Application of bifidobacterium longum microbial agent in preparation of medicine for preventing and treating demyelinating disease and regulating intestinal flora

Abstract

The invention discloses application of a bifidobacterium longum microbial agent in preparation of a medicine for preventing and treating demyelinating disease and regulating intestinal flora. The bifidobacterium longum microbial inoculum is a selenium-rich bifidobacterium longum microbial inoculum. The selenium-rich bifidobacterium longum microbial inoculum and the bifidobacterium longum microbial inoculum can effectively prevent and inhibit demyelination and prevent and improve behavioral abnormalities of demyelination animals including Alzheimer's disease animals, and the selenium-rich bifidobacterium longum microbial inoculum further enhances the action effect of the bifidobacterium longum microbial inoculum; the selenium-rich bifidobacterium longum microbial inoculum can promote myelin sheath regeneration; the selenium-rich bifidobacterium longum microbial inoculum and the bifidobacterium longum microbial inoculum can adjust the intestinal flora of the demyelination animals, and the ability of the bifidobacterium longum microbial inoculum to remodel the intestinal flora is further significantly improved. And a new basis is provided for the selenium-rich bifidobacterium longum microbial agent to prevent and treat the demyelination disease. The selenium-rich bifidobacterium longum microbial inoculum has the effects of both medicines and nutrition functions, not only can exert the efficacy, but also has the characteristic of high safety, and an acute oral toxicity test of rats proves that the selenium-rich bifidobacterium longum microbial inoculum belongs to an actual non-toxic level and can be taken for a long time for prevention or treatment.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a Bifidobacterium longum bacterial agent in preparing medicines for preventing and treating demyelinating diseases and regulating intestinal flora. Background Art

[0002] Myelin is a multi-layered myelin-rich sheath produced by oligodendrocytes arranged around the axons of neurons in the central nervous system. It plays the role of insulation and protection of axons and neurons, and can significantly improve the efficiency of axons in transmitting nerve signals. The dynamic changes of the myelin sheath in the central nervous system are believed to change the speed and intensity of nerve impulse transmission, thereby improving the synchronization between neurons and participating in the regulation of brain function, which is called myelin plasticity. Studies have found that myelin plasticity is involved in the complex functions of the brain, such as learning and memory, emotions, movement, and vision (Annu Rev Neurosci. 2018; 41: 61-76.). However, myelin lesions and loss can lead to abnormal neurological function and cause demyelinating diseases. Multiple sclerosis (MS) is a common demyelinating disease of the central nervous system. Due to the different areas affected, the symptoms are diverse. MS patients may experience impaired motor skills, mental abnormalities, and cognitive defects (Handb Clin Neurol. 2017; 145: 263-283.). The prevalence of MS in my country has been increasing year by year (Lancet Reg Health West Pac. 2020; 1: 100010.).

[0003] Recent studies have reported that, in addition to MS, myelin loss has been found in the brains of patients with Alzheimer's disease (AD) (Neuron. 2021; 109(14): 2292-2307.e5.; Nature. 2023; 618(7964): 349-357.) and major depressive disorder (MDD) (J Clin Invest. 2022; 132(8): e152187.). Studies have found that demyelination occurs in the early stages of AD, and the reduction of myelin occurs earlier than the degeneration of neuronal axons. In animal experiments, the learning and memory abilities of AD mice were restored after myelin damage was repaired using genetic or pharmacological methods (Neuron. 2021; 109(14): 2292-2307.e5.). On the other hand, the administration of pro-myelination drugs to depressed mice was found to effectively alleviate myelin damage in the hippocampus and significantly improve the depressive-like behavior of mice (J Clin Invest. 2022; 132(8): e152187.). In addition, the antidepressant ketamine can exert an antidepressant effect by promoting the differentiation of oligodendrocyte precursor cells and repairing myelin (Mol Psychiatry. 2023.). In summary, the above studies show that myelin changes are closely related to the occurrence and development of cognitive deficits and depression, and myelin damage and loss are important pathological features of MS, AD and MDD. Therefore, maintaining myelin integrity and promoting myelin health will be of great significance for the prevention and treatment of demyelinating diseases.

[0004] Cuprizone (CPZ) can induce demyelination in multiple brain regions such as the corpus callosum, hippocampus and cortex of rodents, showing behavioral changes such as cognitive deficits, impaired motor skills and abnormal emotions. It can simulate the neuropathological and behavioral changes of MS, AD and MDD, and is a classic animal model of demyelination. CPZ chelates copper ions in mitochondrial complex IV, causing oligodendrocyte energy metabolism disorders, thereby inducing oligodendrocyte death and demyelination. When CPZ administration is stopped, the myelin sheath of CPZ model mice can be spontaneously restored (Mol Neurodegener. 2022; 17(1): 34.). Therefore, this model can be used to study the mechanism of demyelination and myelin regeneration.

[0005] Studies have found that the intestinal microbiota of patients with MS, AD, and MDD are all abnormal to varying degrees. Clinically, the intestinal flora of MS patients is significantly different from that of healthy people, among which 61 bacteria have differential abundance and 31 bacteria are enriched in MS cases. These abnormalities are directly related to blood inflammatory markers (Genome Med. 2023; 15(1): 1.). Another study further confirmed that compared with normal healthy people, the content of methane in the breath of MS patients is significantly increased, which is consistent with the observed increase in the relative abundance of Methanobacterium in the intestine of MS patients (Nat Commun. 2016; 7: 12015.). In the course of AD disease, imbalance of intestinal flora leads to abnormal increase of phenylalanine and isoleucine in peripheral blood, which in turn induces differentiation and proliferation of peripheral proinflammatory Th1 cells and infiltration into the brain. The Th1 cells that enter the brain and the inherent M1 microglia in the brain are activated together, leading to the occurrence of AD-related neuroinflammation (Cell Res. 2019; 29(10): 787-803.). The role of GV-971 in AD is to reduce neuroinflammation in the brain by reshaping the balance of intestinal flora, reducing the accumulation of peripheral related metabolites phenylalanine / isoleucine, and reducing Aβ deposition and Tau protein phosphorylation, thereby improving cognitive impairment and achieving the effect of treating AD (Cell Res. 2019; 29(10): 787-803.). In terms of depression, abnormal changes in microbial composition and metabolites in MDD patients can lead to the destruction of intestinal flora homeostasis, causing intestinal barrier dysfunction and inflammatory response (Cell Mol Gastroenterol Hepatol. 2021; 11(5): 1463-1482.), and intestinal toxic metabolites can lead to increased systemic translocation through the damaged intestinal barrier, exacerbating the systemic inflammatory response associated with the pathogenesis of depression (Psychoneuroendocrinology. 2018; 98: 52-60.). The vagus nerve also plays an important role in transmitting microbial signals from the intestine to the brain in patients with depression. In clinical practice, vagus nerve stimulation therapy has been approved for the treatment of refractory depression (Am J Psychiatry. 2017; 174(7): 640-648.). The above evidence shows that intestinal microorganisms play an important role in the pathogenesis and progression of MS, AD and MDD. Existing studies have shown that probiotic therapy has a positive effect on controlling the disease progression of MS, AD and MDD, and is a potential method to regulate intestinal flora imbalance to intervene in the treatment of MS, AD and MDD (Gut Microbes.2021;13(1):1943289.; Int J Mol Sci.2022;23(9):4494.; Front Psychiatry.2022;13:879491.).However, probiotic therapy alone has the limitation of insufficient efficacy. Therefore, it is necessary to develop live microbial therapy with higher efficacy.

[0006] Selenium-enriched Bifidobacterium longum is an enhanced probiotic obtained through selenium-enriched fermentation. The conversion of selenium by Bifidobacterium longum can reduce selenium toxicity and improve selenium bioavailability (Biol Trace Elem Res. 2020; 197(2): 599-605.), and the addition of selenium can also enhance the stability and antioxidant potential of Bifidobacterium longum (J Trace Elem MedBiol. 2017; 40: 1-9.), and the two can synergistically integrate or improve their respective functions. However, the selenium-enriched longum Bifidobacterium agents reported in current studies are all based on their ability to regulate intestinal microecology and intestinal metabolism, further significantly enhancing the anti-tumor (Asian Pac J Cancer Prev. 2014; 15(8): 3681-6.) and hypoglycemic (R Soc Open Sci. 2018; 5(12): 181156.) effects of bifidobacteria, as well as improving the original effects of bifidobacteria in improving ulcerative colitis (Front Microbiol. 2022; 13: 955112.) and irritable bowel syndrome (Food Funct. 2023; 14(11): 5355-5374.).

[0007] At present, there is no literature reporting on the preventive and therapeutic effects of selenium-enriched Bifidobacterium longum on demyelination. The preventive and therapeutic effects of selenium-enriched Bifidobacterium longum on demyelination, as well as the regulatory effects of oral selenium-enriched Bifidobacterium longum on intestinal flora in demyelination models are still unclear.

[0008] There is no report on the improvement of selenium-enriched Bifidobacterium longum on demyelination and remyelination. Therefore, the present invention introduces a CPZ-induced demyelination model to evaluate the effect of selenium-enriched Bifidobacterium longum on demyelination and remyelination. It is further verified in the APP / PS1 Alzheimer's disease animal model. Finally, the regulatory effect of selenium-enriched Bifidobacterium longum on the intestinal flora of the demyelination model is explored. Summary of the invention

[0009] In order to solve the problems of the prior art, the present invention provides an application of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating demyelinating diseases and regulating intestinal flora.

[0010] In order to achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0011] In the first aspect, the present application provides an application of a long bifidobacterium bacterial agent in the preparation of a drug for preventing and treating demyelinating diseases. In the second aspect, the present application provides an application of a long bifidobacterium bacterial agent in the preparation of a drug for regulating intestinal flora. In the third aspect, the present application provides a drug for preventing and treating demyelinating diseases or regulating intestinal flora. In the fourth aspect, the present application provides a method for preparing the drug provided by the application.

[0012] The first aspect of the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating demyelinating disease. Original bacterial species name: Bifidobacterium longum; China Industrial Microbiological Culture Collection Administration Center, bacterial species number: CICC 6188.

[0013] A second aspect of the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for regulating intestinal flora.

[0014] Furthermore, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

[0015] Furthermore, the preparation method of the selenium-enriched Bifidobacterium longum bacterial agent comprises the following steps:

[0016] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0017] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0018] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0019] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0020] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0021] f. Ferment for 8-10 hours, during which nitrogen is passed to drive out the air, and 30% glucose is added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum inoculum.

[0022] Furthermore, in step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0023] (1) Add stabilizer to a beaker;

[0024] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0025] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0026] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0027] Furthermore, the Bifidobacterium longum bacterial agent is used to prevent or inhibit demyelination in the central nervous system, and the selenium-enriched Bifidobacterium longum bacterial agent further enhances the inhibitory effect of the Bifidobacterium longum bacterial agent. Demyelinating diseases include any one of multiple sclerosis, Alzheimer's disease or major depression.

[0028] Furthermore, the Bifidobacterium longum bacterial agent is used to promote the remyelination of the central nervous system.

[0029] Furthermore, Bifidobacterium longum inoculum can regulate the intestinal flora of demyelinating animal models, reduce harmful flora, and increase beneficial flora. Selenium-enriched Bifidobacterium longum inoculum further significantly enhances the ability of Bifidobacterium longum inoculum to reshape the intestinal flora.

[0030] Furthermore, harmful bacteria include Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus, and Eisenbergiella; beneficial bacteria include Dubosiella, Lactobacillus, Faecalibaculum, Bifidobacterium, Coriobacteriaceae_UCG-002, and Enterorhabdus.

[0031] The third aspect of the present application provides a drug for preventing and treating demyelinating diseases or regulating intestinal flora, comprising a Bifidobacterium longum agent or a selenium-enriched Bifidobacterium longum agent as an active ingredient.

[0032] The fourth aspect of the present application provides a method for preparing the drug for use, comprising the following steps: (1) adding selenium to a culture solution containing Bifidobacterium longum, culturing in an incubator, and collecting the mixed bacterial solution to obtain selenium-enriched Bifidobacterium longum;

[0033] (2) mixing the selenium-enriched Bifidobacterium longum with a protective agent;

[0034] (3) Depending on the desired dosage form, the selenium-enriched Bifidobacterium longum is resuspended in skimmed milk and processed into an oral solution, lyophilized powder or capsule.

[0035] Beneficial effects: Application of the Bifidobacterium longum agent of the present invention in the preparation of drugs for preventing and treating demyelinating diseases and regulating intestinal flora. Selenium-enriched Bifidobacterium longum agent and Bifidobacterium longum agent can prevent or inhibit demyelination of the central nervous system, prevent or improve behavioral abnormalities in demyelination models and Alzheimer's disease models, and selenium-enriched Bifidobacterium longum agent further enhances the inhibitory effect of Bifidobacterium longum agent; selenium-enriched Bifidobacterium longum agent can promote regeneration of myelin in the central nervous system; selenium-enriched Bifidobacterium longum agent and Bifidobacterium longum agent can regulate the intestinal flora in the demyelination model, reduce harmful bacteria, and increase beneficial bacteria, and selenium-enriched Bifidobacterium longum agent further significantly enhances the ability of Bifidobacterium longum agent to reshape the intestinal flora. Compared with the prior art, the present invention has the following advantages:

[0036] (1) Myelin protection effect: Selenium-enriched Bifidobacterium longum and Bifidobacterium longum can effectively protect or inhibit demyelination in the central nervous system, and selenium-enriched Bifidobacterium longum further enhances the effect of Bifidobacterium longum.

[0037] (2) Promote myelin regeneration: The selenium-enriched Bifidobacterium longum bacterial agent of the present invention can promote myelin regeneration after demyelination, and has a positive effect on the recovery of damaged nerve function. Therefore, the selenium-enriched Bifidobacterium longum bacterial agent can be used to prepare drugs for preventing and treating demyelinating diseases.

[0038] (3) Regulating intestinal flora: Selenium-enriched Bifidobacterium longum and Bifidobacterium longum can regulate the intestinal flora of demyelinating animal models and reduce harmful flora, such as Desulfovibrio and Christensenellaceae_R-7_group. At the same time, it increases beneficial flora, such as Faecalibaculum and Bifidobacterium, thereby improving the balance of intestinal microorganisms. Selenium-enriched Bifidobacterium longum further significantly enhances the ability of Bifidobacterium longum to reshape intestinal flora. Therefore, selenium-enriched Bifidobacterium longum can be used to prepare drugs to improve and regulate the intestinal flora of demyelinating diseases.

[0039] (4) Improvement of various behavioral abnormalities: In the demyelination model, the selenium-enriched Bifidobacterium longum inoculum can significantly improve the motor coordination ability of mice, which is better than a single Bifidobacterium longum inoculum; in addition, the selenium-enriched Bifidobacterium longum inoculum can also significantly improve the cognitive dysfunction and depressive-like behavior of the demyelination model. In the Alzheimer's disease model, the selenium-enriched Bifidobacterium longum inoculum can significantly improve the learning and memory ability of mice and alleviate the cognitive function defects of Alzheimer's disease mice. The selenium-enriched Bifidobacterium longum inoculum further enhances the effect of the Bifidobacterium longum inoculum. Provide new live microbial therapy: Compared with a single Bifidobacterium longum inoculum, the selenium-enriched Bifidobacterium longum inoculum has a stronger ability to protect myelin and regulate intestinal flora, providing a new therapy for the treatment of demyelinating diseases.

[0040] (5) Safety and long-term use: Bifidobacterium longum has a good history of safe use and compliance, and has been included in China's "List of Bacteria that Can Be Used in Food" and the EU Safety Qualification (QPS) list, indicating that its safety is recognized. Selenium-enriched Bifidobacterium longum has very good safety, and the acute oral toxicity test in rats has proved that it is actually non-toxic. Selenium-enriched Bifidobacterium longum has both medicinal and nutritional functions, is highly safe, and is suitable for long-term use to prevent or treat demyelinating diseases. Flexibility in dosage adjustment: The dosage of the active ingredient in the drug can be adjusted according to the patient's specific situation to achieve the best therapeutic effect and safety.

[0041] (6) Reducing medical expenses: Since the preparation cost of the selenium-enriched Bifidobacterium longum bacterial agent is low, using this strain as a drug ingredient can reduce the medical expenses of patients to a certain extent. Expanding the scope of application: The application of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention in the preparation of drugs for the prevention and treatment of demyelinating diseases expands the application scope of live microbial drugs in the field of the treatment of demyelinating diseases of the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The effect of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention on the weight change of demyelinated mice; C57BL / 6 mice were divided into four groups, ND: normal diet group, CPZ: normal diet + CPZ group, BL+CPZ: Bifidobacterium longum bacterial agent + CPZ group, Se-BL+CPZ: selenium-enriched Bifidobacterium longum bacterial agent + CPZ group, and C57BL / 6 mice were fed with 0.2% CPZ for 5 weeks; the data are expressed as Mean±SEM, and the differences between the groups are expressed as *p<0.05, **p<0.01, ***p<0.001, wherein:

[0043] Figure 1 A is the change curve of the average body weight of each group of mice every 7 days;

[0044] Figure 1B is the statistical difference of the average body weight change of each group of mice per week in the present invention.

[0045] Figure 2 The present invention shows the effect of the selenium-enriched Bifidobacterium longum bacterial agent on myelin changes and behavioral changes in demyelinated mice; C57BL / 6 mice were divided into four groups, ND: normal diet group, CPZ: normal diet + CPZ group, BL+CPZ: Bifidobacterium longum bacterial agent + CPZ group, Se-BL+CPZ: selenium-enriched Bifidobacterium longum bacterial agent + CPZ group, and C57BL / 6 mice were fed with 0.2% CPZ for 5 weeks; the data are expressed as Mean ± SEM, and the differences between the groups are expressed as *p<0.05, **p<0.01, ***p<0.001, wherein:

[0046] Figure 2 A is the MBP immunohistochemical staining diagram of each group of mice in the present invention, MBP marks myelin basic protein, which is used to characterize demyelination;

[0047] Figure 2 B is a statistical diagram of quantitative analysis of MBP immunohistochemical staining of each group of mice in the present invention;

[0048] Figure 2 C is a statistical diagram of the quantitative analysis of the falling latency of each group of mice in the acute rotarod test (4-40 rpm, 120 s) of the present invention;

[0049] Figure 2 D is a statistical diagram of the quantitative analysis of the rotation speed of each group of mice when they fall in the acute rotarod test (4-40 rpm, 120 s) of the present invention;

[0050] Figure 2 E is a statistical diagram of quantitative analysis of the falling latency of each group of mice in the chronic rotarod test (4-40 rpm, 240 s) of the present invention;

[0051] Figure 2 F is a statistical diagram of the quantitative analysis of the rotation speed of each group of mice when they fell in the chronic rotarod test (4-40 rpm, 240 s) of the present invention;

[0052] Figure 2 G is a statistical graph showing the quantitative analysis of the gripping force of each group of mice in the gripping force experiment of the present invention.

[0053] Figure 3The effect of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention on the changes in myelin regeneration in demyelinated mice; C57BL / 6 mice were divided into three groups, ND: normal diet group, CPZ: first fed with a standard basal feed containing 0.2% CPZ for 5 weeks, and then changed to a standard basal feed for 2 weeks, Se-BL+CPZ: selenium-enriched Bifidobacterium longum bacterial agent + CPZ group; data are expressed as Mean±SEM, and differences between groups are expressed as *p<0.05, **p<0.01, ***p<0.001, wherein:

[0054] Figure 3 A is a flowchart of the myelin regeneration experiment of the present invention;

[0055] Figure 3 B is the MBP immunohistochemical staining diagram of each group of mice of the present invention, MBP marks myelin basic protein, which is used to characterize demyelination;

[0056] Figure 3 C is a statistical diagram of quantitative analysis of MBP immunohistochemical staining of each group of mice in the present invention.

[0057] Figure 4 The present invention shows the effects of the selenium-enriched Bifidobacterium longum bacterial agent on changes in myelin sheath in hippocampus / cortex, neuronal damage and learning and memory deficits in demyelinating mice; C57BL / 6 mice were divided into three groups, ND: normal diet group, CPZ: normal diet + CPZ group, Se-BL + CPZ: selenium-enriched Bifidobacterium longum bacterial agent + CPZ group, and C57BL / 6 mice were fed with 0.2% CPZ for 5 weeks; data are expressed as Mean ± SEM, and differences between groups are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, wherein:

[0058] Figure 4 A is the MBP immunohistochemical staining image of the hippocampus / cortex of each group of mice in the present invention. MBP marks myelin basic protein and is used to characterize demyelination;

[0059] Figure 4 B is a statistical diagram of quantitative analysis of MBP immunohistochemical staining in the hippocampus of each group of mice in the present invention;

[0060] Figure 4 C is a quantitative analysis statistical diagram of MBP immunohistochemical staining of the cortex of each group of mice in the present invention;

[0061] Figure 4 D is the Nissl staining image of the hippocampus / cortex of each group of mice in the present invention. Nissl staining marks neurons and is used to characterize neuronal damage;

[0062] Figure 4 EG is a statistical diagram of Nissl staining quantitative analysis of hippocampus / cortex of each group of mice in the present invention;

[0063] Figure 4 H is the motion trajectory diagram of each group of mice in the water maze experiment of the present invention;

[0064] Figure 4 IK is a quantitative analysis statistical chart of the number of times each group of mice reached the platform, the distance traveled in the quadrant where the platform is located, and the time they stayed in the quadrant where the platform is located in the water maze experiment of the present invention.

[0065] Figure 5 The effect of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention on the depressive-like behavior of demyelinating mice; C57BL / 6 mice were divided into three groups, ND: normal diet group, CPZ: normal diet + CPZ group, Se-BL + CPZ: selenium-enriched Bifidobacterium longum bacterial agent + CPZ group, and C57BL / 6 mice were fed with 0.2% CPZ for 5 weeks; data are expressed as Mean ± SEM, and differences between groups are expressed as *p < 0.05, **p < 0.01, ***p < 0.001, wherein:

[0066] Figure 5 A is a schematic diagram of the sugar water preference experiment of the present invention;

[0067] Figure 5 B is a statistical diagram of quantitative analysis of sucrose preference index of each group of mice in the sugar water preference experiment of the present invention;

[0068] Figure 5 C is a schematic diagram of the tail suspension experiment of the present invention;

[0069] Figure 5 D is a statistical diagram of quantitative analysis of the immobility time of each group of mice in the tail suspension experiment of the present invention.

[0070] Figure 6 The present invention is an effect of the selenium-enriched Bifidobacterium longum inoculum on the changes of cortex and hippocampal myelin sheath in APP / PS1 Alzheimer's disease mice; the experiment was divided into five groups: a wild-type control group (WT), an APP / PS1 AD model group (AD), a Bifidobacterium longum inoculum intervention group (BL+AD), a selenium-enriched Bifidobacterium longum inoculum intervention group (Se-BL+AD) and a GV-971 intervention group (GV-971+AD), and the data were expressed as Mean±SEM, and the differences among the groups were expressed as *p<0.05, **p<0.01, and ***p<0.001, wherein:

[0071] Figure 6 A is the MBP immunohistochemical staining image of the cortex and hippocampus of each group of mice in the present invention. MBP marks myelin basic protein and is used to characterize demyelination;

[0072] Figure 6 BF is the statistical diagram of quantitative analysis of MBP immunohistochemical staining of the cortex and hippocampus of each group of mice in the present invention.

[0073] Figure 7 The present invention is an effect of the selenium-enriched Bifidobacterium longum inoculum on cortical and hippocampal neuronal damage in APP / PS1 Alzheimer's disease mice; the experiment was divided into five groups: a wild-type control group (WT), an APP / PS1 AD model group (AD), a Bifidobacterium longum inoculum intervention group (BL+AD), a selenium-enriched Bifidobacterium longum inoculum intervention group (Se-BL+AD) and a GV-971 intervention group (GV-971+AD), and the data were expressed as Mean±SEM, and the differences among the groups were expressed as *p<0.05, **p<0.01, and ***p<0.001, wherein:

[0074] Figure 7 A is the NeuN immunofluorescence staining image of the cortex and hippocampus of each group of mice in the present invention. NeuN marks neurons and is used to characterize neuronal damage;

[0075] Figure 7 BE are statistical diagrams of quantitative analysis of NeuN immunofluorescence staining in the cortex and hippocampus of each group of mice in the present invention.

[0076] Figure 8 The effect of the selenium-enriched Bifidobacterium longum inoculum of the present invention on learning and memory impairment in APP / PS1 Alzheimer's disease mice; the experiment was divided into five groups: a wild-type control group (WT), an APP / PS1 AD model group (AD), a Bifidobacterium longum inoculum intervention group (BL+AD), a selenium-enriched Bifidobacterium longum inoculum intervention group (Se-BL+AD) and a GV-971 intervention group (GV-971+AD), and the data were expressed as Mean±SEM, and the differences between the groups were expressed as *p<0.05, **p<0.01, and ***p<0.001, wherein:

[0077] Figure 8 A is a flow chart of the water maze experiment of the present invention;

[0078] Figure 8 B and C are statistical graphs showing the quantitative analysis of the escape latency of each group of mice in the water maze experiment of the present invention;

[0079] Figure 8 D is the motion trajectory diagram of each group of mice in the water maze experiment of the present invention;

[0080] Figure 8 EG is a quantitative analysis statistical chart of the number of times each group of mice reached the platform, the distance traveled in the quadrant where the platform is located, and the time they stayed in the quadrant where the platform is located in the water maze experiment of the present invention;

[0081] Figure 8 H is a graph showing the changes in search strategies of mice in each group in the water maze experiment of the present invention;

[0082] Figure 8I is a flow chart of a new object recognition experiment of the present invention;

[0083] Figure 8 J is a quantitative analysis statistical graph of the novel object recognition index of each group of mice in the novel object recognition experiment of the present invention.

[0084] Fig. 9 The present invention explores the effect of selenium-enriched Bifidobacterium longum on the changes in intestinal flora of demyelinated mice based on 16s rRNA sequencing; C57BL / 6 mice were divided into four groups, ND: normal diet group, CPZ: normal diet + CPZ group, BLC: Bifidobacterium longum + CPZ group, SBC: selenium-enriched Bifidobacterium longum + CPZ group, and C57BL / 6 mice were fed with 0.2% CPZ for 5 weeks; data are expressed as Mean ± SEM, and differences between groups are expressed as *p<0.05, **p<0.01, ***p<0.001, where:

[0085] Fig. 9 A is a heat map of relative abundance of species at the genus level in the microbiome of each group of mice of the present invention;

[0086] Fig. 9 BN is a quantitative analysis statistical chart of the relative abundance of Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus, Eisenbergiella; Dubosiella, Lactobacillus, Faecalibaculum, Bifidobacterium, Coriobacteriaceae_UCG-002, and Enterorhabdus in each group of mice of the present invention. DETAILED DESCRIPTION

[0087] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0088] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0089] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0090] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0091] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0092] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise specified, all reagents are conventional biochemical reagents.

[0093] The first aspect of the embodiment of the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating demyelinating disease. Original bacterial species name: Bifidobacterium longum; China Industrial Microbiological Culture Collection Administration Center, bacterial species number: CICC 6188.

[0094] A second aspect of the embodiments of the present application provides a use of a Bifidobacterium longum agent in the preparation of a drug for regulating intestinal flora.

[0095] In some embodiments, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

[0096] Selenium-enriched Bifidobacterium longum inoculum is obtained by selenium-enriched fermentation of Bifidobacterium longum inoculum. In particular, the preventive and therapeutic effects of selenium-enriched Bifidobacterium longum inoculum on demyelination, as well as regulating the balance of intestinal microorganisms in demyelination models, increasing the relative abundance of beneficial intestinal microorganisms, and reducing the relative abundance of harmful microorganisms, have been studied, thereby protecting the body.

[0097] The selenium-enriched Bifidobacterium longum bacterial agent of the present invention can be applied to the prevention and treatment of demyelinating diseases. Experimental results show that the selenium-enriched Bifidobacterium longum bacterial agent and the Bifidobacterium longum bacterial agent can inhibit the demyelination of the central nervous system, and the selenium-enriched Bifidobacterium longum bacterial agent further enhances the inhibitory effect of the Bifidobacterium longum bacterial agent; the selenium-enriched Bifidobacterium longum bacterial agent can promote the regeneration of myelin in the central nervous system; the selenium-enriched Bifidobacterium longum bacterial agent and the Bifidobacterium longum bacterial agent can regulate the intestinal flora of the demyelination model, reduce harmful bacteria, and increase beneficial bacteria, and the selenium-enriched Bifidobacterium longum bacterial agent further significantly enhances the ability of the Bifidobacterium longum bacterial agent to reshape the intestinal flora.

[0098] The invention provides a new basis for preventing and treating demyelinating diseases with a selenium-enriched Bifidobacterium longum bacterial agent. The selenium-enriched Bifidobacterium longum bacterial agent can be used to prepare drugs for preventing and treating demyelinating diseases and to prepare drugs for improving and regulating intestinal flora of demyelinating diseases.

[0099] Compared with the prior art, the application of the Bifidobacterium longum agent of the present invention in the preparation of drugs for preventing and treating demyelinating diseases and regulating intestinal flora has the following advantages:

[0100] (1) In the demyelination model and Alzheimer's disease model, the selenium-enriched Bifidobacterium longum inoculum exerted the best myelin protection effect. Long-term oral administration of the selenium-enriched Bifidobacterium longum inoculum significantly protected and inhibited myelin loss and significantly improved the behavioral abnormalities of the model mice. Compared with a single Bifidobacterium longum inoculum, the selenium-enriched Bifidobacterium longum inoculum exhibited a more excellent myelin protection effect, which may be closely related to the synergistic fusion of selenium and Bifidobacterium longum inoculum.

[0101] (2) Selenium-enriched Bifidobacterium longum has a stronger ability to regulate intestinal flora. Compared with a single Bifidobacterium longum agent, selenium-enriched Bifidobacterium longum has more advantages in increasing beneficial bacteria and reducing harmful bacteria.

[0102] (3) Selenium-rich Bifidobacterium longum bacterial agent takes into account both the role of medicine and functional health care. It can not only exert its medicinal effect, but also has the characteristics of high safety. It can be taken for a long time for treatment or prevention. The acute oral toxicity test of rats proves that it is actually non-toxic. The main active ingredients contained are selenium and Bifidobacterium longum bacterial agent, which has been used in clinical practice. It is conducive to reducing the preparation cost of drugs for the treatment of demyelinating diseases, thereby reducing the medical expenses of patients to a certain extent.

[0103] In some embodiments, the Bifidobacterium longum bacterial agent is used to prevent and inhibit demyelination in the central nervous system, and the selenium-enriched Bifidobacterium longum bacterial agent further enhances the inhibitory effect of the Bifidobacterium longum bacterial agent.

[0104] In some embodiments, the selenium-enriched Bifidobacterium longum bacterial agent is used to promote remyelination in the central nervous system.

[0105] In some embodiments, the demyelinating disease is multiple sclerosis.

[0106] In some embodiments, the demyelinating disease is Alzheimer's disease.

[0107] In some embodiments, the demyelinating disease is major depressive disorder.

[0108] In some embodiments, the Bifidobacterium longum bacterial agent is used to promote remyelination in the central nervous system.

[0109] In some embodiments, the Bifidobacterium longum agent can regulate the intestinal flora of a demyelination animal model, reduce harmful flora, and increase beneficial flora. The selenium-enriched Bifidobacterium longum agent further significantly enhances the ability of the Bifidobacterium longum agent to reshape the intestinal flora.

[0110] In some embodiments, harmful bacteria include Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus, and Eisenbergiella; beneficial bacteria include Dubosiella, Lactobacillus, Faecalibaculum, Bifidobacterium, Coriobacteriaceae_UCG-002, and Enterorhabdus.

[0111] The preparation method of the selenium-enriched Bifidobacterium longum bacterial agent comprises the following steps:

[0112] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0113] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0114] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0115] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0116] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0117] f. Ferment for 8-10 hours, during which nitrogen is passed to drive out the air, and 30% glucose is added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum inoculum.

[0118] In some embodiments, in step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0119] (1) Add stabilizer to a beaker;

[0120] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0121] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0122] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0123] In some embodiments, in step (1), the stabilizer is epigallocatechin gallate (EGCG).

[0124] In some embodiments, in step (1), the stabilizer is chitosan.

[0125] In some embodiments, in step (1), the stabilizer is gum arabic.

[0126] In some embodiments, in step (1), the stabilizer is bovine serum albumin (BSA).

[0127] In some embodiments, in step (1), the stabilizer is sodium hyaluronate.

[0128] In some embodiments, in step (2), the reducing agent is reduced glutathione GSH.

[0129] In some embodiments, in step (2), the reducing agent is vitamin C (Vc).

[0130] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.1 mM to 40 mM (for EGCG).

[0131] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.01% to 0.7% (for chitosan).

[0132] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.04% to 0.4% (for gum arabic).

[0133] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.4 mg / ml to 60 mg / ml (for BSA).

[0134] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.02% to 0.25% (for sodium hyaluronate).

[0135] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-50 mM (GSH).

[0136] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0137] In step (3), the final concentration of sodium selenite in the reaction system is 5 mM.

[0138] In some embodiments, in step (1), the molecular weight of the stabilizer is in the range of 100 KD - 1000 KD (for chitosan).

[0139] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for gum arabic).

[0140] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for sodium hyaluronate).

[0141] In step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0142] In some embodiments, the peak value of the particle size distribution of the nano-selenium prepared by the above steps can be precisely controlled within a range of 20 nm-60 nm, and the particle size is mainly distributed within a range of 10 nm-150 nm.

[0143] In terms of feeding strategy, the present invention adopts a constant rate flow addition method to ensure a uniform supply of nutrients throughout the fermentation process and avoid bacterial growth inhibition or waste due to insufficient or excessive nutrition.

[0144] The selenium-rich long bifidobacterium bacterial agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body. The content of nano-selenium in the bacterial agent is 0.5-2.9 micrograms / gram, and the number of active colonies of long bifidobacterium is 1×10 9 Up to 5×10 10 CFU / g.

[0145] In some embodiments, the Bifidobacterium longum bacterial preparation is in the form of a tablet.

[0146] In some embodiments, the Bifidobacterium longum bacterial agent is a lyophilized powder.

[0147] In some embodiments, the Bifidobacterium longum bacterial preparation is a capsule.

[0148] In some embodiments, the Bifidobacterium longum agent is a dairy product.

[0149] In some embodiments, the Bifidobacterium longum inoculum is stirred yogurt.

[0150] In some embodiments, the Bifidobacterium longum bacterial agent is in the form of set yogurt.

[0151] A third aspect of the embodiments of the present application provides a drug for preventing and treating demyelinating diseases or regulating intestinal flora, comprising a Bifidobacterium longum agent or a selenium-enriched Bifidobacterium longum agent as an active ingredient.

[0152] In some embodiments, the pharmaceutical dosage form is an oral solution.

[0153] In some embodiments, the pharmaceutical dosage form is a lyophilized powder.

[0154] In some embodiments, the pharmaceutical dosage form is a capsule.

[0155] In some embodiments, the pharmaceutical dosage form is made of a selenium-enriched Bifidobacterium longum bacterial agent and excipients acceptable to mammals.

[0156] The fourth aspect of the present application provides a method for preparing the drug for use, comprising the following steps:

[0157] (1) adding selenium to a culture solution containing Bifidobacterium longum, culturing in an incubator, and collecting the mixed bacterial solution to obtain selenium-enriched Bifidobacterium longum;

[0158] (2) mixing the selenium-enriched Bifidobacterium longum with a protective agent;

[0159] (3) Depending on the desired dosage form, the selenium-enriched Bifidobacterium longum is resuspended in skimmed milk and processed into an oral solution, lyophilized powder or capsule.

[0160] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific medicament of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0161] Example 1

[0162] The invention discloses an application of a Bifidobacterium longum bacterial agent in preparing a medicine for preventing and treating demyelinating disease.

[0163] The invention discloses an application of a Bifidobacterium longum bacterial agent in preparing a drug for regulating intestinal flora. The Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

[0164] The selenium-enriched Bifidobacterium longum inoculum is prepared by fermenting the Bifidobacterium longum inoculum with selenium. The preventive and therapeutic effects of the selenium-enriched Bifidobacterium longum inoculum on demyelination are particularly studied.

[0165] The selenium-enriched Bifidobacterium longum bacterial agent has been proven to be practically non-toxic through an acute oral toxicity test in rats, and meets food safety requirements.

[0166] Example 2

[0167] The difference between Example 2 and Example 1 is that the selenium-enriched Bifidobacterium longum bacterial agent is used to promote the regeneration of myelin sheath in the central nervous system.

[0168] Example 3

[0169] The difference between Example 2 and Example 1 is that the selenium-enriched Bifidobacterium longum bacterial agent and the Bifidobacterium longum bacterial agent are used to prevent and inhibit demyelination of the central nervous system, and the selenium-enriched Bifidobacterium longum bacterial agent further enhances the prevention and inhibition effect of the Bifidobacterium longum bacterial agent.

[0170] Example 4

[0171] The invention discloses an application of a selenium-enriched Bifidobacterium longum bacterial agent in preparing a drug for regulating intestinal flora.

[0172] The present invention regulates the intestinal microbial balance of the demyelination model, increases the relative abundance of beneficial intestinal microorganisms, and reduces the relative abundance of harmful microorganisms, thereby protecting the body.

[0173] Example 5

[0174] The difference between Example 5 and Example 1 is that the demyelinating disease is multiple sclerosis.

[0175] Example 6

[0176] The difference between Example 6 and Example 1 is that the demyelinating disease is Alzheimer's disease.

[0177] Example 7

[0178] The difference between Example 7 and Example 1 is that the demyelinating disease is major depression.

[0179] The selenium-enriched Bifidobacterium longum bacterial agent and the Bifidobacterium longum bacterial agent can regulate the intestinal flora of the demyelination animal model, reduce harmful flora, and increase beneficial flora. The selenium-enriched Bifidobacterium longum bacterial agent further significantly enhances the ability of the Bifidobacterium longum bacterial agent to reshape the intestinal flora.

[0180] Example 8

[0181] Harmful bacteria include Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus, and Eisenbergiella.

[0182] Example 9

[0183] The beneficial bacteria include Dubosiella, Lactobacillus, Faecalibaculum, Bifidobacterium, Coriobacteriaceae_UCG-002, and Enterorhabdus.

[0184] Example 10

[0185] The drug dosage form is oral solution.

[0186] Embodiment 11

[0187] The drug dosage form is freeze-dried powder.

[0188] Example 12

[0189] The dosage form of the drug is capsule.

[0190] Example 13

[0191] The drug dosage form is prepared from selenium-enriched Bifidobacterium longum bacterial agent and excipients acceptable to mammals.

[0192] Embodiment 14

[0193] A method for preparing a drug of the present invention comprises the following steps: (1) adding selenium to a culture solution containing a long bifidobacterium bacterial agent, culturing in an incubator, and collecting the mixed bacterial solution to obtain a selenium-enriched long bifidobacterium bacterial agent;

[0194] (2) mixing the selenium-enriched Bifidobacterium longum bacterial agent with a protective agent;

[0195] (3) Depending on the desired dosage form, the selenium-enriched Bifidobacterium longum bacterial agent is resuspended in skimmed milk and processed into an oral solution, lyophilized powder or capsule.

[0196] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific medicament of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0197] Test Example 1

[0198] Selenium-enriched Bifidobacterium longum prevents and inhibits myelin loss and promotes myelin regeneration in a demyelination model

[0199] 1. Experimental Methods

[0200] In order to verify the pharmacological effects of selenium-enriched Bifidobacterium longum on demyelination and remyelination, we constructed a Cuprizone (CPZ)-induced demyelination and remyelination animal model and adopted preventive and therapeutic administration to evaluate whether selenium-enriched Bifidobacterium longum can prevent or treat the disease.

[0201] 1. Experimental animals: C57BL / 6J male mice, 6-7 weeks old, weighing 20-22 g, were purchased from Shanghai Experimental Animal Research Center and raised in an SPF environment. All animal experimental operations were in compliance with the experimental animal management regulations and ethical requirements of the Animal Management Committee of Nanjing University.

[0202] 2. Experimental reagents: Cuprizone (Sigma-Aldrich), AIN93G standard feed (Nantong Trophy Feed Technology Co., Ltd.).

[0203] 3. Experimental drugs: The selenium-enriched Bifidobacterium longum bacterial agent described in the present invention is prepared in this laboratory by fermenting the Bifidobacterium longum bacterial agent through selenium enrichment.

[0204] 4. Establishment of CPZ-induced demyelination model and intervention with selenium-enriched Bifidobacterium longum: To establish the CPZ-induced demyelination model, CPZ was added to the standard feed at a ratio of 0.2%, and mice were fed for 35 days to induce demyelination. On the day of the Cuprizone-induced demyelination model, the mice were randomly divided into four groups, namely: ND: normal diet group, standard conventional diet; CPZ: normal diet + CPZ group, standard conventional diet containing 0.2% CPZ; BL + CPZ: Bifidobacterium longum + CPZ group, standard conventional diet containing 0.2% CPZ and intervention with Bifidobacterium longum; Se-BL + CPZ: Selenium-enriched Bifidobacterium longum + CPZ group, standard conventional diet containing 0.2% CPZ and intervention with selenium-enriched Bifidobacterium longum. Until the end of the experiment.

[0205] 5. Establishment of CPZ-induced myelin regeneration model and intervention with selenium-enriched Bifidobacterium longum inoculum: To establish the CPZ-induced myelin regeneration model, CPZ was added to the standard feed at a ratio of 0.2%, and the mice were fed for 5 weeks, and then changed to standard feed for 2 weeks to induce myelin regeneration. The mice were randomly divided into three groups, namely: ND: normal diet group, standard conventional diet; CPZ: normal diet + CPZ group, standard conventional diet containing 0.2% CPZ for 5 weeks, and then changed to standard conventional diet for 2 weeks; Se-BL + CPZ: Selenium-enriched Bifidobacterium longum inoculum + CPZ group, standard conventional diet containing 0.2% CPZ for 5 weeks + standard conventional diet for 2 weeks, and at the end of the third week, selenium-enriched Bifidobacterium longum inoculum was intervened. Until the end of the experiment.

[0206] 6. Body weight measurement and drug administration: The body weight of each experimental animal was measured at 9:00-10:00 every morning. Each mouse was orally gavaged with 1.5×10^9 CFU of Bifidobacterium longum or selenium-enriched Bifidobacterium longum daily. Before gavage, the strain was resuspended in PBS solution.

[0207] 7. Rotarod test: The rotarod test is used to detect the motor coordination ability of rodents. The experimental method is provided in reference XX. The animals are placed in the room where the instrument is located in advance, and the experiment is carried out after 1-2 hours of adaptation. Turn on the power switch of the instrument and set the instrument parameters to 4-40rpm, duration 120s (acute rotarod test) and 4-40rpm, duration 240s (chronic rotarod test). Place the animals on the rotarod in each channel in turn. After no animals fall, click start and record the latency to fall and the rotation speed when falling. Repeat 2-3 times for each mouse, and record the average value as the measurement value. The animals rest for at least 30 minutes between each test to avoid fatigue.

[0208] 8. Grip test: The grip test is designed to evaluate the motor function and forelimb control of rodents, and is often combined with the motor coordination test in the rotating rod test. Bring the animal to the test room at least 1 hour before the test to adapt to the environment. Gently take the animal out of the test cage, gently grasp its tail, allow the animal's forelimbs to grasp the grip plate of the grip tester, and at the same time, the tail is gently pulled back and parallel. When the maximum force is applied to the experimental animal, record the reading on the grip tester. Repeat 3-5 times for each mouse, and record the average value as the measurement value. The animal rests for at least 15 minutes between each test.

[0209] 9. Brain removal by perfusion: (1) After weighing the experimental mouse, inject anesthetic into the abdominal cavity. After confirming anesthesia, fix the mouse on the operating table, open the abdominal cavity, and completely expose the heart. (2) Prepare syringes and intravenous needles, 4℃ pre-cooled PBS buffer and 4% paraformaldehyde. Use straight-tipped forceps in the right hand to lift the heart from the apex of the heart, and use ophthalmic scissors in the left hand to make a small incision in the right atrium. Use straight-tipped forceps in the left hand to lift the apex of the heart, and pick up the syringe with 20-30ml PBS in the right hand and insert it parallel to the left and right midlines of the heart. The depth is best to insert into the aorta, and a good perfusion effect can also be obtained if the needle remains in the left ventricle. Push PBS at a uniform speed. If the needle is inserted at an incorrect angle or too deep, withdraw the needle and reinsert it in time, which generally does not affect the perfusion effect. When the outflowing fluid becomes clear and the liver is completely bleeding, the perfusion can be stopped. (3) After successful perfusion, quickly remove the brain tissue and fix it in 4% paraformaldehyde at 4℃ overnight. (4) Paraffin embedding and sectioning were then performed according to standard operating procedures.

[0210] 10. Immunohistochemical staining: After paraffin sections are dewaxed and hydrated, antigen retrieval is performed. The tissue sections are placed in a retrieval box filled with citric acid antigen retrieval buffer (pH 6.0) in a microwave oven for antigen retrieval. Then the sections are placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark to block endogenous peroxidase. 3% BSA is added to the tissue circle to evenly cover the tissue and blocked at room temperature for 30 minutes (the primary antibody is from goat, which is blocked with rabbit serum, and other sources are blocked with BSA). Gently shake off the blocking solution, add the primary antibody (MBP, rabbit antibody, 1:500) prepared in a certain ratio to the sections, and lay the sections flat in a humidified box and incubate overnight at 4°C. (A small amount of water is added to the humidified box to prevent the antibody from evaporating). The next day, the slides are placed in PBS and shaken on a decolorizing shaker for washing 3 times. After the sections are slightly dried, the secondary antibody (HRP-labeled goat anti-rabbit, 1:200) of the corresponding species to the primary antibody is added to the circle to cover the tissue and incubate at room temperature for 50 minutes. Wash with PBS, shake the slices slightly dry, and then add freshly prepared DAB color development solution in the circle. Control the color development time under a microscope, rinse the slices with tap water to stop the color development, dehydrate and seal the slices. Observe and take pictures under a microscope, collect images, and use ImageJ for quantitative analysis.

[0211] 2. Experimental Results

[0212] like Figure 1 As shown in A, as the feeding time of mice increased, the average body weight of mice in the ND group gradually increased, while the body weight of mice in the CPZ model group decreased significantly. There was a significant difference in the average body weight of mice in the ND group and the CPZ group at 1, 2, 3, 4, and 5 weeks after the start of the experiment ( Figure 1 B; p<0.05). Compared with the CPZ model group and the Bifidobacterium longum inoculum treatment group, the body weight of mice in the selenium-enriched Bifidobacterium longum inoculum treatment group was significantly increased at the 1st, 4th, and 5th weeks ( Figure 1 B; p<0.05), the body weight of mice in the Bifidobacterium longum inoculum treatment group was not significantly different from that in the CPZ model group. The above results indicate that the selenium-enriched Bifidobacterium longum inoculum can improve the weight loss of CPZ model mice.

[0213] like Figure 2 As shown in A, the myelin sheath of the corpus callosum of the ND group mice was stained darker, more evenly distributed, and had a dense structure. The myelin sheath of the corpus callosum of the CPZ model group mice was severely damaged, with lighter and uneven staining. The MBP immunohistochemistry positive area was significantly lower than that of the ND group ( Figure 2 B; p<0.05). In contrast, compared with the CPZ model, the myelin loss of mice in the Bifidobacterium longum or Selenium-enriched Bifidobacterium longum treatment groups was restored to varying degrees, and the MBP immunohistochemical positive area was significantly increased ( Figure 2 B; p<0.05). Compared with the group treated with Bifidobacterium longum, the MBP immunohistochemical positive area in the group treated with selenium-enriched Bifidobacterium longum was significantly increased ( Figure 2 B; p<0.05). The above results show that the selenium-enriched Bifidobacterium longum bacterial agent can inhibit CPZ-induced demyelination and has the best effect in improving demyelination.

[0214] The rotarod and gripping test evaluates the motor coordination ability of mice by measuring the latency to fall, the rotation speed when falling and the gripping force. The results of acute and chronic rotarod studies showed that after 5 weeks of CPZ feeding, the latency to fall and the rotation speed when falling of mice were significantly reduced compared with the ND group ( Figure 2 CF, p<0.05), which may be related to the abnormal motor function caused by demyelination of the corpus callosum. After 5 weeks of intervention with Bifidobacterium longum or Selenium-enriched Bifidobacterium longum, the falling latency and the speed of falling of the mice in the Bifidobacterium longum or Selenium-enriched Bifidobacterium longum treatment groups were significantly restored compared with the CPZ model group ( Figure 2 CF, p<0.05). Compared with the group treated with Bifidobacterium longum, the mice in the group treated with Selenium-enriched Bifidobacterium longum showed a more significant increase in the latency and rotation speed when falling ( Figure 2 CF, p<0.05). The grip force experiment further confirmed the above results ( Figure 2G). The above data show that the selenium-enriched Bifidobacterium longum inoculum can improve the motor coordination ability of demyelinated mice, which is stronger than the Bifidobacterium longum inoculum.

[0215] To evaluate the remyelination of each group of mice, the coronal sections of the corpus callosum were stained with MBP immunohistochemistry. MBP is used to detect myelin protein components and is sensitive to myelin loss, which is used to evaluate myelin regeneration. Clear MBP-positive fibers were visible in the corpus callosum of ND mice, and the staining on the corpus callosum was dense and regular. In contrast, the MBP staining of mice exposed to CPZ for 5 weeks and restored to a normal diet for 2 weeks became lighter and irregular ( Figure 3 B), further quantitative analysis showed that the MBP immunohistochemical positive area in the CPZ model group was significantly reduced compared with the ND group ( Figure 3 C, p<0.05). Interestingly, compared with the CPZ model group, the MBP labeling in the group treated with selenium-enriched Bifidobacterium longum inoculum was more significant 2 weeks after cessation of CPZ, and oral administration of selenium-enriched Bifidobacterium longum inoculum significantly increased the MBP-positive staining area of ​​the corpus callosum ( Figure 3 C, p<0.05). These results indicate that selenium-enriched Bifidobacterium longum can promote myelin regeneration in CPZ mice.

[0216] In summary, the present invention has carried out the effect of preventing, suppressing and intervening in the treatment of demyelinating diseases by selenium-rich Bifidobacterium longum inoculum and Bifidobacterium longum inoculum in two models of preventing and suppressing CPZ-induced demyelination model (CPZ-induced demyelination model and intervention of selenium-rich Bifidobacterium longum inoculum) and treating CPZ-induced demyelination model (CPZ-induced myelin regeneration model and intervention of selenium-rich Bifidobacterium longum inoculum). Selenium-rich Bifidobacterium longum inoculum and Bifidobacterium longum inoculum can significantly prevent and suppress CPZ-induced demyelination, improve the motor coordination ability of demyelination model, and selenium-rich Bifidobacterium longum inoculum further enhances the effect of Bifidobacterium longum inoculum. Selenium-rich Bifidobacterium longum inoculum has more advantages in exerting myelin protection and improving the ability of behavioral abnormalities. On the other hand, selenium-rich Bifidobacterium longum inoculum can also promote the remyelination of the central nervous system.

[0217] Test Example 2

[0218] Selenium-enriched Bifidobacterium longum inoculum prevents and improves cognitive dysfunction in demyelinating models by preventing and inhibiting hippocampal / cortical demyelination and repairing neuronal damage

[0219] 1. Experimental Methods

[0220] In order to explore the effect of selenium-enriched Bifidobacterium longum on the cognitive function of the demyelinating model, water maze, Nissl staining and MBP immunohistochemistry experiments were used to evaluate the effects of selenium-enriched Bifidobacterium longum on the learning and memory ability, hippocampal / cortical neurons and myelin damage of the demyelinating model.

[0221] 1. Nissl staining: paraffin sections are dewaxed until hydrated, tissue sections are placed in Nissl stain for 5 minutes, washed with tap water, slightly differentiated with 0.1% acetic acid, washed with tap water to terminate the reaction, and the degree of differentiation is controlled under a microscope. After washing with tap water, the sections are placed in an oven to dry. The sections are placed in clean xylene for 10 minutes and sealed with neutral gum. Finally, the microscope is used to collect and photograph.

[0222] 2. Water maze: The water maze pool is filled with water, heated, and the temperature is controlled at 22±1℃. Take the experimental animals to the room where the water maze is located 1h in advance to adapt to the experimental environment, set the software parameters, and add the experimental animal information. Put the animals in the corresponding area of ​​the water maze and start the water maze experiment. The water maze experiment is divided into a positioning navigation experiment and a space exploration experiment; Positioning navigation experiment: a total of 5 days, tested at a fixed time every day, each experimental animal enters the water from 4 different quadrants twice a day, each time lasting 1 minute, and the interval between each two trainings is more than 30min. If a mouse fails to find the platform within 1 minute, the escape latency is recorded as 60 seconds, and then it is guided to find the platform and let the mouse stay on the platform for 15 seconds. After each experiment, the animal is taken out of the water maze, the surface water is wiped off, and it is put back in the cage. After the positioning navigation experiment on the 5th day, that is, on the 6th day, the platform is removed, and the space exploration experiment is carried out. The quadrant farthest from the platform is selected as the animal's entry point for free exploration for 1 minute. The number of times the experimental animals crossed the platform, the distance they traveled in the target quadrant, and the time they stayed in the target quadrant were calculated using computer software.

[0223] 2. Experimental Results

[0224] The hippocampus is the most important area for regulating learning and memory in the brain, and myelinated fibers are also distributed. Literature reports in recent years have shown that myelin is closely related to the occurrence and development of learning and memory. Next, MBP immunohistochemical staining was used to detect the content of myelin basic protein in hippocampal and cortical tissues. Figure 4 As shown in A, the MBP staining in the hippocampus and cortex of the ND group mice was uniform, and the MBP-positive fibers were dense. In contrast, compared with the ND group, the CPZ model mice showed obvious demyelination in the hippocampus and cortex. It is worth noting that after the intervention of the selenium-enriched Bifidobacterium longum inoculum, an increase in MBP-positive fibers was observed in the hippocampus and cortex. Further quantitative analysis of the sections found that the MBP immunohistochemical positive staining area of ​​the mice treated with the selenium-enriched Bifidobacterium longum inoculum was significantly higher than that of the CPZ model group ( Figure 4 B and C, p<0.05). The above results suggest that selenium-enriched Bifidobacterium longum can improve CPZ-induced demyelination damage in the hippocampus and cortex.

[0225] Nissl staining was used to evaluate the effects of selenium-enriched Bifidobacterium longum on CPZ-induced damage to hippocampal and cortical neurons. Figure 4As shown in D, the arrangement of neurons in the dentate gyrus (DG), CA1 region and cortex of the hippocampus in the ND group was relatively linear and the staining was darker, indicating that Nissl bodies were abundant. Compared with the ND group, the neurons in the hippocampus and cortex of the CPZ group were disordered and irregular in layering, and the staining was lighter; further quantitative analysis revealed that the number of neurons marked by Nissl staining was significantly reduced ( Figure 4 DG, p<0.05). In contrast, Figure 4 DG, mice treated with selenium-enriched Bifidobacterium longum restored the irregular arrangement of hippocampal and cortical neurons, and the staining became darker; interestingly, the number of neurons in the hippocampus and cortex was also significantly improved.

[0226] Next, the water maze experiment was used to evaluate the learning and memory abilities of each group of experimental mice. Figure 4 HK showed that compared with the ND group, the number of times the CPZ model mice crossed the platform, the time they stayed in the quadrant where the platform was located, and the distance they marched were significantly reduced. Compared with the CPZ group, the number of times the mice crossed the platform, the time they stayed in the quadrant where the platform was located, and the distance they marched were significantly increased after the intervention of the selenium-enriched Bifidobacterium longum inoculum ( Figure 4 HK, p<0.05). This result shows that the selenium-enriched Bifidobacterium longum agent can improve the learning and memory ability of the demyelination model.

[0227] In summary, the present invention also found that the selenium-enriched Bifidobacterium longum bacterial agent can also prevent and improve cognitive dysfunction in demyelinating model mice by preventing and inhibiting demyelination of the hippocampus / cortex and repairing neuronal damage.

[0228] Test Example 3

[0229] Selenium-enriched Bifidobacterium longum prevents and improves depressive-like behavior in a demyelination model

[0230] 1. Experimental Methods

[0231] 1. Sugar water preference experiment: Based on the fact that rodents have a strong innate desire for sweets, when they are provided with two drinking water devices that can be freely chosen, namely, containing sucrose solution and ordinary water, they will selectively drink the sweet sucrose solution. Detecting the preference of experimental animals for sucrose solution in this way can be used as a useful means to evaluate the symptoms of anhedonia and depression in experimental animals. Specifically, one day before the experiment, a bottle of aqueous solution containing 1% sucrose and a bottle of normal drinking water were placed in the cage, and the mice drank freely to adapt to the sugar water, and the positions were changed every 12 hours. At 9 o'clock in the morning on the day of the experimental test, the mice were deprived of water for 12 hours. At 9 o'clock in the evening, the aqueous solution containing 1% sucrose and normal drinking water were weighed and placed in the cage for the mice to drink freely. At 1 o'clock in the afternoon of the next day, the two bottles of water in each cage were taken out and weighed, and the mass of drinking water and sucrose water consumed by the experimental mice within 16 hours was recorded. The sucrose preference index calculation formula is:

[0232]

[0233] 2. Tail suspension test: In the tail suspension test, mice in normal physiological state will show the behavior of struggling to survive and getting rid of restraints. In the depressed state, mice will give up struggling to survive and remain motionless. Therefore, the time of immobility is used as an indicator to evaluate depressive behavior. The specific experimental operation is: stick the tip of the tail of the experimental mouse 1-2cm with medical tape, and hang it upside down on the balance bar to ensure that the tip of its nose is 15-20cm off the ground. The hanging time is 6 minutes, and the time of immobility within 4 minutes is recorded.

[0234] 1. Experimental Results

[0235] In the present invention, the sugar water preference test and the tail suspension test were used to evaluate the effect of the selenium-enriched Bifidobacterium longum bacterial agent on the depressive-like behavior of demyelinated mice. Figure 5 As shown in the figure, compared with the ND group, the sucrose preference index of mice in the CPZ model group was significantly decreased and the immobility time in the tail suspension test was significantly increased (p<0.05), indicating that after CPZ-induced myelin damage, mice showed depressive-like behavior. It is worth noting that compared with the CPZ group, the sucrose preference index of mice in the selenium-enriched Bifidobacterium longum inoculum group was significantly increased (p<0.05), and the ability to experience pleasure was increased. Interestingly, consistent with the results of the sugar water preference experiment, the immobility time of mice in the tail suspension test after intervention with the selenium-enriched Bifidobacterium longum inoculum was significantly decreased (p<0.05). The above results show that the selenium-enriched Bifidobacterium longum inoculum can alleviate the depressive-like behavior of demyelinated mice and has an antidepressant effect.

[0236] Test Example 4

[0237] Selenium-enriched Bifidobacterium longum prevents and improves cognitive deficits in Alzheimer's disease models by preventing and inhibiting hippocampal / cortical demyelination and repairing neuronal damage

[0238] 1. Experimental Methods

[0239] Water maze, novel object recognition, NeuN immunofluorescence staining and MBP immunohistochemistry experiments were used to evaluate the effects of selenium-enriched Bifidobacterium longum on the learning and memory ability, hippocampal / cortical neurons and myelin damage in the Alzheimer's disease model.

[0240] 1. Experimental animals: 4-month-old male APP / PS1 double transgenic Alzheimer's disease (AD) mice (B6C3-Tg (APPswe, PS1dE9)) and their littermates of wild-type mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. All experimental mice were placed in an SPF-grade breeding environment, and the mice were free to eat and drink. All mouse feeding management and experimental operation procedures strictly complied with the experimental animal management regulations and ethical requirements of the Animal Management Committee of Nanjing University.

[0241] 2. Experimental grouping and drug administration: The experimental mice were randomly divided into the following 5 groups: wild-type control group (WT), APP / PS1AD model group (AD), Bifidobacterium longum inoculum intervention group (BL+AD), selenium-enriched Bifidobacterium longum inoculum intervention group (Se-BL+AD) and GV-971 intervention group (GV-971+AD). Each mouse in the probiotic intervention group was gavaged with 1.5×10^9 CFU of Bifidobacterium longum or selenium-enriched Bifidobacterium longum inoculum per day. The intervention dose of GV-971 was 100 mg / kg / d. The control group and the model group were gavaged with the same dose of PBS. Intervention started at 4 months of age, once a day, for 6 consecutive months.

[0242] 2. Immunofluorescence staining: paraffin sections are dewaxed to water, and then antigen repair is performed. After immersion, the repaired sections are immersed in 3% hydrogen peroxide, blocked at room temperature for 30 minutes in the dark, and then washed with distilled water. After the sections are slightly dried, use a tissue pen to draw circles around the tissue, add 10% serum consistent with the source of the secondary antibody, and incubate at 37°C for 30 minutes. Shake off the serum, dilute the first antigen solution with 10% serum, prepare the first antibody working solution (NeuN, rabbit antibody, 1:1000), add an appropriate amount of the first antibody working solution to each section, and incubate at 4°C overnight. The next day, take the sections out of the refrigerator, place them at room temperature for 15 minutes (rewarming), rinse them 3 times with TBST, and then immerse them 3 times, each time for 3 minutes. Dilute the second antigen solution with TBST to prepare the second antibody working solution (Alexa Fluor 594 labeled goat anti-rabbit, 1:300), add an appropriate amount of the second antibody working solution to each section, and incubate at 37°C for 45 minutes. Rinse with TBST 3 times, and then immerse and wash 3 times, each time for 3 minutes. Then, add an appropriate amount of DAPI working solution (prepared with DAPI stock solution 1:500) to each slice, stain the nucleus for 5 minutes in the dark, and then rinse with TBST. Finally, seal the slice with anti-fluorescence quenching sealing agent. Then collect images with a fluorescence microscope.

[0243] 3. New object recognition: The new object recognition experiment is divided into two stages: the familiarization stage and the test stage. The familiarization stage lasts for two days. On the first day, the experimental animals are placed in an experimental box without objects and allowed to move freely for 5 minutes. On the second day, two identical objects (cubic wooden blocks) are placed in the experimental box, and then the animals are placed in the experimental box with their backs facing the objects at an equal distance from the two objects, allowing them to explore freely within 5 minutes. After each experiment, the experimental box is cleaned and disinfected to remove the odor left by the animals. In the test stage, that is, the third day, the animals are put back into the experimental box. At this time, a familiar object is retained in the experimental box, and another familiar object is replaced with a new object (a cylindrical wooden block of similar size). The animals are allowed to explore freely again for 5 minutes. Observe and record the exploration time of the animals around the familiar objects and new objects, and calculate the new object recognition index.

[0244] 2. Experimental Results

[0245] MBP immunohistochemical staining was used to observe the pathological changes of myelin in the cortex and hippocampus. Figure 6 As shown in A, the MBP-positive fibers in the cortex and hippocampus of WT mice were dense, and the myelin sheath was intact and uniform without damage. In contrast, compared with the WT group, the MBP-positive fibers in the cortex and hippocampus of AD model mice were sparse and less, and the myelin sheath was damaged and incomplete. Further quantitative statistics showed that the area of ​​MBP-positive area was significantly reduced ( Figure 6 BF, p<0.05), indicating myelin function defects in AD mice. However, after intervention with Bifidobacterium longum or selenium-enriched Bifidobacterium longum or GV-971, myelin damage in the cortex and hippocampus was restored to varying degrees, and the MBP-positive area was significantly increased compared with the AD group mice. Compared with Bifidobacterium longum or GV-971, the MBP-positive area in the cortex and hippocampus was significantly increased after intervention with selenium-enriched Bifidobacterium longum ( Figure 6 BF, p<0.05), indicating that the selenium-enriched Bifidobacterium longum bacterial agent has the best ability to delay myelin sheath damage and loss.

[0246] Myelin deficiency is an important factor in neuronal loss and dysfunction. Next, we used NeuN immunofluorescence staining to assess changes in neuronal loss in the cortex and hippocampus. Figure 7As shown in AE, the mature neurons labeled by NeuN in the cortex and hippocampus of WT mice were evenly distributed and had strong fluorescent staining, indicating that the neuronal function was intact. In contrast, the number of mature neurons labeled by NeuN in the CA1, CA3, and DG regions of the cortex and hippocampus of AD mice was scarce, unevenly distributed, and the staining became lighter. Further quantitative analysis found that compared with the WT group, the number of mature neurons labeled by NeuN in the cortex and hippocampus of AD mice was significantly reduced (p<0.05). Compared with the AD model group, the number of mature neurons labeled by NeuN in the cortex and hippocampus of AD mice treated with Bifidobacterium longum or selenium-enriched Bifidobacterium longum was observed to increase to varying degrees (p<0.05). Interestingly, compared with the Bifidobacterium longum group, mice treated with selenium-enriched Bifidobacterium longum had significantly more NeuN-positive cells in the cortex and hippocampus DG region (p<0.05), showing the characteristics of large number and close arrangement, and full staining morphology. This result shows that the selenium-enriched Bifidobacterium longum can better restore the number of neurons in AD mice. Similar to the selenium-enriched Bifidobacterium longum agent, GV-971 intervention was also able to increase the number of neurons in the hippocampus and cortex of AD mice (p<0.05).

[0247] In order to further explore the effect of selenium-enriched Bifidobacterium longum on the cognitive function of AD mice after improving myelin and neuronal damage, water maze and novel object recognition experiments were used to detect the learning and memory abilities of each group of mice. Figure 8 As shown in B and C, during five consecutive days of acquisition training, we found that the escape latency of WT mice to find the underwater platform gradually shortened, while the escape latency of AD model mice only changed slightly, and the escape latency remained longer, indicating that the spatial positioning and memory ability of AD mice to the platform position declined, leading to blind search for the platform. There were significant differences in the escape latency of WT and AD mice on days 2, 4, and 5, respectively (p<0.05). Compared with the AD model group, there was no statistical difference in the escape latency of mice after intervention with Bifidobacterium longum inoculum in the first four days, but there was a significant improvement on the fifth day (p<0.05). Interestingly, the escape latency of mice in the selenium-enriched Bifidobacterium longum inoculum group on the fourth day was significantly improved compared with the AD model group, and there was a significant difference on the fourth and fifth days (p<0.05). The mice in the GV-971 group also had significant differences on the fourth and fifth days compared with the AD model group (p<0.05). The day after acquisition training, we removed the platform and conducted exploration training, such as Figure 8As shown in DG, we can see the water maze motion trajectory diagrams among the five groups. It can be observed that the motion trajectories of the WT and other three groups of mice treated with drug intervention in the quadrant where the platform is located are more than those of the AD model group. Further quantitative analysis showed that compared with the AD group, the number of times the mice in the BL+AD, Se-BL+AD and GV-971+AD groups crossed the platform, as well as the distance and time spent in the target quadrant were significantly increased (p<0.05). It is worth noting that compared with the BL+AD group, the number of times the mice in the Se-BL+AD group crossed the platform increased significantly (p<0.05), indicating that the improvement effect of the non-genetically modified selenium-enriched Bifidobacterium longum inoculum was stronger. On the other hand, after the intervention of the selenium-enriched Bifidobacterium longum inoculum, the classic edge-type search strategy of AD mice was changed to a trend / random type ( Figure 8 H).

[0248] Next, the water maze test results were further verified using a novel object recognition experiment. The novel object recognition experiment is a learning and memory test method that uses the principle that animals have an innate tendency to explore new objects. This method allows mice to conduct learning and memory tests in a free-moving state, which can more closely simulate human learning and memory behavior. Figure 8 As shown in Figure 1, compared with the WT group, AD mice did not show a tendency towards new objects, and the novel object recognition index decreased significantly (p<0.05). Compared with AD mice, mice in the Bifidobacterium longum group showed a certain tendency towards new objects, and the novel object recognition index was significantly improved (p<0.05). Interestingly, mice after intervention with the selenium-enriched Bifidobacterium longum were more inclined to explore new objects, and the novel object recognition index was further improved (p<0.05), which was comparable to the WT group. Similar to the selenium-enriched Bifidobacterium longum group, the novel object recognition index of mice in the GV-971 treatment group, our positive control drug, was also significantly enhanced (p<0.05). The above results indicate that Bifidobacterium longum and selenium-enriched Bifidobacterium longum can significantly improve cognitive dysfunction in AD mice, and the selenium-enriched Bifidobacterium longum has the best effect and improves learning and memory ability. Its efficacy is comparable to that of GV-971, or even better than that of GV-971.

[0249] In summary, the present invention further found that the selenium-enriched Bifidobacterium longum inoculum can also prevent and improve the cognitive function defects of the Alzheimer's disease model by preventing and inhibiting hippocampal / cortical demyelination and repairing neuronal damage, and the selenium-enriched Bifidobacterium longum inoculum further enhances the effect of the Bifidobacterium longum inoculum.

[0250] Test Example 5

[0251] Selenium-enriched Bifidobacterium longum regulates intestinal flora in a demyelination model

[0252] 1. Experimental Methods

[0253] In order to explore the regulatory effect of selenium-enriched Bifidobacterium longum on the intestinal flora of the demyelination model, after the model was established, 16S rRNA sequencing technology was used to evaluate the changes in the intestinal flora of each group of mice.

[0254] 1. Feces collection: CPZ-induced demyelination model was established and Bifidobacterium longum or selenium-enriched Bifidobacterium longum was used for intervention. At 8:00 a.m. on the second day after the last oral gavage of each group of mice, each mouse was placed in an autoclaved non-litter cage to allow free defecation, and the feces formed were quickly collected in a sterilized EP tube, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0255] 2.16S rRNA sequencing:

[0256] The genomic DNA of mouse feces was extracted using MagPure Soil DNALQ Kit (Magan) according to the instructions. The concentration and purity of DNA were detected by NanoDrop 2000 (Thermo Fisher Scientific) and agarose gel electrophoresis, and the extracted DNA was stored at -20°C. The extracted genomic DNA was used as a template, and specific primers with barcodes and Takara Ex Taq high-fidelity enzyme were used to perform PCR amplification of the bacterial 16S rRNA gene. The universal primers 343F (5'-TACGGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3') (World J Gastroenterol. 2010; 16 (33): 4135-4144.) were used to amplify the V3-V4 variable region of the 16S rRNA gene. The PCR amplification product was detected by agarose gel electrophoresis. Then it was purified using magnetic beads, and the purified product was used as a second-round PCR template, and a second-round PCR amplification was performed. The purified products were purified again using magnetic beads, and the purified products were quantified by Qubit, and then the concentration was adjusted for sequencing. The Illumina NovaSeq 6000 sequencing platform was used for sequencing, and 250bp double-end reads were generated. The QIIME2 software was used to perform relative abundance statistics for each sample, and bacterial phylum to genus classification.

[0257] 2. Experimental Results

[0258] The present invention uses 16S rRNA to detect the composition of the intestinal flora in mouse feces. Fig. 9 The 16S rRNA sequencing results of the feces of mice in each group. Fig. 9As shown in A, at the genus level, the relative abundance of opportunistic pathogens increased after CPZ treatment, and the imbalance of intestinal microorganisms was alleviated to a certain extent after treatment with selenium-enriched Bifidobacterium longum or Bifidobacterium longum. Fig. 9 As shown in BH, selenium-enriched Bifidobacterium longum inoculum or Bifidobacterium longum inoculum significantly reduced the relative abundance of conditionally pathogenic bacterial genera (p<0.05), including Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus and Eisenbergiella. Compared with the Bifidobacterium longum inoculum treatment group, the relative abundance of Desulfovibrio and Christensenellaceae_R-7_group in the selenium-enriched Bifidobacterium longum inoculum treatment group decreased more (p<0.05). It is worth noting that the relative abundance of Desulfovibrio and Christensenellaceae_R-7_group is also abnormally increased in the intestinal flora of clinical multiple sclerosis patients (Nat Rev Neurol. 2022; 18(9):544-558.). As Fig. 9 As shown in IN, compared with the ND and CPZ groups, the relative abundance of the potential beneficial bacteria Faecalibaculum and Bifidobacterium increased significantly after treatment with Bifidobacterium longum inoculum (p<0.05). Compared with the ND, CPZ and Bifidobacterium longum inoculum groups, the relative abundance of potential beneficial bacteria such as Bifidobacterium, Lactobacillus, Dubosiella, Coriobacteriaceae_UCG-002 and Enterorhabdus increased significantly after treatment with selenium-enriched Bifidobacterium longum inoculum (p<0.05). However, there was no significant difference in the relative abundance of potential beneficial bacteria Lactobacillus, Dubosiella, Coriobacteriaceae_UCG-002 and Enterorhabdus in the Bifidobacterium longum inoculum treatment group compared with the ND and CPZ groups.

[0259] The above results show that selenium-enriched Bifidobacterium longum and Bifidobacterium longum can regulate the balance of intestinal microorganisms, increase the relative abundance of beneficial bacteria, and reduce the relative abundance of conditional pathogens, thereby protecting the body. Selenium-enriched Bifidobacterium longum has more advantages than Bifidobacterium longum in reshaping the homeostasis of intestinal flora.

[0260] Experimental conclusion: Selenium-enriched Bifidobacterium longum and Bifidobacterium longum can effectively prevent and inhibit myelin loss, prevent and improve behavioral abnormalities in demyelination models and Alzheimer's disease models. Selenium-enriched Bifidobacterium longum further enhances the preventive, inhibitory and therapeutic effects of Bifidobacterium longum. In the remyelination animal model, selenium-enriched Bifidobacterium longum can also promote myelin regeneration. Selenium-enriched Bifidobacterium longum and Bifidobacterium longum can regulate the intestinal microbial balance of the demyelination model, increase the relative abundance of beneficial bacteria, reduce the relative abundance of conditional pathogens, and improve the disordered intestinal flora structure. Selenium-enriched Bifidobacterium longum further significantly enhances the ability of Bifidobacterium longum to reshape the intestinal flora. Compared with a single Bifidobacterium longum, selenium-enriched Bifidobacterium longum has a stronger ability to protect myelin and regulate intestinal flora, thus providing a new live microbial therapy for the treatment of demyelinating diseases.

[0261] As can be seen from the above, the experimental results of the above embodiments show that the selenium-enriched Bifidobacterium longum bacterial agent can prevent and inhibit myelin loss and promote myelin regeneration and regulate the intestinal microbial balance of the demyelination model, and the selenium-enriched Bifidobacterium longum bacterial agent further significantly improves the effect of the Bifidobacterium longum bacterial agent. Therefore, it can be confirmed that the selenium-enriched Bifidobacterium longum bacterial agent of the present invention can be used to prepare a drug for preventing and treating demyelinating disease; prepare a drug for improving and regulating the intestinal flora of demyelinating disease.

[0262] Test Example 6

[0263] Acute oral toxicity test of selenium-enriched Bifidobacterium longum

[0264] 1. Experimental methods:

[0265] Experimental animals: 20 healthy adult SPF SD rats, half male and half female, with a weight of 187-205g for females and 206-220g for males, provided by Nanjing Medical University, production license number SCXK (Su) 2021-0001, certificate number: 202335660. The experimental animals were raised in the barrier system of this center at a temperature of 20-26°C and a relative humidity of 40%-70%, and the experimental animal use license number is SYXK (Su) 2020-0006. The experimental mouse maintenance feed was provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., production license: Su Feed Certificate (2019) 01008.

[0266] Dose selection and test substance administration method: The acute toxicity test of rats was carried out using the limit method, and the dose was 10000 mg / kg·bw. The animals were quarantined for 3 days. The animals were fasted for 16 hours before the test and had free access to water. The test sample was prepared with purified water at a concentration of 500 mg / mL. The gavage volume was 20 mL / kg·bw, and the sample was given once. The dose of the test substance was 10000 mg / kg·bw. After sampling, the animals were observed for 14 consecutive days, and the symptoms of poisoning and death were recorded. The animals were weighed once on the 0th, 7th, and 14th days. At the end of the observation period, the animals underwent gross anatomical examination.

[0267] Main instruments: JJ10 electronic balance, T1000 electronic balance.

[0268] Test method: The acute toxicity test in rats was carried out using the limit method.

[0269] 2. Experimental Results

[0270] During the experimental observation period, the animals in each group had normal diet and activities, grew well, and no poisoning symptoms or deaths were observed. At the end of the observation period, no abnormal pathological changes were observed in the gross autopsy of the animals. The effects of the selenium-enriched Bifidobacterium longum inoculant on the body weight and mortality of rats are shown in Table 1.

[0271] Table 1 Effects of selenium-enriched Bifidobacterium longum on acute oral toxicity in rats

[0272]

[0273] Experimental conclusion: No obvious poisoning symptoms were observed after oral administration of the selenium-enriched Bifidobacterium longum inoculum to animals, no significant effect on the weight gain of animals, and no animal deaths during the observation period. Acute oral toxicity test of selenium-enriched Bifidobacterium longum in female and male rats LD 50 Greater than 10000 mg / kg·bw, according to the acute toxicity dose classification standard, the sample is actually non-toxic.

[0274] The above describes the basic principle, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. Use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating demyelinating disease.

2. Use of a Bifidobacterium longum bacterial agent in the preparation of a drug for regulating intestinal flora.

3. The use according to claim 1 or 2, characterized in that: The Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

4. The use according to claim 3, characterized in that: The preparation method of the selenium-enriched Bifidobacterium longum bacterial agent comprises the following steps: a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter; b. Sterilize at 121°C for 20 minutes and then cool to room temperature; c. Add 30mL-180mL of seed solution as 1%-6% inoculum; d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5; e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L; f. Ferment for 8-10 hours, during which nitrogen is passed to drive out the air, and 30% glucose is added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum inoculum.

5. The use according to claim 4, characterized in that: In step (e), the method for preparing the nano-selenium solution comprises the following steps: (1) Add stabilizer to a beaker; (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved; (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring; (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

6. The use according to claim 1, characterized in that: The Bifidobacterium longum bacterial agent is used for preventing and inhibiting demyelination of the central nervous system; the demyelinating disease includes any one of multiple sclerosis, Alzheimer's disease or major depression.

7. The use according to claim 1, characterized in that: The Bifidobacterium longum bacterial agent is used to promote regeneration of myelin in the central nervous system; the Bifidobacterium longum bacterial agent can regulate the intestinal flora of a demyelination animal model, reduce harmful flora, increase beneficial flora, and has the ability to reshape the intestinal flora.

8. The use according to claim 1, characterized in that: The harmful bacteria include Desulfovibrio, Christensenellaceae_R-7_group, Helicobacter, Lachnoclostridium, Oscillibacter, Anaerotruncus, and Eisenbergiella; the beneficial bacteria include Dubosiella, Lactobacillus, Faecalibaculum, Bifidobacterium, Coriobacteriaceae_UCG-002, and Enterorhabdus.

9. A drug for preventing and treating demyelinating diseases or regulating intestinal flora, characterized in that The method comprises the Bifidobacterium longum bacterial agent according to claim 1 or 2 or the selenium-enriched Bifidobacterium longum bacterial agent according to claim 3 as an active ingredient.

10. A method for preparing the medicine for use according to claim 9, characterized in that The method comprises the following steps: (1) adding selenium to a culture solution containing Bifidobacterium longum, culturing in an incubator, and collecting the mixed bacterial solution to obtain selenium-enriched Bifidobacterium longum; (2) mixing the selenium-enriched Bifidobacterium longum with a protective agent; (3) Depending on the desired dosage form, the selenium-enriched Bifidobacterium longum is resuspended in skimmed milk and processed into an oral solution, lyophilized powder or capsule.

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