CN115887504A

CN115887504A - Application of bifidobacterium longum CCFM1029 in relieving Parkinson's disease

Info

Publication number: CN115887504A
Application number: CN202211129707.3A
Authority: CN
Inventors: 田丰伟; 李田甜; 于雷雷; 翟齐啸; 赵建新; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2023-04-04
Anticipated expiration: 2042-09-16
Also published as: CN115887504B

Abstract

The invention discloses an application of bifidobacterium longum CCFM1029 in relieving Parkinson's disease, and belongs to the technical field of microorganisms and medicines. The bifidobacterium longum CCFM1029 provided by the invention has the effect of relieving the Parkinson's disease, and is specifically reflected in that: (1) Remarkably relieving MPTP-induced mouse motor dysfunction, including improving agility, limb movement coordination and balance ability of mice; (2) The method has the advantages that the level of monoamine and metabolite thereof in striatum of the mice induced by MPTP is obviously improved and increased, the neuroprotective effect is achieved, and the striatum of the mice is protected from DA neuron loss induced by MPTP; (3) The brain antioxidant level of MPTP-induced mice is obviously increased, and the oxidative stress induced by MPTP is relieved; (4) The content of short-chain fatty acid in intestinal tracts of MPTP-induced mice is remarkably improved; and (5) effectively improving the intestinal flora structure of the MPTP-induced mice. Therefore, the bifidobacterium longum CCFM1029 has a great application prospect in preparing products for preventing and/or relieving Parkinson's disease.

Description

Application of bifidobacterium longum CCFM1029 in relieving Parkinson's disease

Technical Field

The invention relates to an application of bifidobacterium longum CCFM1029 in relieving Parkinson's disease, belonging to the technical field of microorganisms and medicines.

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease, occurring at about 1% in people over the age of 65, and is characterized by motor dysfunction due to loss of midbrain dopaminergic neurons, including stiffness, resting tremor, gait disturbance and bradykinesia. The pathogenic mechanisms of PD may be associated with genetic factors, oxidative stress, neuroinflammation and metabolic disorders, which lead to misfolding and aggregation of alpha synuclein. PD patients suffer from motor and non-motor symptoms, usually showing symptoms of gastrointestinal dysfunction and constipation several years before the onset of motor symptoms, and more than 80% of PD patients suffer from various non-motor Gastrointestinal (GI) symptoms such as constipation, nausea and vomiting, which are closely related to alpha-synuclein-related neurodegeneration in the Enteric Nervous System (ENS). In recent years, the gut-brain axis of interaction or interference between the gastrointestinal tract, ENS, the Central Nervous System (CNS) and the intestinal microbiota (GM) has become one of the research hotspots for the pathogenesis of PD. Many clinical studies have found altered gut microbiome composition in PD patients, suggesting that gut dysbiosis plays a key role in exacerbating the pathogenesis of PD. Currently, there is no treatment for curing or improving parkinson's disease. Levodopa is a gold standard drug, but has significant side effects with long-term administration. Therefore, there is an urgent need to find ideal therapeutic drugs and new therapies.

Probiotics are living microorganisms that confer a health benefit to the host, and may improve the physical and mental health of the host by affecting the microbial-gut-brain axis homeostasis. A particular probiotic strain capable of modulating neurotransmitters, neurotrophic factors and behaviour is called "probiotic". A PD patient consuming a complex probiotic product (containing Lactobacillus acidophilus, bifidobacterium bifidum, lactobacillus reuteri and Lactobacillus fermentum) for 12 weeks can improve PD motor symptoms, inhibit oxidative stress, regulate insulin and lipid metabolism (disclosed in Tamtaji OR, taghizadeh M et al, clinical and metabolic stress to biological administration in peptide with Parkinson's disease: A randomised, double-indebl, heart-controlled trial. In Nutr.2019 Jun;38 (3): 1031-1035. Paper); it was found that daily administration of probiotic formulation SLAB51 (containing Streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus) can rescue 6-hydroxydopamine induced dopaminergic neuronal death and improve motor dysfunction in the substantia nigra and striatum of PD model mice (published in Castelli V, d 'Angelo M et al. Effects of the biological formation SLAB51 in vitro and in vivo parkin's disease models aging (Albany NY). 2020 Mar9 (5): 4641-4659.; long-term administration of probiotic cocktail preparations (Bifidobacterium bifidum, bifidobacterium longum, lactobacillus rhamnosus GG, lactobacillus plantarum LP28, and lactococcus lactis subspecies) has a neuroprotective effect on dopaminergic neurons and further alleviates the worsening of Motor dysfunction in MitoParkPD mice (published in Hsieh TH, kuo CW et al. Probiotics Alleviate the Progressive resolution of Motor Functions in a Mouse Model of park's disease. Brain Sci.2020 Apr 1 (4): 206. Thesis). These reports are supportive evidence of the positive effects of ingestion of probiotics by PD patients; however, the mechanisms of probiotic mixtures are complex, including antioxidant, immune response, gut microbial regulation, etc., and secondly, probiotic strain specificity and host conditions, etc., may influence the final clinical outcome. Therefore, it is necessary to perform single strain development validation to confirm the health promoting effect of a specific probiotic in each case.

The bifidobacterium is an important beneficial microorganism in intestinal tracts, and has various important physiological functions of biological barrier, nutrition, anti-tumor effect, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like on human health. In recent years, the efficacy and prospect of the bifidobacterium in neurodegenerative diseases have been paid more attention and explored by more people. Bifidobacterium breve CCFM1025 is a promising candidate strain for psychotic relief from depression and related gastrointestinal disorders (published in Tian P, chen Y, zhu H et al. Bifidobacterium breve CCFM1025 ingredients major depressing disorder and tryptophan metabolism: A random approved clinical trial. Brain Behavv Immun.2022 Feb;100 233-241. Paper); bifidobacterium breve strain A1 has therapeutic potential for the prevention of cognitive impairment in Alzheimer's disease (published in Kobayashi Y, sugahara H et al. Therapeutic potential of Bifidobacterium breve strain A1 for predicting cognitive impairement in Alzheimer's disease Sci Rep.2017 (1): 13510. Article); oral Administration of Probiotic Bifidobacterium breve strain A1[ MCC1274] is capable of restoring Memory decline and cognitive dysfunction in a Mouse Model of Parkinson's disease Induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (published in Ishii T, furuka H et al. Organic Addition of biological Bipolar bacterium expression of Abstract high probability fusion of absorption high probability expression of neuropin MPTP-Induced Motor Model of Parkinson's disease. Biomedicines.2021 Feb 8; 167. Article). However, no report has been made on the neuroprotective effect of a single strain of Bifidobacterium longum on Parkinson's disease and the alleviation of motor dysfunction thereof.

Disclosure of Invention

In order to provide a Bifidobacterium longum (Bifidobacterium longum) which can prevent and/or alleviate parkinson's disease and is safe without side effects, the invention provides a Bifidobacterium longum (Bifidobacterium longum) CCFM1029, wherein the Bifidobacterium longum CCFM1029 is deposited in the Guangdong province collection of microbial cultures in 2018, 10 and 11 days, and the deposit number is GDMCC No:60461, deposited at No. 59, no. 5, of the institute of Pimpom Tourette, 100, guangzhou, and published in the patent No. CN 109652349A.

The invention provides application of the Bifidobacterium longum (Bifidobacterium longum) CCFM1029 in preparing products for preventing and/or relieving Parkinson's disease, wherein the application is not aimed at treating the disease.

In one embodiment of the invention, the product comprises at least one of the following functions:

(1) Remarkably relieving MPTP-induced mouse motor dysfunction, including improving agility, limb movement coordination and balance ability of mice;

(2) The method has the advantages that the level of monoamine and metabolite thereof in striatum of the mice induced by MPTP is obviously improved and increased, the neuroprotective effect is achieved, and the striatum of the mice is protected from DA neuron loss induced by MPTP;

(3) The brain antioxidant level of MPTP-induced mice is obviously increased, and the oxidative stress induced by MPTP is relieved;

(4) The content of short-chain fatty acid in intestinal tracts of MPTP-induced mice is remarkably improved;

(5) Effectively improve the intestinal flora structure of MPTP induced mice.

In one embodiment of the invention, the product is a pharmaceutical product.

In one embodiment of the invention, the viable count of bifidobacterium longum in the product is not less than 5 x 10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

In one embodiment of the present invention, the pharmaceutical product comprises Bifidobacterium longum (Bifidobacterium longum) CCFM1029, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.

In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and/or an additive.

In one embodiment of the invention, the excipient comprises a binder, a filler, a disintegrant and/or a lubricant.

In one embodiment of the invention, the additive comprises a solubilizer, a cosolvent and/or a preservative.

In one embodiment of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

It is a second object of the present invention to provide a microbial preparation containing said Bifidobacterium longum (Bifidobacterium longum) CCFM1029.

In one embodiment of the present invention, the microbial preparation is prepared by inoculating the Bifidobacterium longum (Bifidobacterium longum) CCFM1029 into a culture medium in an inoculum size of 2-4% (v/v) of the total mass of the culture medium, and culturing at 37 ℃ for 18 hours to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the thalli with normal saline for 3 times, and then resuspending with a freeze-drying protective agent to obtain a resuspension solution; and (4) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the leavening agent.

In one embodiment of the present invention, the mass ratio of the lyoprotectant to the microbial cells is 2.

In one embodiment of the invention, the lyoprotectant comprises 130g/L of skimmed milk powder.

In one embodiment of the invention, the culture medium comprises 87.7% of water, 10% of skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract.

In one embodiment of the invention, the pH of the medium is 6.8.

Has the advantages that:

1. the invention screens Bifidobacterium longum (Bifidobacterium longum) CCFM1029, the Bifidobacterium longum CCFM1029 has the function of relieving the Parkinson's disease, and the specific expression is as follows:

(3) The brain antioxidant level of the MPTP-induced mice is obviously increased, and the oxidative stress induced by the MPTP is relieved;

(5) Effectively improve the intestinal flora structure of MPTP induced mice.

2. Bifidobacterium longum (Bifidobacterium longum) is one of probiotics, and the product of Bifidobacterium longum CCFM1029 of the invention does not cause side effects to patients after long-term use, and has high safety.

Biological material preservation

Bifidobacterium longum (Bifidobacterium longum) CCFM1029, which is taxonomically named Bifidobacterium longum and is deposited in Guangdong province collection of microorganisms in 11 th 10 th 2018 with the deposit number GDMCC No:60461, the preservation address is No. 59 building No. 5 building of Michelia Tokyo 100, guangzhou province scientific microbiological research institute.

Drawings

FIG. 1: histograms of behavioral testing of different groups of mice, in which:

a is the climbing test result of the mice of different groups;

b is the balance beam test result of different groups of mice;

c is the test result of the rotating rod of the mice of different groups

FIG. 2: bars of striatal monoamine and its metabolite content for different groups of mice, where:

a is the striatum DOPA content of different groups of mice;

b is the striatum DA content of mice of different groups;

c is the striatum DOPAC content of different groups of mice;

d is the striatum HVA content of mice of different groups;

e is the striatum 5-HA content of mice of different groups;

f is the striatum 5-HIAA content of mice of different groups

FIG. 3: brain antioxidant content in different groups of mice, wherein:

a is the content of CAT in brain tissue of mice of different groups;

b is the content of SOD in the brain tissue of mice of different groups;

c is the content of GSH in brain tissue of mice of different groups;

FIG. 4: short-chain fatty acid content in feces of mice of different groups, wherein:

a is the content of acetic acid in the excrement of mice of different groups;

b is the content of propionic acid in the excrement of mice of different groups;

c is the content of butyric acid in the excrement of mice of different groups;

d is the content of valeric acid in the excrement of mice of different groups

E is the total short-chain fatty acid content in the excrement of mice of different groups;

FIG. 5 is a schematic view of: diversity analysis of intestinal flora of mice of different groups

A is the alpha diversity-Shannon index of intestinal flora of different groups of mice;

b is the alpha diversity-Simpson index of intestinal flora of different groups of mice;

FIG. 6: different groups of mice intestinal flora difference belong to relative abundance maps, wherein:

a is the relative abundance of the intestinal flora of the mice of different groups;

b is the relative abundance of the intestinal flora Lactobacillus of the mice of different groups;

c is the relative abundance of intestinal flora Bacteroides of mice of different groups;

d is the relative abundance of Shigella intestinal flora of different groups of mice;

Detailed Description

Chemicals such as tryptone and yeast powder, which are described in the following examples, were purchased from the national institute of pharmacy; the mice referred to in the following examples were male SPF (Specific pathogen free) grade C57BL/6J mice 6-8 weeks old, purchased from Jiangsu Jiejicaokang Biotech, inc.; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine, MPTP), levodopa (L-DOPA), benserazide, referred to in the following examples, were purchased from MCE; dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT) referred to in the examples below were purchased from Sigma-Aldrich; the Superoxide Dismutase (SOD), catalase (CATalase), and reduced Glutathione (Glutathione, GSH) kits involved in the following examples were purchased from Nanjing Biotech research institute, and the paraformaldehyde involved in the following examples was purchased from Wuhanseville Biotech, inc.; the Fast DNA Spin Kit for Feces Kit referred to in the following examples was purchased from MP Biomedicals.

The media involved in the following examples are as follows:

MRS solid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder, 2g/L of diammonium hydrogen citrate and K ₂ PO ₄ ·3H ₂ O 2.6g/L、MgSO ₄ ·7 H ₂ O 0.1g/L、MnSO ₄ 0.05g/L, tween 80 mL/L, agar 20g/L, cysteine hydrochloride 0.5g/L.

MRS liquid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder, 2g/L of diammonium hydrogen citrate and K ₂ PO ₄ ·3H ₂ O 2.6g/L、MgSO ₄ ·7 H ₂ O 0.1g/L、MnSO ₄ 0.05g/L, tween 80 mL/L and cysteine hydrochloride 0.5g/L.

The preparation of the bifidobacterium longum suspensions referred to in the following examples is as follows:

marking Bifidobacterium longum CCFM1029 on MRS solid culture medium (containing 0.05% cysteine), and culturing at 37 deg.C for 48 hr to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium (containing 0.05 percent of cysteine), culturing for 24 hours at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated solution into an MRS liquid culture medium (containing 0.05% of cysteine) according to the inoculation amount of 2% (v/v), and culturing at 37 ℃ for 24 hours to obtain a bacterial solution; centrifuging the bacterial liquid at 8000g for 10min to obtain Bifidobacterium longum thallus; washing Bifidobacterium longum with physiological saline, and suspending in 200g/L glycerol solution to obtain a solution with a bacterial concentration of 1 × 10 ¹⁰ CFU/mL to obtain bacterial suspension, and storing the bacterial suspension at-80 ℃ for later use.

Example 1: bifidobacterium longum CCFM1029 for relieving MPTP-induced mouse motor dysfunction

The method comprises the following specific steps:

1. animal experiment grouping: 50 healthy female C57BL/6 mice of 6-8 weeks of age with a body weight of 18-20 ug were randomly divided into 5 groups of 10 mice each, 5 groups were: blank Control group (Control), modeling group (MPTP), positive Control group (L-DOPA), bifidobacterium longum CCFM1029 dry Control group, and Bifidobacterium longum Y1 dry Control group.

1. Blank control group: in adaptation period of 1-7 days, drinking water and diet freely; the normal saline is 200 mu L of gastric lavage daily for 8-41 days, and the normal saline with the same volume as MPTP in the abdominal cavity injection and molding set is injected into the abdominal cavity in 33-37 days, wherein the volume is 0.1mL/20g of body weight.

2. Building a module: in the adaptation period of 1-7 days, water and diet are freely drunk; the preparation is administered by intraabdominal injection with normal saline 200 μ L for 8-41 days, and intraperitoneal injection with 30mg/kg MPTP (dissolved in sterilized normal saline) for 33-37 days, wherein the volume is 0.1ml/20g body weight.

3. Positive control group: in the adaptation period of 1-7 days, water and diet are freely drunk; the normal saline is 200 mu L per day for 8-33 days, MPTP (dissolved in sterilized normal saline) is injected into the abdominal cavity for 30mg/kg for 33-37 days, and 100mg/kg L-DOPA +25mg/kg Bensenazide is injected into the stomach for 33-41 days, wherein the stomach volume is 0.1mL/20g body weight per time.

4. Bifidobacterium longum CCFM1029 intervention group: in adaptation period of 1-7 days, drinking water and diet freely; gavage for 8-41 days with a dose of 1 × 10 of 200 μ L per day ⁹ CFU/mL Bifidobacterium longum CCFM1029, 30mg/kg MPTP (dissolved in sterilized normal saline) is intraperitoneally injected for 33-37 days, and the injection volume is 0.1mL/20g body weight.

5. Bifidobacterium longum Y1 intervention group: in the adaptation period of 1-7 days, water and diet are freely drunk; gavage for 8-41 days with a dose of 1 × 10 of 200 μ L per day ⁹ CFU/mL Bifidobacterium longum Y1, 30mg/kg MPTP (dissolved in sterilized normal saline) is intraperitoneally injected for 33-37 days, and the injection volume is 0.1mL/20g body weight.

2. And (3) behavioral testing: evaluating the agility and the sensory dyskinesia of the mice by using a climbing rod experiment; evaluating the movement coordination and balance of the mice by a balance beam experiment; the coordination of limb movements of the mice was tested in a rotarod experiment. All mice received behavioral training once a day starting on days 28-30. Behavioral testing was performed 24 hours after the last MPTP injection on day 37, with the behavioral testing method specified below:

1. pole climbing experiment

In the experiment, a mouse is placed on a wooden rough small ball, the lower end of the ball is connected with a round wooden stick with the length of 50cm, the section diameter of 1cm and the rough surface, and the lower end of the wooden stick is placed in a mouse cage. On each training day, the mice were first placed in a rod cage in a familiar environment, then placed head-up on a wooden stick 15cm from the bottom of the cage 3 times, followed by training at 30cm and 50cm (rod top). On the test day, each mouse was placed head up on the top of the pole on a wooden ball, and when climbing from the wooden ball to the wooden stick head down, the time at that time was recorded as a with a stopwatch, and when climbing to the lowest end of the wooden stick, the time at that time was recorded as B, and the time taken for the mouse to climb the entire wooden stick was recorded as C, C = a-B, and if the mouse could not turn down or continuously fall off the pole, it was recorded as 60s. Each mouse was tested 3 times (1 min interval between each trial) and the average time to climb the rod 3 times was taken as a statistical indicator.

2. Balance beam test

The experimental device is a wood stick with the length of 50cm, the width of 0.8cm and the height of 0.8cm, the wood stick is erected on two columns which are 50cm away from the height of an experimental table board, one end of the wood stick is a starting point, and a black box is placed at the other end of the wood stick to serve as a terminal point. On each training day, mice were placed in a black box for 5min to familiarize with the environment, then placed 5cm from the black box and trained to go to the endpoint 3 times, followed by 15cm, 30cm and 50cm balance beam training. On the day of testing, the time required for the mice to cross the balance from the beginning end of the stick and climb into the end black box was recorded. If the mouse falls half way or turns back to the starting point, the test is repeated. If the mouse failed to reach the end point within 60s or fell off the stick, it was recorded as 60s. Each mouse was tested 3 times (1 min interval between tests), and the average time of 3 out-of-balance trees was used as a statistical index.

3. Rod rotation test

This experiment was tested by a mouse rotating rod fatigue tester. The fatigue rod rotating instrument consists of a plastic rod with the diameter of 6cm and the length of 36cm, an accelerating rod with a rough surface is arranged 20cm above the bottom (diving platform), and the accelerating rod is divided into 5 equal parts by 5 discs (with the diameter of 25 cm), so that 5 mice can walk on the rod simultaneously. An accelerating rotor mode (2-20 r/min, 10-level speed) is adopted. The mice were placed on an accelerating bar at a speed of 2r/min and gradually increased to 20r/min. The time from when the mouse was placed on the post to when it fell off the post was recorded as the fall time. Mice were trained 3 times at a 10rpm spin rate on day 28 for 180s each, then at a 20rpm spin rate on day 29 and a 30rpm spin rate on day 30. On the day of testing, each mouse was tested at a 30rpm rotation speed, 3 times per mouse (1 min interval between tests), and the mean residence time in the rotor was taken as a statistical indicator for 3 times.

3. Results of the experiment

As can be seen from fig. 1A-C, the total descent time in the pole climbing test and the total walking time in the balance beam test of the MPTP model mice were significantly increased and the total residence time in the rotarod test was significantly shortened (all p < 0.0001) compared to the blank control group. Compared with the model building group, the positive control group and the bifidobacterium longum CCFM1029 intervention group both significantly reduce MPTP-induced PD mouse movement dysfunction on climbing poles (p <0.0001, p-knot-0.0001), balance trees (p <0.0001, p-knot-0.01) and rotating rod tests (p <0.001, p-knot-0.01), and the effect of the bifidobacterium longum CCFM1029 intervention group is close to that of the positive control group; bifidobacterium longum Y1 failed to alleviate the motor performance of PD mice in the behavioural test, and the bifidobacterium longum Y1 intervention group had a tendency to aggravate the motor dysfunction of PD mice compared to the model group of mice in the climbing pole test and the balance beam test.

The experimental results show that bifidobacterium longum CCFM1029 has the effect of relieving MPTP-induced PD mouse motor dysfunction.

Example 2: bifidobacterium longum CCFM1029 increases MPTP-induced levels of mouse monoamines and metabolites thereof

The method comprises the following specific steps:

1. animal experiment grouping: the same as in example 1.

2. Detection of monoamines and their metabolites: detecting by high performance liquid chromatography. More than 10mg of the striatum specimen of each group of mice was weighed, 100. Mu.L of 0.1M or 0.2M perchloric acid solution was added to each 10mg of sample, and after the sample was crushed with a 1mL syringe tip, it was further crushed to a tissue homogenate state using an ultrasonic crusher. The homogenized sample was centrifuged at 15000rpm 15min at 4 ℃ and after centrifugation, the supernatant was filtered through a 0.22 μm aqueous filter to the vial liner.

The HPLC adopts a Waters Atlantis T3 chromatographic column, the mobile phase is acetonitrile-water-PBS, the flow rate is 1.0mL/min, the column temperature is 30 ℃, and the sample injection amount is 20 muL. The conditions for fluorescence detection are an excitation wavelength of 280nm and an absorption wavelength of 320nm.

3. Results of the experiment

As can be seen from FIGS. 2A-F, the levels of DOPA, DA, 5-HT, DOPAC, 5-HIAA, and HVA in the striatum of the mice in the model group were significantly reduced compared to the placebo group, all statistically significant (all p < 0.01). Compared with the modeling group, the L-DOPA treatment group obviously improves the levels of striatum DOPA, DA, 5-HT, DOPAC, 5-HIAA and HVA, and has statistical significance (all p is less than 0.01); compared with the modeling group, the bifidobacterium longum CCFM1029 intervention group remarkably improves the levels of DOPA (p < 0.01), DA (p < 0.001), DOPAC (p < 0.05), HVA (p < 0.001), 5-HT (p < 0.0001) and 5-HIAA (p < 0.01), and respectively improves the levels by 2.47 times, 2.04 times, 1.70 times, 3.10 times, 2.87 times and 2.15 times, and has statistical difference. These results indicate that ingestion of bifidobacterium longum CCFM1029 significantly restores MPTP-induced reductions in DA, 5-HT and their metabolites; intervention with bifidobacterium longum Y1 did not have the effect of alleviating MPTP-induced striatal neurotransmitter decline in mice.

Therefore, the experimental results show that intervention of bifidobacterium longum CCFM1029 has a neuroprotective effect on MPTP-induced mice, and the level of MPTP-induced mouse striatal monoamine and metabolite thereof is obviously improved.

Example 3: bifidobacterium longum CCFM1029 increases brain antioxidant level in MPTP-induced mice and reduces MPTP-induced oxidative stress

The method comprises the following specific steps:

1. animal experiment grouping: the same as in example 1.

2. Detection of brain tissue antioxidants

The levels of Superoxide Dismutase (SOD), catalase (CAT), and reduced Glutathione (GSH) were measured using a test kit. The mouse brain tissue is subjected to ultrasonic treatment and cracking in physiological saline. After centrifugation, the supernatant was analyzed using the test kit according to the manufacturer's instructions. The level of antioxidant in brain tissue of all mice was estimated from standard activities of the antioxidant and then normalized to protein concentration and expressed as enzyme units per mg protein.

3. Results of the experiment

As can be seen from fig. 3A-C, brain CAT, GSH levels were significantly reduced in the model-made mice compared to the blank control group (all p < 0.01); the positive control group and the bifidobacterium longum CCFM1029 intervention group remarkably improve the reduction of the MPTP induction on the brain CAT and GSH content in mice, and the difference is 2.33 times and 1.99 times, 2.50 times and 2.07 times higher than that of the model-making group respectively, and has statistical significance (all p is less than 0.05). However, there was no significant difference in brain SOD levels in MPTP-induced mice in the positive control group and bifidobacterium longum CCFM1029 (fig. 3B), and intervention of bifidobacterium longum Y1 did not have the effect of alleviating brain antioxidant level reduction in MPTP-induced mice.

These results indicate that CCFM1029 enhances the antioxidant level and antioxidant capacity of MPTP-induced brain in mice, and that this antioxidant capacity is likely mediated by CAT and GSH.

Example 4: effect of Bifidobacterium longum CCFM1029 on MPTP-induced mouse short-chain fatty acid content

The method comprises the following specific steps:

1. animal experiment grouping: the same as in example 1.

2. Determination of short-chain fatty acid content in feces

Placing the collected mouse feces in liquid nitrogen, transferring to a box at-80 ℃, taking out before detecting the content of short-chain fatty acid, carrying out vacuum freeze drying, accurately weighing 0.05g of lyophilized feces sample, dissolving in 0.5mL of saturated sodium chloride solution, soaking for 30min, homogenizing by a tissue homogenizer, adding 0.02mL of sulfuric acid with the concentration of 10%, oscillating for 30s, and accurately adding 0.8mL of ethyl acetate into the feces solution in a ventilation cabinetThe ether solution was shaken for 30 seconds and then centrifuged for 15min (8000g, 4 ℃), and the supernatant was transferred to a centrifugal tube containing 0.3g of anhydrous sodium sulfate，Shaking, centrifuging for 15min (8000g, 4 deg.C), collecting supernatant, transferring into gas volumetric flask, and detecting short chain fatty acid content by GC-MS, with the detection result shown in figure.

3. Results of the experiment

As can be seen from fig. 4A-D, the levels of acetic acid (p < 0.001), propionic acid (p < 0.001), butyric acid (p < 0.001), and valeric acid (p < 0.0001) in the colon of the mice in the molding group showed significant decrease compared to the blank control group, and all were statistically significant, which resulted in a significant decrease in the total SCFAs content of the molding group (p < 0.0001) (as shown in fig. 4E). Compared with the modeling group, the positive control group obviously improves the acetic acid (p < 0.001) and propionic acid contents (p < 0.01) in the MPTP-induced mouse excrement, and has no statistical difference compared with the modeling group although the contents of butyric acid and valeric acid are increased; acetic acid, propionic acid, butyric acid and valeric acid levels in the mouse feces of the bifidobacterium longum CCFM1029 dry group are remarkably increased (all p is less than 0.001), the levels are respectively increased by 2.52 times, 2.07 times, 2.76 times and 2.44 times compared with the manufacturing module, the total concentration of SCFAs in the feces of the mice of the CCFM1029 dry group is also remarkably increased (p is less than 0.0001), and the acetic acid level in the feces of the mice is only remarkably increased by the intervention of bifidobacterium longum Y1 (p is less than 0.01).

Example 5: effect of Bifidobacterium longum CCFM1029 on MPTP-induced intestinal flora diversity of mice

The method comprises the following specific steps:

1. animal experiment grouping: the same as in example 1.

2. After a Fast DNA Spin Kit for Feces Kit is adopted to extract the metagenome of fecal bacteria of each group of mice, the sequence of the 1qs V3-V4 region is subjected to PCR amplification, and the diversity of intestinal flora in a fecal sample is obtained by a second-generation sequencer. The results are shown in FIG. 5.

3. Results of the experiment

As shown in fig. 5A-B, shannon and Simpson indices of the molded groups were significantly reduced (all P < 0.001) compared to the blank control group; compared with the modeling group, the positive control group and the bifidobacterium longum Y1 intervention group improve the Shannon index and the Simpson index, but have no statistical difference compared with the modeling group; compared with the model-making group, the bifidobacterium longum CCFM1029 intervention group significantly reversed MPTP-induced reduction in alpha diversity (all P < 0.0001), and Shannon and Simpson indexes were increased by 1.40 and 1.24 times, respectively.

Example 6: effect of Bifidobacterium longum CCFM1029 on MPTP-induced intestinal flora composition of mice

The method comprises the following specific steps:

1. animal experiment grouping: the same as in example 1.

2. After a Fast DNA Spin Kit for Feces Kit is adopted to extract the metagenome of the fecal bacteria of each group of mice, the sequence of the 1qs V3-V4 region is subjected to PCR amplification, and then the composition of intestinal flora in a fecal sample is obtained by a second-generation sequencer. The results are shown in FIG. 6.

3. Results of the experiment

Phylogenetic analyses showed that the mouse intestinal flora consisted mainly of five groups, firmicutes, bacteriodes, proteobacteria, verrucomicrobia, actinobacterium. At the genus level, flora differences at the genus level were accomplished using LEfSe analysis. Using LDA score >3.0 and alpha <0.05 as difference analysis threshold, detecting 15 groups of differences in total; as shown in fig. 6, the abundance of Bifidobacterium (Bifidobacterium), lactobacillus (Lactobacillus), bacteroides (Bacteroides), and Shigella (Shigella) was significantly changed in chronic stress state and could be reversed by intervention of Bifidobacterium longum CCFM1029. MPTP treatment significantly reduced the relative abundance of Bifidobacterium, lactobacillus in the mouse gut, significantly increased the relative abundance of Bacteroides and Shigella (all p < 0.05), and Bifidobacterium longum CCFM1029 intervention was able to reverse this change (all p < 0.05). The above experimental results show that CCFM1029 intervention restores the abundance of probiotics and reduces the level of harmful bacteria.

Example 7: application of bifidobacterium longum CCFM1029

The lactobacillus sake CCFM1029 can be used for preparing bacterial powder, and the specific preparation process of the bacterial powder is as follows:

lactobacillus sake CCFM1029 streaked on MRS solid culture mediumCulturing at 37 deg.C for 48 hr to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated solution into a culture medium according to the inoculation amount of 2% (v/v), and culturing at 37 ℃ for 18h to obtain a bacterial solution; centrifuging the bacterial liquid at 8000g for 10min to obtain bacterial mud; washing the bacterial sludge with normal saline for 3 times, and suspending with protectant to 1 × 10 ¹⁰ CFU/mL to obtain a bacterial suspension; pre-culturing the bacterial suspension at 37 deg.C for 60min, and lyophilizing to obtain powder of Lactobacillus sake CCFM 1029;

the preparation method of the culture medium comprises the following steps: dissolving 10% of enzyme hydrolysis skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract by using 87.7% of water based on the total weight of the culture medium, and then adjusting the pH of the solution to 6.8 to obtain a culture medium;

the components of the protective agent comprise: 130g/L skimmed milk powder.

Example 8: application of bifidobacterium longum CCFM1029

Bifidobacterium longum CCFM1029 can be used for preparing capsule product, and the capsule product is prepared by the following steps

Marking Bifidobacterium longum CCFM1029 on MRS solid culture medium, and culturing at 37 deg.C for 48 hr to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated solution into a culture medium according to the inoculation amount of 2% (v/v), and culturing at 37 ℃ for 18h to obtain a bacterial solution; centrifuging the bacterial liquid at 6000r/min for 10min to obtain bacterial sludge; washing the bacterial sludge with normal saline for 3 times, and then resuspending the bacterial sludge with protective agent to the concentration of 1 × 10 ¹⁰ CFU/mL to obtain bacterial suspension; adding the bacterial suspension into a sodium alginate solution with the concentration of 30g/L to reach the concentration of 2 x 10 ⁹ Fully stirring after CFU/mL to uniformly disperse cells of the bifidobacterium longum CCFM1029 in the sodium alginate solution to obtain a mixed solution; extruding the mixed solution into a calcium chloride solution with the concentration of 20g/L to form colloidal particles; standing and solidifying the formed colloidal particles for 30min, and filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; filling the powder into a medicinal capsule to obtain a gelA bladder article;

the preparation method of the culture medium comprises the following steps: 10% of enzymatically hydrolyzed skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract were dissolved using 87.7% of water based on the total weight of the medium, and then the pH thereof was adjusted to 6.8, to obtain a medium.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of Bifidobacterium longum (Bifidobacterium longum) CCFM1029 in preparing a product for preventing and/or relieving Parkinson's disease, wherein the application is not aimed at treating the disease;

the bifidobacterium longum CCFM1029 is preserved in Guangdong province microorganism strain collection center in 2018, 10 and 11 days, and the preservation number is GDMCC No:60461, the preservation address is No. 59 building 5 of No. 100 college of the Pieli Zhonglu, guangzhou city.

2. Use according to claim 1, characterized in that the product comprises at least one of the following functions:

(2) Obviously improve and increase mouse striatum monoamine and its metabolite level induced by MPTP, have effects of neuroprotection, protect mouse striatum to avoid DA neuron loss induced by MPTP;

(5) Effectively improve the intestinal flora structure of MPTP induced mice.

3. Use according to claim 1 or 2, wherein the product is a pharmaceutical product.

4. Use according to claim 1 or 2, wherein the viable count of Bifidobacterium longum in the product is not less than 5 x 10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

5. Use according to claim 3, wherein the medicament comprises Bifidobacterium longum (Bifidobacterium longum) CCFM1029, a pharmaceutical carrier and/or a pharmaceutical excipient.

6. Use according to claim 5, wherein the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.

7. Use according to claim 5, wherein the pharmaceutical excipient comprises an excipient and/or an additive.

8. Use according to claim 7, characterized in that the excipient comprises a binder, a filler, a disintegrant and/or a lubricant; the additive comprises a solubilizer, a cosolvent and/or a preservative.

9. The use of claim 3, wherein the medicament is in the form of a powder, granules, capsules, tablets, pills or oral liquid.

10. A microbial preparation comprising Bifidobacterium longum (CCFM 1029).