Application of bifidobacterium longum fungicide in preparation of medicine for preventing and treating Parkinson's disease

Abstract

The invention discloses an application of a bifidobacterium longum microbial inoculum in preparation of a medicine for preventing and treating Parkinson's disease, which proves that selenium-rich bifidobacterium longum and bifidobacterium longum can be used for preventing and treating a Parkinson's disease model for the first time, and the application of the selenium-rich bifidobacterium longum microbial inoculum in 1-methyl-4-phenyl-1, 2, 3, 4-tetramethyl-1, 2, 3, 4-tetramethyl-1, 2, 3, 4- An acute Parkinson's disease model constructed by 2, 3, 6-tetrahydropyridine (MPTP) and a chronic Parkinson's disease model constructed by rotenone verify that the selenium-rich bifidobacterium longum and the bifidobacterium longum can improve central symptoms and peripheral symptoms of the Parkinson's disease. The invention proves that selenium-rich bifidobacterium longum and bifidobacterium longum can comprehensively prevent and treat Parkinson's disease from peripheral and central symptoms for the first time, including acute Parkinson's disease mouse models and chronic Parkinson's disease mouse models. The selenium-rich bifidobacterium longum microbial inoculum has the effects of both medicines and nutrition functions, is high in safety, is proved to be in an actual non-toxic level by an acute oral toxicity test of rats, and can be taken for a long time for prevention or treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to application of a Bifidobacterium longum bacterial agent in preparing a drug for preventing and treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a common degenerative disease of the elderly nervous system, also known as "tremor paralysis", a degenerative disease of the central nervous system common in middle-aged and elderly people. The incidence rate of people over 65 years old is as high as 1.7%, and it is showing a trend of getting younger. The incidence rate of people under 40 years old is increasing year by year (Jankovic J. Journal of Neurology, Neurosurgery, and Psychiatry, 2008, 79 (4): 368-376.). Parkinson's disease is currently incurable, and chemical drugs are mainly used to relieve limb symptoms. However, most patients experience weakened effects and increased side effects when using levodopa for a long time. These treatment methods cannot prevent the deterioration of the disease and the continuous loss of neurons, and increase the burden of life for patients (Lewitt P A. Movement Disorders: Official Journal of the Movement Disorder Society, 2015, 30 (1): 64-72.). Parkinson's symptoms are mainly divided into peripheral symptoms and central symptoms. Peripheral symptoms mainly include gastrointestinal dysfunction, while central symptoms are mainly the loss of dopaminergic neurons leading to severe degeneration of limb motor ability (Kalia LV, Lang AE. Lancet, 2015, 386(9996):896-912.).

[0003] Clinically, Parkinson's peripheral symptoms are mainly manifested in intestinal flora disorders and constipation in the vast majority of patients. The chemical drug Madopar used clinically to treat Parkinson's can cause strong gastrointestinal side effects (Liu H, Chen L, Zhang Z, et al. Acupuncture In Medicine: Journal of the British Medical Acupuncture Society, 2017, 35 (6): 404-412.), so probiotics have become a feasible method for trying to treat and improve Parkinson's symptoms. In preclinical and clinical trials, probiotics have been shown to relieve constipation in Parkinson's patients. Compared with placebo, patients who took bifidobacterium and lactic acid bacteria strains for 3 months had improved quality of life, increased bowel movements, and reduced intestinal defects (Tan AH, Lim SY, Chong KK, et al. Neurology, 2021, 96 (5): e772-e782.). However, these studies are limited to relieving the intestinal symptoms of Parkinson's patients, and no further research and verification of central symptoms has been conducted. There are no reports on probiotic treatment for central nervous system symptoms to date.

[0004] The central symptom of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra and striatum of the brain, which leads to impaired motor ability and gradual loss of limb function in patients. Selenium is a trace element that is essential to human health. There is evidence that selenium plays a vital role in the brain and brain function and can protect Parkinson's dopaminergic neuron damage. But it can also be highly neurotoxic, depending on the intake and form of action (Ingold I, Berndt C, Schmitt S, et al. Cell, 2018, 172 (3) 409-422.e21.; Solovyev N D. Journal of Inorganic Biochemistry, 2015, 153: 1-12.).

[0005] The present invention has developed a selenium-enriched Bifidobacterium longum as a drug for preventing and treating Parkinson's disease. It has been proven in mice that it can comprehensively improve Parkinson's disease's motor disorders and non-motor disorders. Compared with the positive drug Madopar, the selenium-enriched Bifidobacterium longum can protect the motor neurons of Parkinson's mice from damage by starting from the pathogenesis of Parkinson's disease. In addition, it can also protect the intestinal microecology from destruction. In view of the limitations of existing drugs for the treatment of Parkinson's disease, in general, the selenium-enriched Bifidobacterium longum is an ideal biological drug for the prevention and treatment of Parkinson's disease. Summary of the invention

[0006] In order to solve the problems of the prior art, the purpose of the present invention is to provide a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating Parkinson's disease.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: In the first aspect, the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating Parkinson's disease.

[0008] In a second aspect, the present application provides a drug for preventing and treating Parkinson's disease, comprising Bifidobacterium longum or selenium-enriched Bifidobacterium longum as an active ingredient.

[0009] In a third aspect, the present application provides a pharmaceutical composition.

[0010] 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 Parkinson's disease.

[0011] Furthermore, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent. Selenium-enriched Bifidobacterium longum (original bacterial species name: Bifidobacterium longum; bacterial species number: CICC 6188).

[0012] The efficacy of Bifidobacterium longum and its selenium-enriched Bifidobacterium longum in preventing and treating Parkinson's disease was fully verified in two models: an acute Parkinson's disease model constructed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP and a chronic Parkinson's disease model constructed by rotenone. The efficacy of selenium-enriched Bifidobacterium longum was significantly better than that of Bifidobacterium longum.

[0013] Furthermore, the preparation method of the selenium-enriched Bifidobacterium longum bacterial agent comprises the following steps:

[0014] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0015] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0016] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0017] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0018] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0019] f. Ferment for 8-10 hours, during which nitrogen is passed to drive out air, and 30% glucose is added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum inoculum. The preparation method of selenium-enriched Bifidobacterium longum is to enrich the Bifidobacterium longum with selenium or nano-selenium during the fermentation process.

[0020] Furthermore, in step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0021] (1) Add stabilizer to a beaker;

[0022] (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;

[0023] (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;

[0024] (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.

[0025] Furthermore, the selenium-enriched long bifidobacterium bacterial agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein 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.

[0026] The second aspect of the present application provides a drug for preventing and treating Parkinson's disease, comprising Bifidobacterium longum or selenium-enriched Bifidobacterium longum as an active ingredient.

[0027] The third aspect of the present application provides a pharmaceutical composition comprising Bifidobacterium longum or selenium-enriched Bifidobacterium longum and a pharmaceutically acceptable carrier or diluent for preventing and treating Parkinson's disease.

[0028] Beneficial effects: The present invention proves for the first time that selenium-enriched Bifidobacterium longum and Bifidobacterium longum can comprehensively prevent and treat Parkinson's disease from peripheral and central symptoms, including acute Parkinson's disease mouse and chronic Parkinson's disease mouse models. The present invention has high safety and low toxic and side effects.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The present invention is based on the limitation that the chemical drug Madopar is currently used in clinical treatment of Parkinson's disease, which can only improve limb symptoms but cannot fundamentally prevent motor neuron damage. It is found that the selenium-enriched Bifidobacterium longum and Bifidobacterium longum constructed in the laboratory can not only improve the limb symptoms of Parkinson's mice, but also prevent the loss of dopaminergic neurons in the substantia nigra and striatum of Parkinson's mice.

[0031] (2) The present invention is based on the fact that the chemical drug Madopar is currently used clinically to treat Parkinson's disease, which can aggravate the toxic side effects of gastrointestinal dysfunction in Parkinson's patients. It is found that the selenium-enriched Bifidobacterium longum and Bifidobacterium longum constructed by the present invention can not only improve the limb symptoms of Parkinson's mice from the central nervous system, but also improve the intestinal dysfunction of Parkinson's mice from the periphery, and alleviate constipation and intestinal inflammatory infiltration in Parkinson's mice.

[0032] (3) The selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention belong to biological therapy. Parkinson's disease is a type of neurodegenerative disease that is difficult to detect and diagnose in the early stage and progresses rapidly in the later stage. Compared with small molecule chemical drugs, selenium-enriched Bifidobacterium longum has lower safety and is easier to be accepted by patients in the early prevention and treatment of Parkinson's disease. It has little toxicity and side effects and can improve the quality of life of patients. It is expected to become a direct or auxiliary prevention and treatment plan worthy of clinical selection.

[0033] (4) From the perspective of central symptoms, selenium-enriched Bifidobacterium longum can improve the damage of dopaminergic neurons in the brain of mice and improve the motor dysfunction of mice. The neuron protection effect is stronger than that of the current drug for the treatment of Parkinson's disease, Madopar, and the motor function improvement effect is similar to that of Madopar. From the perspective of peripheral symptoms, selenium-enriched Bifidobacterium longum can significantly improve constipation and colitis infiltration in mice, while Madopar aggravates colitis. The preventive and therapeutic effects of selenium-enriched Bifidobacterium longum are significantly better than those of Bifidobacterium longum. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the test examples of the present application, the drawings required for use in the test examples or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some test examples of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1The invention discloses an experimental scheme for verifying the efficacy of the selenium-enriched Bifidobacterium longum in an acute Parkinson's mouse model constructed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Selenium-enriched Bifidobacterium longum (SBM), selenium (SeM), Bifidobacterium longum (BLM) and positive drug Madopar (MDM) are administered by oral gavage starting from Day 0 for 28 days. MPTP for acute Parkinson's modeling is administered by intraperitoneal injection starting from Day 14 to Day 21. Behavioral experiments are performed on Day 0 and Day 28, respectively.

[0036] Figure 2 The selenium-enriched Bifidobacterium longum (SBM) of the present invention can protect the damage of dopaminergic neurons in the striatum and substantia nigra of the brain in the acute Parkinson's mouse model constructed by MPTP, and its effect is stronger than that of selenium (SeM) and Bifidobacterium longum (BLM) taken alone and the positive drug Madopar (MDM); Figure 2 A is the immunohistochemical staining of tyrosine hydroxylase (TH) in the striatum and substantia nigra of MPTP acute parkinsonian mice; Figure 2 B is the grayscale quantification of immunohistochemical staining of TH in the striatum of MPTP acute parkinsonian mice; Figure 2 C is the grayscale quantification of immunohistochemical staining of TH in the substantia nigra of MPTP-induced acute parkinsonian mice.

[0037] Figure 3 The selenium-enriched Bifidobacterium longum of the present invention can improve motor dysfunction in the acute Parkinson's mouse model constructed by MPTP, and its effect is stronger than that of selenium and Bifidobacterium longum taken alone, and is similar to the improvement effect of the positive drug Madopar; Figure 3 A is the mouse grasping strength test, which is used to test the grasping strength of the mouse's forelimbs; Figure 3 B is the mouse rotarod fatigue test, which is used to test the limb movement ability of mice; Figure 3 C, 3D, and 3E are open field experiments for mice, which are used to test the anxiety and motor ability of mice. Figure 3 C is the movement trajectory of mice in the open field experiment. Figure 3 D is the total distance traveled by mice in the open field experiment. Figure 3 E is the quantification of the movement distance of mice in the central area of ​​the open field experiment; Figure 3 F is a mouse climbing pole experiment, which is used to test the motor ability of the mouse's limbs.

[0038] Figure 4 The present invention discloses a drug efficacy verification experimental scheme of selenium-enriched Bifidobacterium longum in a chronic Parkinson's mouse model constructed by rotenone (ROT); selenium-enriched Bifidobacterium longum, selenium, Bifidobacterium longum and positive drug Madopar are administered by gavage starting from Day 0 for 42 days, and rotenone for chronic Parkinson's modeling is administered by gavage starting from Day 0 4 hours later for 42 days; behavioral experiments are performed on Day 0 and Day 43, respectively.

[0039] Figure 5 The selenium-enriched Bifidobacterium longum (SBR) of the present invention can protect the damage of dopaminergic neurons in the striatum and substantia nigra of the brain in the chronic Parkinson's mouse model constructed by ROT, and its effect is stronger than that of selenium (SeR) and Bifidobacterium longum (BLR) taken alone and the positive drug Madopar (MDR); Figure 5 A is the immunohistochemical staining of tyrosine hydroxylase (TH) in the striatum and substantia nigra of ROT chronic parkinsonian mice; Figure 5 B is the grayscale quantification of immunohistochemical staining of TH in the striatum of ROT chronic parkinsonian mice; Figure 5 C is the grayscale quantification of immunohistochemical staining of TH in the substantia nigra of ROT chronic parkinsonian mice.

[0040] Figure 6 The selenium-enriched Bifidobacterium longum of the present invention can improve motor dysfunction in the chronic Parkinson's mouse model constructed by ROT, and its effect is stronger than that of selenium and Bifidobacterium longum taken alone, and is similar to the improvement effect of the positive drug Madopar; Figure 6 A is the mouse grasping strength test, which is used to test the grasping strength of the mouse's forelimbs; Figure 6 B is the mouse rotarod fatigue test, which is used to test the limb movement ability of mice; Figure 6 C, 6D, and 6E are open field experiments for mice, which are used to test the anxiety and motor ability of mice. Figure 6 C is the movement trajectory of mice in the open field experiment. Figure 6 D is the total distance traveled by mice in the open field experiment. Figure 6 E is the quantification of the movement distance of mice in the central area of ​​the open field experiment; Figure 6 F is a mouse climbing pole experiment, which is used to test the motor ability of the mouse's limbs.

[0041] Figure 7 The results of the small intestine propulsion experiment of the selenium-enriched longum bifidobacterium of the present invention in the chronic Parkinson's mouse model constructed by ROT showed that the constipation of mice can be significantly improved, and its effect is stronger than that of the selenium group and the positive drug Madopar group used alone, and is similar to that of the longum bifidobacterium used alone. Figure 7 A is a photo of the extent of small intestine advancement. Figure 7 B is the small intestinal propulsion rate.

[0042] Figure 8 The results of HE staining showed that the selenium-enriched Bifidobacterium longum of the present invention can protect the inflammatory infiltration of colon tissue in the acute Parkinson's mouse model constructed by MPTP, and its effect is stronger than that of selenium and Bifidobacterium longum used alone, while the positive drug Madopar can aggravate the inflammatory infiltration of colon tissue.

[0043] Fig. 9The results of HE staining showed that the selenium-enriched Bifidobacterium longum of the present invention can protect the inflammatory infiltration of colon tissue in the chronic Parkinson's mouse model constructed by ROT, and its effect is stronger than that of selenium and Bifidobacterium longum used alone, while the positive drug Madopar can aggravate the inflammatory infiltration of colon tissue. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in the test examples of the present invention to clearly and completely describe the technical solutions in the test examples of the present invention. Obviously, the test examples described are only part of the test examples of the present invention, not all of the test examples. Based on the test examples in the present invention, all other test examples obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] 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.

[0046] 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.

[0047] It should be understood that in the various test examples 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 test examples of the present application.

[0048] The terms used in the test examples of this application are only for the purpose of describing specific test examples, and are not intended to limit this application. The singular forms "a", "said" and "the" used in the test examples of this application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0049] The weight of the relevant components mentioned in the test example description of this application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the test example description of this application, it is within the scope disclosed in the test example description of this application. Specifically, the mass described in the test example description of this application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0050] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the test examples of this application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0051] Unless otherwise specified, the reagents and materials used in the present invention are prepared by conventional methods or obtained from commercial channels.

[0052] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual points, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0053] The invention is further described in detail below with reference to the accompanying drawings.

[0054] In a first aspect, an embodiment of the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating Parkinson's disease.

[0055] In some embodiments, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent. Use of selenium-enriched Bifidobacterium longum (original strain name: Bifidobacterium longum; strain number: CICC 6188) and Bifidobacterium longum bacterial agent in the preparation of drugs for preventing and treating Parkinson's disease. The present invention first discovered and verified its efficacy in preventing and treating Parkinson's disease.

[0056] The acute Parkinson's disease model constructed by MPTP and the chronic Parkinson's disease model constructed by rotenone were used to more comprehensively verify its efficacy in preventing and treating Parkinson's disease.

[0057] The efficacy of Bifidobacterium longum and its selenium-enriched Bifidobacterium longum in preventing and treating Parkinson's disease was fully verified in two models: an acute Parkinson's disease model constructed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP and a chronic Parkinson's disease model constructed by rotenone. The efficacy of selenium-enriched Bifidobacterium longum was significantly better than that of Bifidobacterium longum.

[0058] The preparation method of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention comprises the following steps:

[0059] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0060] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0061] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0062] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0063] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0064] 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.

[0065] In step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0066] (1) Add stabilizer to a beaker;

[0067] (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;

[0068] (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;

[0069] (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.

[0070] In some embodiments, in step (1), the stabilizer is epigallocatechin gallate (EGCG).

[0071] In some embodiments, in step (1), the stabilizer is chitosan.

[0072] In some embodiments, in step (1), the stabilizer is gum arabic.

[0073] In some embodiments, in step (1), the stabilizer is bovine serum albumin (BSA).

[0074] In some embodiments, in step (1), the stabilizer is sodium hyaluronate.

[0075] In some embodiments, in step (2), the reducing agent is reduced glutathione GSH.

[0076] In some embodiments, in step (2), the reducing agent is vitamin C (Vc).

[0077] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.1 mM to 40 mM (for EGCG).

[0078] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.01% to 0.7% (for chitosan).

[0079] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.04% to 0.4% (for gum arabic).

[0080] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.4 mg / ml to 60 mg / ml (for BSA).

[0081] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.02% to 0.25% (for sodium hyaluronate).

[0082] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-50 mM (GSH).

[0083] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0084] In step (3), the final concentration of sodium selenite in the reaction system is 5 mM.

[0085] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 100KD to 1000KD (for chitosan).

[0086] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for gum arabic).

[0087] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for sodium hyaluronate).

[0088] In step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the Bifidobacterium longum bacterial preparation is in the form of a tablet.

[0093] In some embodiments, the Bifidobacterium longum bacterial agent is a lyophilized powder.

[0094] In some embodiments, the Bifidobacterium longum bacterial preparation is a capsule.

[0095] In some embodiments, the Bifidobacterium longum agent is a dairy product.

[0096] In some embodiments, the Bifidobacterium longum inoculum is stirred yogurt.

[0097] In some embodiments, the Bifidobacterium longum bacterial agent is in the form of set yogurt.

[0098] A second aspect of an embodiment of the present application provides a drug for preventing and treating Parkinson's disease, comprising Bifidobacterium longum or selenium-enriched Bifidobacterium longum as an active ingredient.

[0099] In some embodiments, the pharmaceutical dosage form is an oral solution.

[0100] In some embodiments, the pharmaceutical dosage form is a lyophilized powder.

[0101] In some embodiments, the pharmaceutical dosage form is a capsule.

[0102] In some embodiments, the pharmaceutical dosage form is made of a selenium-enriched Bifidobacterium longum bacterial agent and excipients acceptable to mammals.

[0103] A third aspect of the embodiments of the present application provides a pharmaceutical composition comprising Bifidobacterium longum or selenium-enriched Bifidobacterium longum, and a pharmaceutically acceptable carrier or diluent for preventing and treating Parkinson's disease.

[0104] Example 1

[0105] The first aspect of the embodiments of the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing and treating Parkinson's disease.

[0106] The bifidobacterium longum bacterial agent is a selenium-enriched bifidobacterium longum bacterial agent. The application of the selenium-enriched bifidobacterium longum (original bacterial species name: bifidobacterium longum; bacterial species number: CICC 6188) and the bifidobacterium longum bacterial agent in the preparation of drugs for preventing and treating Parkinson's disease. The present invention first discovered and verified the efficacy of the drug in preventing and treating Parkinson's disease.

[0107] The acute Parkinson's disease model constructed by MPTP and the chronic Parkinson's disease model constructed by rotenone were used to more comprehensively verify its efficacy in preventing and treating Parkinson's disease.

[0108] The efficacy of Bifidobacterium longum and its selenium-enriched Bifidobacterium longum in preventing and treating Parkinson's disease was fully verified in two models: an acute Parkinson's disease model constructed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP and a chronic Parkinson's disease model constructed by rotenone. The efficacy of selenium-enriched Bifidobacterium longum was significantly better than that of Bifidobacterium longum.

[0109] The preparation method of the selenium-enriched Bifidobacterium longum bacterial agent of the present invention comprises the following steps:

[0110] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0111] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0112] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0113] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0114] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0115] 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.

[0116] In step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0117] (1) Add stabilizer to a beaker;

[0118] (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;

[0119] (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;

[0120] (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.

[0121] 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.

[0122] Example 2

[0123] Fermentation of Selenium-Enriched Bifidobacterium longum

[0124] Prepare 3L of culture medium according to Table 1, adjust pH to 6.85, and load into a 5L fermenter. Sterilize at 121℃ for 20min, and cool to room temperature. Prepare according to the formula in Table 1.

[0125] Table 1 Main components of Bifidobacterium longum culture medium

[0126] Element g / L Vc (Sodium Ascorbate) 4 NaCl 2.4 <![CDATA[Na2HPO4]]> 5.8 <![CDATA[KH2PO4]]> 0.528571 glucose 10 Yeast Peptone FP103 (Angel Yeast) 60

[0127] Fermentation parameters of the 5-liter fermenter were as follows: fermentation temperature was 37°C, fermentation tank stirring speed was 30 rpm, and fermentation pH was set at pH 6.0.

[0128] Fermentation process: 60 mL of seed liquid (2% inoculation amount) was added, 20% NaOH solution was used to control the pH to 6.0, and the filtered and sterilized nano-selenium solution (final concentration 20-50 mg / L) was added from the inoculation port on the top of the tank to start fermentation. Nitrogen was passed for 10 minutes before and after inoculation and before and after sampling to remove the air in the tank. After 4 hours of fermentation, 30% glucose was added at a constant rate of 50 mL / h. The function of feed pump 3 was set to "feeding", "flow rate mode-constant speed" was selected, the flow rate was set to 0.9 ml / min, and the speed was set to 10%. After 10 hours of fermentation, the fermentation was stopped and obtained.

[0129] Example 3

[0130] The method for preparing the freeze-dried powder of selenium-enriched Bifidobacterium longum of the present invention comprises the following steps:

[0131] Centrifugal collection: After fermentation, centrifuge at 8000g, 4℃ for 10min to collect the bacteria.

[0132] Preparation of inlet solution: resuspend selenium-enriched Bifidobacterium longum sludge with 20mM PBS, 0.9% NaCl, 5% skimmed milk powder, pH 7.0 sterilization solution, with the ratio of 50ml solution to resuspend 1 gram of sludge.

[0133] Freeze drying: After the liquid enters the box, set the freeze drying program and start freeze drying. After the freeze drying is completed, collect the freeze dried powder, take a certain amount of freeze dried powder, dissolve it with sterile water to a certain concentration, count the live bacteria of selenium-enriched longum Bifidobacterium, and analyze the selenium content in the freeze dried powder by inductively coupled plasma mass spectrometry (ICP-MS).

[0134] Example 4

[0135] The invention discloses a selenium-enriched Bifidobacterium longum and a Bifidobacterium longum bacterial agent for use in preparing drugs for preventing and treating Parkinson's disease. The invention firstly discovers and verifies the efficacy of the drug for preventing and treating Parkinson's disease.

[0136] Bifidobacterium longum (No.: CICC 6188) can improve the motor dysfunction of acute Parkinson's mice induced by MPTP model. Bifidobacterium longum can improve the constipation of chronic Parkinson's mice induced by rotenone model. Selenium-enriched Bifidobacterium longum can improve the damage of dopaminergic neurons in the striatum and substantia nigra of acute Parkinson's mice induced by MPTP model and chronic Parkinson's mice induced by rotenone model. Selenium-enriched Bifidobacterium longum can improve the motor dysfunction of acute Parkinson's mice induced by MPTP model and chronic Parkinson's mice induced by rotenone model. Selenium-enriched Bifidobacterium longum can improve the constipation of chronic Parkinson's mice induced by rotenone model. Selenium-enriched Bifidobacterium longum can improve the colonic inflammatory infiltration of acute Parkinson's mice induced by MPTP model and chronic Parkinson's mice induced by rotenone model.

[0137] Test Example 1

[0138] 1. Construction, prevention and treatment of acute Parkinson's disease mouse model

[0139] like Figure 1, MPTP was used to construct an acute Parkinson's disease model in mice. 8-week-old C57 male mice were selected because Parkinson's disease is more common in males and MPTP is sensitive to 10-week-old C57 mice. The mice were adaptively raised for one week. Starting from Day 0, mice were gavaged with selenium-enriched Bifidobacterium longum for prevention and treatment. The experimental groups were as follows: (1) Wild group (WTM): 150 μL of normal saline was gavaged from Day 0 to Day 28; (2) Acute model group (MPTP): 150 μL of normal saline was gavaged from Day 0 to Day 28; from Day 14 to Day 21, first gavage with selenium-enriched Bifidobacterium longum, and 4 hours later, 30 mg / kg MPTP was intraperitoneally injected; (3) Selenium-enriched Bifidobacterium longum group (SBM): 150 μL of selenium-enriched Bifidobacterium longum (1.0-1.5×10 9 CFU / mouse), and then 4 hours later, intraperitoneal injection of 30 mg / kg MPTP was given from Day 14 to Day 21; (4) Selenium group (SeM): from Day 0 to Day 28, 150 μL of selenium was gavaged (the daily oral dose of nano-selenium was 1 mg Se / kg / BW (body weight) / day), and then from Day 14 to Day 21, nano-selenium was first taken orally, and then 4 hours later, 30 mg / kg MPTP was injected intraperitoneally; (5) Bifidobacterium longum group (BLM): from Day 0 to Day 28, 150 μL of Bifidobacterium longum (1.5×10 9 CFU / mouse), from Day 14 to Day 21, Bifidobacterium longum was first gavaged, and 30 mg / kg MPTP was injected intraperitoneally 4 hours later; (6) Positive drug Madopar group (MDM): from Day 0 to Day 28, 20 mg / kg Madopar was gavaged, from Day 14 to Day 21, Madopar was first gavaged, and 30 mg / kg MPTP was injected intraperitoneally 4 hours later.

[0140] MPTP is prepared with normal saline, and needs to be protected from light and personal protection. Before gavage, resuspend the strain with PBS. Selenium is prepared with normal saline. Madopar is prepared with normal saline.

[0141] 2. Construction and treatment of chronic Parkinson's mouse model

[0142] like Figure 4, rotenone (ROT) was used to construct a mouse chronic Parkinson's model. 8-week-old C57 male mice were selected because Parkinson's disease is more common in males and rotenone is sensitive to 8-week-old C57 mice. The mice were adaptively raised for one week. From Day 0, the mice were treated by gavage with selenium-enriched Bifidobacterium longum. The experimental groups were as follows: (1) Wild group (WTR): 150 μL 0.05% sodium carboxymethyl cellulose was gavaged from Day 0 to Day 42; (2) Chronic model group (ROT): 150 μL normal saline was gavaged from Day 0 to Day 42, and 30 mg / kg rotenone was gavaged 4 hours later; (3) Selenium-enriched Bifidobacterium longum group (SBR): 150 μL selenium-enriched Bifidobacterium longum (1.0-1.5×10 9 CFU / mouse), and then 30 mg / kg rotenone was gavaged 4 hours later; (4) Selenium group (SeR): 150 μL selenium (daily oral nano-selenium dose was 1 mg Se / kg / BW / day) was gavaged from Day 0 to Day 42, and then 30 mg / kg rotenone was gavaged 4 hours later; (5) Bifidobacterium longum group (BLR): 150 μL Bifidobacterium longum (1.0-1.5×10 9 (6) Positive drug Madopar group (MDR): from Day 0 to Day 42, 20 mg / kg of Madopar was given by gavage, and 4 hours later, 30 mg / kg of rotenone was given by gavage.

[0143] Rotenone is prepared with 0.05% sodium carboxymethylcellulose, and it is necessary to avoid light and take personal protection. Before gavage, resuspend the strain with PBS solution. Selenium is prepared with normal saline. Madopar is prepared with normal saline.

[0144] During the experiment, the living conditions and weight of the mice were recorded every day. At the end of the experiment, the mice were subjected to behavioral tests. Finally, the mice were killed by cervical dislocation, and their brain, small intestine and colorectal tissues were taken for subsequent experiments.

[0145] 3. Immunohistochemical staining of brain neurons in acute and chronic Parkinson's mice

[0146] Tyrosine hydroxylase (TH) is the enzyme responsible for catalyzing the conversion of L-tyrosine to DOPA, which is a precursor of dopamine. Therefore, TH is used as a marker of dopaminergic neurons. The mouse was killed by cervical dislocation and fixed on a foam board. The chest cavity was opened to expose the heart. The upper left corner of the heart was cut off, and the syringe needle was inserted from the left apex to the lower right corner. Pre-cooled PBS was added to the syringe and slowly perfused until the liver turned white and the outflowing fluid was basically colorless. Then 4% paraformaldehyde was perfused until the tail was lifted. The brain was removed on ice after decapitation and fixed in 4% paraformaldehyde for 6 hours. The whole mouse brain was cut coronally with a blade to separate the striatum and substantia nigra, and embedded in wax blocks respectively. Cut into 3-4mm slices with a microtome. Bake in a 65℃ oven for 60 minutes. Soak in three cylinders of dewaxing solution (xylene) for 10 minutes each to remove the paraffin embedded in the tissue. Soak in 100%, 100%, 95%, and 75% gradient alcohol for 2 minutes each. Soak in purified water for 2 minutes. Soak in 3% hydrogen peroxide for 5 minutes, and soak in PBS for 2 minutes. Add EDTA repair solution to the repair box, put in the slice, put the repair box in a 100℃ water bath for 20 minutes, cool naturally, and soak in PBS for 2 minutes. Draw 50μL of primary antibody on the slide to cover the tissue, and soak at room temperature for 60 minutes. Soak in PBS three times, 3 minutes each time. Add 50μL of secondary antibody to cover the tissue, and soak at room temperature for 20 minutes. Soak in PBS three times, 3 minutes each time, so that the secondary antibody and the primary antibody can fully bind. Soak in PBS to remove the unbound secondary antibody. Add 50μL DAB working solution to cover the tissue and stain for 5 minutes. Rinse with purified water to stop the color reaction. Add 50μL hematoxylin to cover the tissue, stain for 3 minutes, rinse with purified water, add 50uL acid wash solution for 1-2 seconds, and rinse with pure water. Rinse with running water for 2 minutes to return to blue, and dye the cell nucleus blue. 75%, 95%, 100% gradient alcohol, 2 minutes per tank, and soak in transparent liquid for 3 minutes. Take out the slide and let it dry, drop neutral resin on it, and cover it with a coverslip. The peroxidase in the secondary antibody oxidizes DAB to form a brown-yellow precipitate. Each slice is processed in parallel to ensure the degree of staining. Use the Olympus VS200 whole slide scanning system to scan the stained slices. Use Image J to perform grayscale quantitative analysis of the scanning results. Use Graphpad Prism 9 software for statistical analysis and drawing, and use one-way ANOVA for inter-group comparison. P<0.05 indicates that the results are statistically significant. The experimental results are shown in Figure 2 A and 5A, for quantitative analysis see Figure 2 B&C and 5B&C.

[0147] 4. Acute and chronic Parkinson's disease mouse behavioral experiments

[0148] 4.1 Grip test

[0149] Measure the weight of the mouse. Place the Yiyan YLS-13A grip strength meter horizontally, set up three repeated measurements, grab the mouse's tail, let the mouse's forelimbs naturally grab the grip bar, use the same force to gently pull the mouse's tail back until the instrument shows a reading. Relative grip strength = grip strength reading (N) / body weight (g). See the experimental results. Figure 3 A and 6A.

[0150] 4.2 Rotarod test

[0151] Set the rotarod to 4rpm and place the mouse on the rotating rod for 5 minutes of training. Conduct the formal experiment 24 hours after training. Set the parameters to an initial speed of 5rpm and an acceleration of 20rpm / min until the maximum speed reaches 40rpm and the time is 240s. Place the mouse on the rotarod, start the rotarod and start timing until the mouse falls off the rotarod, and record the time and speed of the fall. When the mouse falls, take it out and put it back in the cage to rest in time. Repeat the test three times, with at least 2 hours between each test. The experimental results are shown in Figure 3 B and 6B.

[0152] 4.3 Open field experiment

[0153] Place the mice in a dark room to acclimate for 2 hours. Turn on the video camera and photoelectric detection equipment. Clean the open field each time before placing the mice in to eliminate the effects of residual mouse odor on their movement and exploration abilities. Place each mouse in the open field at the same position and angle. Turn on the camera and start recording for 5 minutes. The tester should remain still and quiet. At the end of the test, remove the mice from the open field and place them in a new cage. Clean the open field with disinfectant and wait for the disinfectant to dry completely before placing the next mouse in. Use software to analyze the mouse's movement trajectory and distance. See the experimental results. Figure 3 C and 6C, quantitative results of total distance and central area distance Figure 3 D&E and 6D&E.

[0154] 4.4 Pole climbing experiment

[0155] A 3cm diameter, 50cm high wooden pole was placed vertically in a cage familiar to the mouse. The mouse was placed head up on the top of the pole and the time it took for the mouse to climb from the top to the bottom of the pole was recorded. The mouse was trained first and the formal experiment started 24 hours after training. After each experiment, the mouse was taken out and put back into the cage to rest. The experiment was repeated three times with at least 2 hours between each experiment. The experimental results are shown in Figure 3 F and 6F.

[0156] 5. Small Intestinal Propulsion Experiment in Chronic Parkinson's Mice

[0157] The small intestine has peristalsis function. By observing the running distance of ink in the intestine, we can understand the constipation of mice and the effect of drugs on constipation. The mice were fasted for 12 hours, and blue ink was mixed with normal saline. 150μL was absorbed and gavaged. The mice were killed after 20 minutes. The small intestine was removed by dissection, and the total length of the small intestine and the ink propagation length were taken and measured. Propulsion rate = ink propagation length (cm) / total length of small intestine (cm). The experimental results are shown in Figure 7 A, quantitative results are shown in Figure 7 B.

[0158] 6. HE staining of colorectum in acute and chronic Parkinson's mice

[0159] HE staining was used to observe the inflammatory infiltration of the mouse colon. The middle part of the mouse colon was dissected and fixed with paraformaldehyde and embedded in wax. It was cut into 3-4 mm slices using a microtome. Bake in a 65°C oven for 60 min. Soak in three cylinders of xylene for 10 min in each cylinder to remove the paraffin embedded in the tissue. Soak in 100%, 100%, 95%, and 75% gradient alcohol for 2 min in each cylinder. Dehydrate the tissue with gradient alcohol and immerse in xylene. Stain with a hematoxylin aqueous solution, color with acid water and ammonia water, rinse with running water for 1 hour, and place in distilled water. After that, dehydrate in 70% and 90% ethanol, stain with alcohol eosin staining solution for 2 minutes, and drip with gum. Take pictures under a microscope to observe the inflammation of the intestinal tissue. The experimental results are shown in Figure 8 and Fig. 9 .

[0160] Test Example 2

[0161] Evaluation of the efficacy of the selenium-enriched Bifidobacterium longum and Bifidobacterium longum in preventing and treating central nervous system symptoms in acute Parkinson's mice

[0162] In the acute Parkinson's mouse model constructed by MPTP, the present invention evaluated the pharmacodynamics of selenium-enriched Bifidobacterium longum and Bifidobacterium longum in preventing and improving Parkinson's central symptoms; in this experiment, drugs including selenium-enriched Bifidobacterium longum and Bifidobacterium longum were first gavaged for 14 days, and then gavaged and injected with MPTP on the same day to build a model for 7 days, in order to observe the effects of drug prevention and treatment, especially the preventive effect. First, brain pathology was used to verify that the selenium-enriched bacteria SBM group can improve the loss of dopaminergic neurons in the striatum and substantia nigra caused by MPTP, such as Figure 2As shown in A, the efficacy of selenium-enriched Bifidobacterium longum (SBM) is greater than that of selenium (SeM) and Bifidobacterium longum (BLM) used alone, as well as the positive drug Madopar (MDM). Madopar is currently one of the most widely used drugs in the clinical treatment of Parkinson's disease and Parkinson's syndrome. The experimental results show that the drugs used in the clinic to treat Parkinson's disease cannot fundamentally prevent the damage to motor neurons. Secondly, a series of behavioral experiments verified that the selenium-enriched Bifidobacterium longum (SBM) group can alleviate the motor dysfunction of acute Parkinson's mice, such as Figure 3 As shown in A, the results of the gripping test showed that MPTP can cause the forelimb gripping power of mice to weaken. Selenium-enriched Bifidobacterium longum can significantly alleviate the phenomenon of weakened gripping power of mice. Its efficacy is slightly stronger than that of the positive drug Madopar (MDM). Bifidobacterium longum (BLM) used alone did not significantly alleviate the phenomenon of weakened gripping power of mice. The rotarod test characterizes the limb strength and motor ability of mice by observing the time it takes for mice to fall from the rotarod. Figure 3 The results of the B rotating rod test showed that selenium-enriched Bifidobacterium longum could relieve limb fatigue in acute PD mice, significantly increase exercise time, and its efficacy was similar to that of the positive drug Madopar (MDM). Selenium (SeM) and Bifidobacterium longum (BLM) used alone failed to relieve exercise fatigue in acute PD mice. The open field test can reflect the depression of mice through the path of mice's activities in the central area, and reflect the exercise status of mice through the total distance of mice's activities. Figure 3 C is the movement trajectory of mice in each group. It can be found that selenium-enriched Bifidobacterium longum can significantly improve the motor ability and depression of acute PD mice. Figure 3 D&E quantitative analysis showed that selenium-enriched bacteria significantly increased the movement path of acute PD mice, and Bifidobacterium longum could also improve the movement ability of acute PD mice, but the efficacy of selenium-enriched Bifidobacterium longum was significantly stronger than that of Bifidobacterium longum (BLM) and positive drug Madopar (MDM). The above shows that both Bifidobacterium longum and selenium-enriched Bifidobacterium can improve the movement disorder of acute PD mice, but selenium-enriched Bifidobacterium longum can prevent and treat Parkinson's disease by protecting motor neurons more fundamentally and more effectively from the central nervous system.

[0163] Test Example 3

[0164] Evaluation of the efficacy of the selenium-enriched Bifidobacterium longum in treating central nervous system symptoms in chronic Parkinson's mice

[0165] In order to study the comprehensiveness of the treatment of Parkinson's disease with selenium-enriched Bifidobacterium longum, the present invention also evaluated the pharmacodynamics of the intervention and treatment of selenium-enriched Bifidobacterium longum in improving the central symptoms of Parkinson's disease in a chronic Parkinson's disease mouse model constructed by rotenone. Figure 5As shown in A, firstly, brain pathology verified that rotenone (ROT) can cause neuronal loss in the substantia nigra and striatum of normal mice, and selenium-enriched Bifidobacterium longum (SBR) can improve the loss of dopaminergic neurons in the striatum and substantia nigra caused by rotenone. However, selenium (SeR) and Bifidobacterium longum (BLR) used alone and the positive drug Madopar (MDR) cannot prevent the loss of dopaminergic neurons in the striatum and substantia nigra of mice. Secondly, behavioral experiments also verified that selenium-enriched Bifidobacterium longum (SBR) can alleviate the motor dysfunction of chronic Parkinson's mice, such as Figure 6 As shown in A, the results of the gripping strength experiment showed that ROT would lead to weakened gripping strength of mice's forelimbs, and selenium-enriched Bifidobacterium longum could significantly alleviate the weakened gripping strength of mice. However, Bifidobacterium longum (BLR) and selenium (SeR) and positive drugs used alone did not significantly alleviate the weakened gripping strength of chronic PD mice. Figure 6 The results of the B rotating rod test showed that selenium-enriched Bifidobacterium longum can relieve limb fatigue in chronic PD mice, significantly increase exercise time, and its efficacy is similar to that of the positive drug Madopar (MDR). Selenium (SeR) and Bifidobacterium longum (BLR) used alone failed to relieve exercise fatigue in chronic PD mice. Figure 6 C is the movement trajectory of mice in the open field experiment. It can be found that selenium-enriched Bifidobacterium longum can significantly improve the motor ability and depression of chronic PD mice. Figure 6 D&E quantitative analysis showed that selenium-enriched bacteria significantly increased the movement path of chronic PD mice, and the efficacy of selenium-enriched Bifidobacterium longum was stronger than the positive drug Madopar (MDR). Behavioral experiments showed that selenium-enriched Bifidobacterium longum can improve the motor dysfunction of chronic PD mice. Combined with Experimental Example 2, it can be shown that selenium-enriched Bifidobacterium longum can prevent and treat acute and chronic Parkinson's disease by protecting motor neurons more fundamentally from the central nervous system.

[0166] Test Example 4

[0167] Evaluation of the efficacy of the selenium-enriched Bifidobacterium longum in preventing and treating peripheral symptoms in acute Parkinson's mice

[0168] In addition to the central symptoms of Experimental Examples 2 and 3, the present invention also verifies the intestinal peripheral symptoms of selenium-enriched Bifidobacterium longum. Figure 8 As shown, through H&E staining, the present invention found that severe inflammatory infiltration of colon tissue occurred in the acute Parkinson's mice constructed by MPTP, and the intestinal tissue morphology was severely damaged. The selenium-enriched longum Bifidobacterium and longum Bifidobacterium of the present invention can alleviate the inflammatory infiltration of colon tissue. The effect of selenium-enriched longum Bifidobacterium is stronger than that of selenium and longum Bifidobacterium used alone, while the positive drug Madopar will aggravate the inflammatory infiltration ( Figure 8 ). This indicates that selenium-enriched Bifidobacterium longum can protect the intestinal damage of acute Parkinson's disease.

[0169] Test Example 5

[0170] Evaluation of the efficacy of the selenium-enriched Bifidobacterium longum in treating peripheral symptoms and treating chronic Parkinson's mice

[0171] Clinically, it is found that most Parkinson's patients are accompanied by constipation. In the chronic Parkinson's mice constructed by rotenone, Figure 7 As shown in the small intestinal propulsion experiment, the present invention also found that the rotenone group (ROT) mice had constipation symptoms, and the treatment with Bifidobacterium longum (BLR) and Selenium-enriched Bifidobacterium longum (SBR) could relieve the constipation of Parkinson's mice, while the mice in the positive drug group (MDR) had severe constipation like the rotenone group. Fig. 9 As shown, H&E staining also found that severe inflammatory infiltration of colon tissue occurred in rotenone-induced chronic Parkinson's mice (ROT). The Bifidobacterium longum (BLR) and selenium-enriched Bifidobacterium longum (SBR) of the present invention can alleviate the inflammatory infiltration of colon tissue. The effect of selenium-enriched Bifidobacterium longum is stronger than that of selenium (SeR) and Bifidobacterium longum (BLR) used alone. However, the positive drug Madopar (MDR) also aggravates the inflammatory infiltration ( Fig. 9 ). Combined with Experimental Example 4, it is shown that both Bifidobacterium longum and Selenium-enriched Bifidobacterium longum can protect the intestinal damage of Parkinson's mice, but Selenium-enriched Bifidobacterium longum improves acute and chronic Parkinson's symptoms more comprehensively and effectively from the peripheral protection of intestinal homeostasis.

[0172] The selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention can be used to prepare possible drugs for preventing and treating acute and chronic Parkinson's disease, but the selenium-enriched Bifidobacterium longum can more comprehensively improve the symptoms of the Parkinson's disease mouse model from both central and peripheral aspects.

[0173] Test Example 6

[0174] Acute oral toxicity test of the selenium-enriched Bifidobacterium longum of the present invention

[0175] 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.

[0176] 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.

[0177] Main instruments: JJ10 electronic balance, T1000 electronic balance.

[0178] Test method: The acute toxicity test in rats was carried out using the limit method.

[0179] Experimental results: 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 anatomical examination of the animals. The effects of nano-selenium-enriched Bifidobacterium longum on the body weight and mortality of rats are shown in Table 1.

[0180] Table 1 Effects of nano-selenium Bifidobacterium longum on acute oral toxicity in rats

[0181]

[0182] Experimental conclusion: No obvious poisoning symptoms were observed after oral administration of nano-selenium-enriched Bifidobacterium longum to animals, no significant effect on animal weight gain, and no animal died during the observation period. Acute oral toxicity test of nano-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.

[0183] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above test examples, and the above test examples 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 Parkinson's disease.

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

3. The use according to claim 2, 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.

4. The use according to claim 3, 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.

5. The use according to claim 4, characterized in that: 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, wherein 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.

6. A drug for preventing and treating Parkinson's disease, characterized in that The method comprises the Bifidobacterium longum bacterial agent according to claim 1 or the selenium-enriched Bifidobacterium longum bacterial agent according to claim 3 as an active ingredient.

7. A pharmaceutical composition, characterized in that The method comprises Bifidobacterium longum or the selenium-enriched Bifidobacterium longum according to claim 3, and a pharmaceutically acceptable carrier or diluent for preventing and treating Parkinson's disease.

Images