention deficit hyperactivity disorder, and providing new candidates for the prevention and treatment of ADHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a brief flow chart of the zebrafish experimental groups and experiments.

[0022] Figure 2 are photos of bacterial growth in different groups.

[0023] Figure 3 It is a statistical chart of the development indicators of each group of fry (including mortality and deformity rates).

[0024] Figure 4 This is a graph showing the trajectory of zebrafish spontaneous activity detected at 5 dpf.

[0025] Figure 5 This is a comparison of the spontaneous activity speed of zebrafish in the Lactobacillus sanfrancisco colonization group (MC) and other groups (* p <0.05, ** p <0.01).

[0026] Figure 6 It is a marker of noradrenergic nerves in each group of zebrafish dβh and dopaminergic neuron markers th Quantitative gene expression (* p <0.05, *** p <0.001).

[0027] Figure 7 Comparison of the spontaneous activity speed of zebrafish in the atomoxetine-treated group (TO) and other groups (5 dpf, *** p <0.001).

[0028] Figure 8 This is a comparison of the movements of the San Francisco Lactobacillus treatment group (FMT-AA) and the control group (FMT-AC). The right picture is a schematic diagram of the trajectory, and the left picture is a statistical result diagram ( *p<0.05 , **p<0.01 ).

[0029] Fig. 9 This is the 5-CSRTT test results of the San Francisco Lactobacillus treatment group (FMT-AA) and the control group (FMT-AC) ( *p ＜0.05, **p＜0.01 ).

[0030] Fig.10 This is the Beta diversity (PCoA) analysis result of each group of mice and wild-type mice (WT) after microbiota transplantation and treatment.

[0031] Fig.11 This is a graph showing the results of Lactobacillus abundance analysis in each group of mice and wild-type mice (WT) after microbiota transplantation and treatment. DETAILED DESCRIPTION

[0032] The present invention will be described clearly and completely below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by those of ordinary skill in the art based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0033] Unless otherwise stated, all terms used herein should be understood as common meanings known in the art. Other more specific definitions of certain terms used in this application are described below, and are intended to be uniformly applied throughout the specification and the scope of the invention patent application, unless the definition explicitly listed otherwise provides a broader definition. Throughout the specification and the scope of the invention patent application, the word "include" and its variants are not intended to exclude other technical features, additives, components or steps. In addition, the word "include" covers the situation of "consisting of". By reading the specification, other objects, advantages and features of the present invention will become apparent to those skilled in the art, or can be learned by implementing the present invention. In addition, the present invention covers all possible combinations of the specific and specific embodiments described herein.

[0034] In this specification and the accompanying invention patent scope, the indefinite article term ("a", "an") and the definite article term ("the") in the singular form include plural indicators. Unless otherwise clearly indicated by the context. The indefinite article term "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably herein. In addition, the "and / or" used in this article should be regarded as a clear disclosure that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" (for example, "A and / or B") used in the phrase in this article is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" (for example, "A, B and / or C") used in the phrase is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure relates. Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers defining the ranges. The headings provided herein are not limitations on the various aspects or aspects of the present disclosure, which can be obtained by reference to the entire specification as a whole. Therefore, the terms defined directly below are more fully defined by reference to the entire specification.

[0035] As mentioned above, previous studies on the gut microbiota of ADHD patients have confirmed that the gut microbiota is closely related to the occurrence of ADHD, but most studies focus on identifying differential bacteria at the genus or family level. Therefore, there is an urgent need for more in-depth studies on the gut microbiota of ADHD patients to provide new solutions or new evidence for the prevention and / or treatment of ADHD through gut microbes. To fill the above gaps, the inventors adopted strict inclusion and exclusion criteria and used shotgun metagenomic sequencing to comprehensively analyze the composition of the gut microbiota in the fecal samples of all participants to explore the differences and roles of the gut microbiota in ADHD patients and healthy controls (HCs). The inventors recruited 188 participants, including 94 drug-naive patients diagnosed with ADHD and 94 HCs. Metagenomic sequencing of fecal samples was performed by the Beijing Genomics Institute using the MGISEQ 2000 platform and PE150 mode. Compared with other sequencing methods (such as 16S rRNA sequencing), shotgun metagenomic sequencing can provide more reliable microbial information and extend the classification resolution to the species level.

[0036] Subsequently, this study used linear discriminant analysis (LEfSe) and statistical analysis of metagenomic profiles (STAMP) algorithms to distinguish and analyze the intestinal microorganisms in different experimental groups. LEfSe and STAMP are two analytical methods used to distinguish and locate different intestinal microorganisms between different experimental groups. LEfSe identifies microbial taxa with statistically significant differences, while STAMP provides more statistical tests and effect size measurements. The combination of these two methods can provide a more comprehensive and in-depth analysis. This study used LEfSe analysis to identify microbial taxa with significant differences between the two groups, and then used STAMP analysis to verify the statistical significance of different taxa and calculate the effect size.

[0037] By combining the results of LEfSe and STAMP analyses, we identified seven species with significant differences between the ADHD and HC groups, five of which were significantly more abundant in the HC group, including Lactobacillus sanfranciscous ( Lactobacillus sanfranciscensis ), Pseudomonas (Paraprevotella xylaniphila) Bacteroides (Bacteroides zoogleoformans), Bacteroides heparinolyticus (Bacteroides heparinolyticus), Prevotella rumenicola (Prevotella ruminicola) , two species were significantly more abundant in the ADHD group, including Haemophilus parainfluenzae ( Haemophilus parainfluenzae salivarius ( Streptococcus salivarius ). Although five strains were significantly more abundant in the HC group compared to the ADHD group, four of them are anaerobes and cannot survive in water for long periods of time. Only Lactobacillus sanfranciscoensis (a facultative anaerobe) could survive under aerobic conditions.

[0038] On the basis of the above experimental results, the inventors further transplanted the low-abundance candidate strain (Lactobacillus sanfrancisco) found in ADHD patients into zebrafish and mouse models for in vivo experiments. The study found that supplementation with Lactobacillus sanfrancisco corrected the overactive behavior in zebrafish (GF group). In addition, human intestinal microbiota with Lactobacillus sanfrancisco deficiency characteristics can induce overactive behavior in mice, and such behavioral manifestations can be eliminated by supplementation with Lactobacillus sanfrancisco, confirming the relationship between ADHD and the abundance of Lactobacillus sanfrancisco in intestinal microorganisms, thereby completing the present invention.

[0039] Therefore, in a first aspect, the present invention provides Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis ) for use in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder.

[0040] The inventors found that Lactobacillus sanfrancisco has the effect of treating and preventing ADHD, and then found the relationship between ADHD and the abundance of Lactobacillus sanfrancisco in intestinal microorganisms, thus proving that ADHD can be treated and prevented by supplementing Lactobacillus sanfrancisco. In the study, the inventors tested Lactobacillus sanfrancisco isolated from the intestinal microbiota of fecal samples of several healthy control groups and Lactobacillus sanfrancisco deposited in the American Type Culture Collection with the deposit number ATCC27651 ( Lactobacillus_sanfranciscensis ) (Weiss and Schillinger 1984, DSM 20451 T , Leibniz Institute DSMZ, Germany), found that there was no significant difference in the effects of Lactobacillus sanfrancisco from different sources. Since Lactobacillus sanfrancisco is a common probiotic in the human intestine, those skilled in the art should understand that Lactobacillus sanfrancisco from other sources can also achieve similar effects. The present invention is not limited to a specific Lactobacillus sanfrancisco strain that has the function of diagnosing, treating and preventing ADHD, but has discovered the relationship between ADHD and the abundance of Lactobacillus sanfrancisco in intestinal microorganisms.

[0041] In an experiment using a mouse model that is closer to the human intestinal microbiota, the inventors divided the mice into three groups, namely a group colonized with healthy bacteria (FMT-H), an ADHD sample group colonized with high-abundance pathogenic bacteria (Haemophilus parainfluenzae and Streptococcus salivarius) (FMT-B), and an ADHD sample group colonized with low-abundance probiotics (Lactobacillus sanfrancisco deficiency) (FMT-A). Both colonization with high-abundance pathogenic bacteria and low-abundance probiotics can cause overactive behavior in mice, but after supplementing the two groups of mice with Lactobacillus sanfrancisco, only the overactive behavior of mice in the low-abundance probiotic group disappeared, while there was no change in the high-abundance pathogenic bacteria group.

[0042] In the present invention, Lactobacillus sanfranciscosi can be used to prepare various products for preventing and / or treating attention deficit hyperactivity disorder, including functional foods or drugs, such as dietary supplements, solid beverages, and dairy products.

[0043] Since Lactobacillus sanfrancisco is derived from the intestinal microbiota, the products prepared using it are preferably administered through the gastrointestinal tract. Therefore, all product types or pharmaceutical dosage forms suitable for administration through the gastrointestinal tract are suitable for the present invention. In some embodiments, the dosage forms of the drug include pills, powders, capsules, tablets, suspensions and granules.

[0044] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0045] In a second aspect, the present invention provides a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder, the microbial preparation comprising Lactobacillus sanfrancisco ( Lactobacillus_sanfranciscensis ).

[0046] In this article, the so-called "microecological preparations" are live microbial preparations made from normal microorganisms or substances that promote the growth of microorganisms. In other words, all preparations that can promote the growth and reproduction of normal microorganisms and inhibit the growth and reproduction of pathogenic bacteria are called "microecological preparations."

[0047] In some embodiments, the content of Lactobacillus sanfrancisco is at least 1×10 8 CFU / dose.

[0048] In some embodiments, the microbial preparation is administered gastrointestinal tract.

[0049] Those skilled in the art will appreciate that the above descriptions related to attention deficit hyperactivity disorder in the first aspect of the present invention are all applicable to the second aspect of the present invention, and therefore will not be repeated here.

[0050] The present invention will be further described below by the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. Example

[0051] Materials and methods

[0052] 1. Zebrafish Experiment Design

[0053] All zebrafish experiments were performed using wild-type AB zebrafish. Zebrafish were raised at a density of 8-10 / L at 28°C, and germ-free zebrafish larvae were cultured using the aseptic development of zebrafish embryos.

[0054] To investigate how the microbiota influences behavior, juvenile fish were divided into five groups of 200 each, as follows: germ-free group (GF), conventionally reared group (CV), conventionally reared group with sterile treatment (CVZ), microbial colonization group (MC), and atomoxetine treatment group (TO). The CV group was reared in normal zebrafish culture medium without any sterile treatment, while the GF, CVZ, MC, and TO groups were sterile treated. After sterile treatment, embryos were collected into cell culture bottles containing either germ-free zebrafish culture medium (GF, MC, and TO groups) or regular zebrafish culture medium (CVZ group). At 3.5 days post fertilization, germ-free zebrafish in the MC group were placed in culture medium containing Lactobacillus sanfrancisco. On day 5 post fertilization, behavioral tests were performed on zebrafish in each group. On the day of behavioral testing, the TO group was treated with 10 μmol / L atomoxetine for 1 hour before behavioral testing and analysis together with other groups. Figure 1 The grouping and brief process of the experiment are shown.

[0055] 2. Sterile Derivation of Zebrafish Embryos

[0056] Refer to the existing methods for culturing germ-free zebrafish

[24] Table 1 shows the timetable for constructing germ-free zebrafish embryos. On the afternoon of the day before spawning (day -1), healthy adult zebrafish females and males were placed at the two ends of a separated breeding cage. On day 0, at the beginning of the light period, the females and males were transferred to sterile water together, and the females and males were allowed to chase each other to spawn. The fertilized eggs were collected within 60 minutes to ensure that all fertilized eggs were at the same developmental stage. The fertilized eggs were transferred to a culture dish containing antibiotic culture medium (containing 250 mg / mL amphotericin B, 5 μg / mL kanamycin, and 100 μg / mL ampicillin) and surface sterilized for 6 hours. Subsequently, they were rinsed with 0.1% polyvinylpyrrolidone-iodine (PVP-I) and 0.003% sodium hypochlorite for surface sterilization, and then the fertilized eggs were placed in a sterile cell culture bottle (the bottle mouth was a 0.2 μm filter membrane, which could filter microorganisms in the air to ensure a sterile environment in the culture bottle) and incubated at 28°C. Embryos were checked daily for sterility and viability.

[0057] Table 1. Timeline of steps for derivation of germ-free zebrafish embryos

[0058] To confirm the microbial status of each group, daily testing was performed to ensure sterility and to characterize the microbiota of the zebrafish larvae groups. 10 μL of culture medium from each group's culture flask was inoculated on nutrient agar (LB) plates and cultured overnight at 28°C. In addition, before behavioral testing, 50 zebrafish larvae from each group were ground under sterile conditions and evenly spread on nutrient agar plates for overnight culture to further confirm sterility. The results showed that no bacterial growth was observed in the plates of the GF group, while bacterial growth was observed in the plates of the CV and CVZ groups ( Figure 2 ).

[0059] The developmental indicators of each group of fry were then evaluated before behavioral testing ( Figure 3 ), no difference was found, which shows that behavioral detection is feasible.

[0060] 3. Strain Cultivation

[0061] The inventors isolated Lactobacillus sanfrancisco from the intestinal microbiota of fecal samples from several healthy control groups. The isolation steps are briefly described as follows: dilute the mixed feces, use MRS agar medium, a selective medium commonly used for lactic acid bacteria culture, to culture monoclonal bacteria, complete preliminary screening by observing the morphology of the monoclonal culture, and then culture it in MRS medium at 30°C, and confirm the culture by rRNA sequencing. In addition to isolating Lactobacillus sanfrancisco from the intestinal microbiota of fecal samples from healthy control groups, the inventors also tested Lactobacillus sanfrancisco, which is deposited in the American Type Culture Collection with the deposit number ATCC27651 ( Lactobacillus_sanfranciscensis ) (Weiss and Schillinger 1984, DSM 20451T, Leibniz Institute DSMZ, German) was purchased from Bioscibio.

[0062] Lactobacillus sanfranciscoensis was cultured in MRS medium at 30°C. The culture medium was centrifuged at 8000 rpm for 5 minutes, the bacteria were collected, and washed twice with sterile PBS. The bacterial concentration was 1×10 5 CFU / mL. When selecting the concentration of Lactobacillus sanfrancisco, due to the lack of relevant literature on the colonization of Lactobacillus sanfrancisco in zebrafish larvae, the inventors referred to the relevant literature on the colonization of other strains in zebrafish larvae, and on this basis, adopted 1×10 5 Up to 1×10 8 The results showed that there was no significant difference in the effect of Lactobacillus sanfrancisco from different sources. The concentration of Lactobacillus sanfrancisco was finally selected as 1×10, which had the least effect on the development of zebrafish larvae. 5 For the consistency of the experiment, Lactobacillus sanfrancisco purchased from Biosai Biotech was used in the examples of this application. Since Lactobacillus sanfrancisco is a common probiotic in the human intestine, those skilled in the art can understand that Lactobacillus sanfrancisco from other sources can also achieve similar effects, which are included in the scope of the present invention.

[0063] 4. Behavioral Detection

[0064] At 5 dpf, 96 larvae from each group were randomly selected for behavioral analysis. Larvae were placed in each well of a 96-well plate (4 groups of 24 larvae were placed in each 96-well plate for a total of 4 experiments), and the speed and distance of movement over a period of time were analyzed using a zebrafish behavior analysis system (DanioVision with EthoVision XT 14.1324, Noldus, Netherlands). The first 15 minutes of the measurement was an adaptation phase, and the behavioral trajectory and speed were recorded for the next 45 minutes. This was repeated 3 times.

[0065] 5. Real-time PCR for gene expression of noradrenergic and dopaminergic neural markers

[0066] Real-time PCR was used to detect noradrenergic neuron markers in each group of zebrafish dβh and dopaminergic neuron markers th Quantitative gene expression of ADHD. ADHD is associated with dysfunction of monoamine neurons (including norepinephrine neurons and dopamine neurons). Tyrosine hydroxylase (TH) is a key enzyme that synthesizes dopamine using tyrosine as a substrate, and dopamine β-hydroxylase (DβH) catalyzes the conversion of DA to NE. Therefore, the expression levels of TH and DβH can be used as markers of monoamine neuron activity. Total RNA was extracted using the Trizol method. β-actin was used as an internal reference. The specific primer sequences are shown in Table 2.

[0067] Table 2. Primer sequences used for real-time PCR

[0068] 6. Atomoxetine treatment

[0069] Atomoxetine is an effective drug for the clinical treatment of ADHD. In the experimental preparation stage, atomoxetine powder (TOCRIS Company, the United States) was dissolved in ddH2O. Before the behavioral test, the zebrafish larvae in the TO group were transferred to 10 μmol / L atomoxetine culture medium for 1 hour, and then the behavioral analysis was performed together with the other groups.

[0070] 7. Mouse Experiment Design

[0071] Four-week-old male C57BL / 6J mice (n=60), weighing 9 to 13 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed at 22 to 23°C with a 12-hour light / dark cycle. After a 1-week acclimation period, mice were given free access to food and water. Subsequently, mice were orally administered a mixture of four antibiotics (ABX) for 7 days: ampicillin (200 mg / kg / day, Solarbio Cat#A6920), vancomycin (100 mg / kg / day, VIANEX SA PLANTC), neomycin (200 mg / kg / day, SIGMA Cat# N6386), and metronidazole (200 mg / kg / day, SIGMA Cat# M1547). [25，26] This regimen resulted in an enlarged cecum and a significant reduction in the gut microbiota in mice (demonstrated by fecal 16S rRNA analysis).

[0072] Subsequently, the mice were divided into groups and received fecal microbiota transplantation (FMT) from different sources: colonized healthy bacteria group (FMT-H, n=13), colonized high-abundance pathogenic bacteria (Haemophilus parainfluenzae and Streptococcus salivarius) ADHD sample group (FMT-B, n=18) and colonized low-abundance probiotics (Lactobacillus sanfrancisco-deficient) ADHD sample group (FMT-A, n=20). Lactobacillus sanfrancisco (FMT-BA / FMT-AA group, 1x10^8 / mouse / day), butyrate (FMT-BB / FMT-AB group, 20 mg / 0.5 ml) or PBS (FMT-BC / FMT-AC group) were added to the drinking water of mice in the FMT-B group and the FMT-A group, respectively. Because there was no previous reference for direct colonization of Lactobacillus sanfrancisco, the inventors referred to Lactobacillus reuteri ( Lactobacillus reuteri ) colonization concentration (Sgritta et al., 2019). After two rounds of colonization, each mouse was subjected to behavioral analysis using the open field test (open field test box volume 40 × 40 × 25 cm). In addition, the 5-CSRTT was used to evaluate the attention and impulsivity levels of the mice. Compared with mice with unrestricted food intake, the mice were deprived of food to maintain 85% of their body weight. This task involves training mice to recognize the location of visual stimuli in order to obtain food rewards. The training was carried out in seven consecutive stages, each with specific criteria that the mice had to meet for two consecutive days before they could enter the next stage.

[37] . Typical training sessions lasted 30 min or 100 trials, whichever came first. In correct trials, the latency of the correct response was defined as the time interval from the onset of visual stimulation to the time when the subject poked its nose into the cue hole, which can reflect the mouse's attention level. If the mouse poked its nose into an unlit hole it was recorded as an incorrect response. If the mouse failed to indicate the cue location within the specified time limit, it was recorded as an omission. In addition, nose poking that occurred before the cue presentation was classified as a premature response, which can reflect the mouse's impulsiveness level. Throughout the experiment, the weight changes of mice were monitored before and after ABX treatment and before and after bacterial colonization in each group. In addition, fecal 16S rRNA of mice was monitored at baseline, after antibiotic treatment, after transplantation, and after treatment.

[0073] 8.16S rRNA gene analysis

[0074] DNA extraction: approximately 100-200 mg of each meta-sample was transferred to a centrifuge tube containing grinding beads and 1 mL of ATL / PVP-10 buffer; the sample was homogenized using a grinder and incubated at 65°C for 20 min; the lysate was centrifuged at 14,000 × g for 5 min and the supernatant was transferred to a new centrifuge tube; 0.6 mL of PCI buffer was added and then vortexed for 15 seconds to achieve protein precipitation; the mixture was centrifuged at 18,213 × g for 10 min and the resulting supernatant was transferred to a deep-well plate containing magnetic beads and binding solution (600 μL of wash buffer (containing magnetic beads) + 20 μL of proteinase K + 5 μL of RNase A), washed successively with wash buffer 1 (700 μL), wash buffer 2 (700 μL) and wash buffer 3 (700 μL), and finally eluted with 100 μL of elution buffer; the purified DNA was collected in a 1.5 mL tube for further analysis. Construction and sequencing of amplicon libraries: The 16S rRNA gene consists of 9 variable regions and 10 conserved regions; variable regions such as V3–V4 were amplified to characterize bacterial phylogeny and community diversity; sequencing primers were designed for these regions and combined with fusion primers for amplification and high-throughput sequencing. Library construction: Genomic DNA was quantified and PCR amplified using region-specific primers. Amplified products were purified and their quality was tested using standard protocols. Sequencing libraries were prepared and validated before sequencing on the Illumina platform to ensure high accuracy and high coverage. Raw sequencing reads were processed to generate clean data: Primer and adapter sequences were trimmed using cutadapt v2.6. Low-quality reads were removed using a sliding window method (30 bp window, average quality < 20). Reads shorter than 75% of the original length after trimming were discarded. Reads containing ambiguous bases (n) or low-complexity sequences (≥10 consecutive identical bases) were removed (software used: iTools Fqtools fqcheck v0.25, cutadapt v2.6, readfq v1.0); the DADA2 method in QIIME2 was used to generate amplicon sequence variants (ASVs) with 100% sequence similarity. The process includes: importing filtered paired-end reads using the QIIME tool. De-noising and constructing feature tables using QIIME DADA2 denoising pairs. Exporting feature tables using QIIME tools for downstream analysis; classifying representative sequences of each OTU against the reference database using RDP ClassifierV2.2 with a confidence threshold of 0.6.

[0075] After obtaining the genus-level ASV, the data were standardized to ensure comparability between samples. R4.4.1 was then used for downstream analysis of the standardized data. Alpha diversity analysis: Indicators such as the Shannon and Simpson indices were calculated to assess diversity within the sample, reflecting richness and evenness; Beta diversity analysis: Principal coordinate analysis (PCoA) based on the Bray-Curtis distance visualized the differences in microbial community composition between different groups; Genus-level bar charts: The relative abundance of the genus was calculated and visualized as a stacked bar chart to show the microbial composition of samples and groups; Target genus comparison: The relative abundance of target genera (such as Lactobacillus) between groups was statistically compared using the Wilcoxon rank sum test, and significant differences were highlighted.

[0076] 9. Behavioral Data Analysis

[0077] Behavioral data were collected using a video tracking system (video behavior analysis software Ethovision XT, Noldus Information Technology, American). Statistical analysis was performed using Prism 10.1.2 with unpaired t-test. Python 3.9 (available from https: / / github.com / sraorao / animapp_desktop) was used.

[29] Create a model diagram.

[0078] Experimental Results

[0079] 1. Supplementation with Lactobacillus sanfranciscous corrects hyperactive behavior in zebrafish models

[0080] according to Figure 4 , Figure 5 and Table 3. At 5 dpf, the GF group showed hyperactive behavior compared with the CV and CVZ groups. To confirm whether the hyperactive behavior of zebrafish in the GF group is a phenotype of ADHD, the inventors used real-time PCR to detect noradrenergic neural markers in zebrafish in the CV, CVZ, and GF groups. dβh and dopaminergic neuron markers th Quantitative expression of . Figure 6 It can be seen that although there was no significant difference in th expression among the groups, the expression of dβh in the GF group zebrafish was significantly downregulated compared with other groups. This indicates that the activity of the noradrenal system of the germ-free zebrafish in the GF group decreased.

[0081] Next, the inventors tried to treat the GF group with the ADHD drug (atomoxetine) to determine the effectiveness of atomoxetine in correcting the behavior of zebrafish. At 5 dpf, zebrafish in the TO group were immersed in 10 μmol / L atomoxetine for 1 hour before behavioral analysis. The results are shown in Figure 7 The locomotor activity of the TO group was significantly decreased compared with the GF group, but was not significantly different from that of the CV group, indicating that atomoxetine was effective in the TO group.

[0082] The inventors tried to colonize the different strains found in clinical samples into GF zebrafish and found that after colonization with Lactobacillus sanfrancisco, the spontaneous activity speed of the GF group decreased, with no significant difference from the CV and CVZ groups, and the hyperactive behavior disappeared, indicating that Lactobacillus sanfrancisco colonization may help improve the hyperactive behavior of ADHD. Through the experimental results of the MC group, the inventors found that supplementing with Lactobacillus sanfrancisco corrected the overactive behavior of the GF group ( Figure 4 , Figure 5 and Table 3).

[0083] Table 3 Comparison of the spontaneous activity speed of zebrafish in the Lactobacillus sanfrancisco colonization group and other groups

[0084] Note: The "*" indicates that compared with the CV group, p <0.05

[0085] Table 4 Comparison of the spontaneous activity speed of zebrafish in the atomoxetine treatment group and other groups

[0086] Note: The "*" indicates that compared with the CV group, * p <0.05

[0087] 2. Effects of Lactobacillus sanfranciscous supplementation on a mouse model colonized with human gut microbiota

[0088] To further validate the effects of Lactobacillus sanfranciscoensis, the inventors used a mouse model that more closely resembles the human intestinal microbiota to verify the results. First, antibiotics (ABX) were administered to 5-week-old male mice, and then the mice were divided into groups and received fecal microbiota transplants from different sources.

[30] : Healthy individuals (FMT-H), ADHD patients with high abundance of Haemophilus parainfluenzae and Streptococcus salivarius (FMT-B), and ADHD patients with low abundance of Lactobacillus sanfranciscous (FMT-A).

[0089] The results showed that compared with FMT-H mice, mice in the FMT-A / FMT-B group showed a significant increase in movement (FMT-B: F=1.78, P=0.031, n=18, FMT-A: F=1.89, P=0.044, n=20). In the first 5 minutes of the open field test, there was no statistically significant difference in the time spent in the center area among the three groups of mice, indicating that there was no statistically significant difference in the anxiety level of the mice. In addition, in the 5-CSRTT, the attention of mice in the FMT-A group was significantly reduced, as shown by a prolonged average latency of correct responses and a decrease in correct responses (t=3.615, P=0.0025; t=2.275, P=0.0380, n=18), while there was no significant statistical difference in impulsive behavior (t=0.225, P=0.8247, n=18).

[0090] Next, to explore the therapeutic potential of probiotic metabolites such as butyrate and Lactobacillus sanfrancisco, both groups were treated with sodium butyrate and Lactobacillus sanfrancisco. The results showed that butyrate treatment did not significantly improve the movement of either group, while supplementation with Lactobacillus sanfrancisco alleviated the hyperactive behavior of mice in the FMT-A group ( Figure 8 ), and at the same time, effectively alleviated the attention deficit of FMT-A group mice, such as Fig. 9 As shown, the average correct response latency of mice in the FMT-A group was significantly reduced (t=2.466, P=0.0333, n=12), and the correct response rate showed an upward trend (t=1.898, P=0.0869, n=12).

[0091] Furthermore, the inventors performed 16S rRNA analysis on fecal samples collected from transplanted and treated mice. The results of β-diversity analysis determined by PCoA showed that the FMT-H mouse population exhibited the most similar microbial community composition to the WT mouse population. Other groups, such as FMT-A and FMT-AA, showed different community structures ( Fig.10 ). Fig.11 An analysis of the abundance of Lactobacillus in the different groups is provided in. Taken together, the results suggest that a reduction in Lactobacillus sanfrancisco in the gut microbiota can lead to ADHD-like symptoms, whereas supplementation with Lactobacillus sanfrancisco in an ADHD model with low abundance of Lactobacillus sanfrancisco can effectively alleviate ADHD-like symptoms.

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Claims

1. Lactobacillus sanfranciscoensis ( Lactobacillus_sanfranciscensis ) for use in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder.

2. The use according to claim 1, wherein the attention deficit hyperactivity disorder is characterized by the absence of Lactobacillus sanfrancisco or a lower abundance of Lactobacillus sanfrancisco compared to healthy individuals.

3. The use according to claim 1 or 2, wherein the product is administered gastrointestinal tract.

4. The use according to claim 1 or 2, wherein the product comprises a functional food or a medicine.

5. The use according to claim 4, wherein the dosage form of the drug is pills, powders, capsules, tablets, suspensions or granules.

6. The use according to claim 4, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

7. A microbial preparation for preventing and / or treating attention deficit hyperactivity disorder, the microbial preparation comprising Lactobacillus sanfrancisco ( Lactobacillus_sanfranciscensis ).

8. The microbial preparation according to claim 7, wherein the content of Lactobacillus sanfrancisco is at least 1×10 8 CFU / dose.

9. The microbial preparation according to claim 7 or 8, wherein the microbial preparation is administered via the gastrointestinal tract.

Images