CN115887504B

CN115887504B - Application of bifidobacterium longum CCFM1029 in relieving parkinsonism

Info

Publication number: CN115887504B
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: 2024-03-26
Anticipated expiration: 2042-09-16
Also published as: CN115887504A

Abstract

The invention discloses an application of bifidobacterium longum CCFM1029 in relieving parkinsonism, and belongs to the technical field of microorganisms and medicines. The bifidobacterium longum CCFM1029 provided by the invention has the effect of relieving the parkinsonism, and is specifically expressed in that: (1) Significantly alleviating MPTP-induced motor dysfunction in mice, including improving mice agility, limb movement coordination, and balance ability; (2) The method has the advantages that the level of the mouse striatum monoamine and the metabolite thereof induced by MPTP is obviously improved, the neuroprotection effect is realized, and the mouse striatum is protected from loss of DA neurons induced by MPTP; (3) Remarkably increases the midbrain antioxidant level of MPTP-induced mice and relieves the oxidative stress induced by MPTP; (4) The short chain fatty acid content in the intestinal tract of an MPTP induced mouse is obviously improved; (5) effectively improving the intestinal flora structure of MPTP-induced mice. Therefore, the bifidobacterium longum CCFM1029 has great application prospect in preparing products for preventing and/or relieving the parkinsonism.

Description

Application of bifidobacterium longum CCFM1029 in relieving parkinsonism

Technical Field

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

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease, occurring at about 1% in people over 65 years old, and is characterized by dyskinesia due to loss of midbrain dopaminergic neurons, including stiffness, resting tremor, gait disorders and bradykinesia. The pathogenic mechanisms of PD may be associated with genetic factors, oxidative stress, neuroinflammation and metabolic disorders that lead to misfolding and aggregation of alpha synuclein. PD patients suffer from motor and non-motor symptoms, often showing symptoms of gastrointestinal dysfunction and constipation a few years before the onset of motor symptoms, 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 α -synuclein-related neurodegenerative changes in the Enteric Nervous System (ENS). In recent years, the intestinal brain axis of interaction or mutual interference between the gastrointestinal tract, ENS, central Nervous System (CNS) and intestinal microbiota (GM) has become one of the focus of research in PD pathogenesis. Many clinical studies have found altered intestinal microbial composition in PD patients, suggesting that intestinal dysbiosis plays a key role in exacerbating PD pathogenesis. At present, no therapeutic method for curing or improving parkinsonism exists. Levodopa is the gold standard drug, but chronic administration of levodopa has significant side effects. Thus, there is an urgent need to find ideal therapeutic drugs and new therapies.

Probiotics are living microorganisms that confer health benefits to the host, and may improve the physical and mental health of the host by affecting the microbial-intestinal-brain axis homeostasis. Specific strains of probiotics capable of modulating neurotransmitters, neurotrophic factors and behavior are referred to as "mental probiotics". The compound probiotic product (containing Lactobacillus acidophilus, bifidobacterium bifidum, lactobacillus reuteri and Lactobacillus fermentum) can be eaten by PD patients for 12 weeks to improve PD movement symptoms, inhibit oxidative stress, and regulate insulin and lipid metabolism (disclosed in Tamtaji OR, taghizadeh M et al clinical and metabolic response to probiotic administration in people with Parkinson's disease: A random, double-blind, placebo-controlled three.Clin Nutr.2019 Jun;38 (3): 1031-1035); it was found that daily administration of the probiotic preparation SLAB51 (containing Streptococcus thermophilus, bifidobacterium longum, bifidobacterium breve, bifidobacterium infantis, lactobacillus acidophilus) can save 6-hydroxydopamine-induced death of dopaminergic neurons in the substantia nigra and striatum of PD model mice and improve motor dysfunction (published in Caselli V, d 'Angelo M et al effects of the probiotic formulation SLAB51 in in vitro and in vivo Parkinson's disease models. Aging (Albany NY). 2020 Mar9;12 (5): 4641-4659); long-term administration of probiotic cocktails (bifidobacterium bifidum, bifidobacterium longum, lactobacillus rhamnosus GG, lactobacillus plantarum LP28 and lactococcus lactis subspecies) has neuroprotective effects on dopaminergic neurons and further reduces exacerbation of MitoParkpD mouse motor dysfunction (published in Hsieh TH, kuo CW et al Probiotics Alleviate the Progressive Deterioration of Motor Functions in a Mouse Model of Parkinson's disease. Brain Sci.2020 Apr 1;10 (4): 206). These reports are supporting evidence of the positive impact of the ingestion of probiotics by PD patients; however, the mechanism of the probiotic mixture is complex, including antioxidant, immune response, intestinal microbial regulation, etc., and secondary probiotic strain specificity and host conditions may affect the final clinical outcome. Therefore, it is necessary to conduct single strain development verification to confirm the health promoting effect of the specific probiotics in each case.

Bifidobacteria are important intestinal beneficial microorganisms, and have various important physiological functions such as biological barrier, nutrition effect, anti-tumor effect, immunity enhancing effect, gastrointestinal tract function improvement, aging resistance and the like for human health. In recent years, the efficacy and prospect of bifidobacteria in neurodegenerative diseases have been emphasized and mined by more people. Bifidobacterium breve CCFM1025 is a promising candidate for the relief of depression and related gastrointestinal disorders (published in Tian P, chen Y, zhu H et al, bifidobacterium breve CCFM1025 attenuates major depression disorder via regulating gut microbiome and tryptophan metabolism: A randomized clinical three. Brain Behav Immun.2022 Feb;100:233-241. Paper); bifidobacterium breve strain A1 has therapeutic potential for preventing cognitive impairment of Alzheimer's disease (published in Kobayashi Y, sugahara H et al, therapeutic potential of Bifidobacterium breve strain A, for preventing cognitive impairment in Alzheimer's disease. Sci Rep.2017 Oct 18;7 (1): 13510. Paper); oral probiotic bifidobacterium breve strain A1[ MCC1274] was able to restore 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -induced memory decline and cognitive dysfunction in the parkinson's disease mouse model (published in Ishii T, furaoka H et al oral Administration of Probiotic Bifidobacterium breve Improves Facilitation of Hippocampal Memory Extinction via Restoration of Aberrant Higher Induction of Neuropsin in an MPTP-Induced Mouse Model of Parkinson's disease.biomedicines.2021 Feb 8;9 (2): 167). However, there have been no reports of a bifidobacterium longum single strain having a neuroprotective effect on parkinson's disease and a motor dysfunction alleviating effect on it.

Disclosure of Invention

In order to provide a bifidobacterium longum (Bifidobacterium longum) capable of preventing and/or alleviating parkinson's disease and safe without side effects, the invention provides a bifidobacterium longum (Bifidobacterium longum) CCFM1029, said bifidobacterium longum CCFM1029 having been deposited at the microorganism strain deposit center in the canton province at 10 months 11 of 2018 under the deposit number GDMCC No:60461, having a deposit address of building 5 from university 100, mitsui, guangzhou, inc., is disclosed in the patent publication No. CN 109652349A.

The invention provides the use of the bifidobacterium longum (Bifidobacterium longum) CCFM1029 described above for the manufacture of a product for the prevention and/or alleviation of Parkinson's disease, said use not being aimed at the treatment of the disease.

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

(1) Significantly alleviating MPTP-induced motor dysfunction in mice, including improving mice agility, limb movement coordination, and balance ability;

(2) The method has the advantages that the level of the mouse striatum monoamine and the metabolite thereof induced by MPTP is obviously improved, the neuroprotection effect is realized, and the mouse striatum is protected from loss of DA neurons induced by MPTP;

(3) Remarkably increases the midbrain antioxidant level of MPTP-induced mice and relieves the oxidative stress induced by MPTP;

(4) The short chain fatty acid content in the intestinal tract of an MPTP induced mouse is obviously 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 present invention, the viable count of bifidobacterium longum in the product is not less than 5×10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

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

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

In one embodiment of the invention, the pharmaceutical excipients comprise excipients and/or additives.

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

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

In one embodiment of the invention, the medicament is in the form of powder, granule, capsule, tablet, pill or oral liquid.

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

In one embodiment of the invention, the preparation method of the microbial preparation comprises inoculating the bifidobacterium longum (Bifidobacterium longum) CCFM1029 into a culture medium according to the inoculum size accounting for 2-4% (v/v) of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing thalli with normal saline for 3 times, and then re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension by vacuum freezing to obtain the starter.

In one embodiment of the invention, the mass ratio of the lyoprotectant to the thalli is 2:1.

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

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

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

The beneficial effects are that:

1. the invention screens out bifidobacterium longum (Bifidobacterium longum) CCFM1029, and the bifidobacterium longum CCFM1029 has the effect of relieving the parkinsonism and is specifically characterized in that:

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

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

Preservation of biological materials

Bifidobacterium longum (Bifidobacterium longum) CCFM1029, taxonomic designation Bifidobacterium longumi, deposited at the cantonese province microbiological bacterial collection center on 10, 11, 2018 under the accession number GDMCC No:60461 the preservation address is building 5 of Guangdong national institute of science and microbiology, guangzhou City, first, china, no. 100, university, 59.

Drawings

Fig. 1: a histogram of behavioral tests of mice of different groups, wherein:

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

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

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

Fig. 2: histogram of striatal monoamine and metabolite content in different groups of mice, wherein:

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

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

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

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

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

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

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

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

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

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

fig. 4: short chain fatty acid content in the faeces of mice of different groups, wherein:

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

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

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

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

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

fig. 5: intestinal flora diversity analysis of mice of different groups

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

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

fig. 6: the intestinal flora difference of different groups of mice is relative abundance pattern, wherein:

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

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

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

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

Detailed Description

Chemicals such as tryptone and yeast powder, which are referred to in the following examples, were purchased from the national drug group; the mice referred to in the examples below were male SPF (Specific pathogen free, without specific pathogen) grade C57BL/6J mice of 6-8 weeks old purchased from Jiangsu Jiex Kangsu Biotech Co., ltd; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), levodopa (L-DOPA), benserazide from MCE company; dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT) were purchased from Sigma-Aldrich company; superoxide dismutase (Superoxide Dismutase, SOD), catalase (CAT) and reduced Glutathione (GSH) kits as referred to in the following examples were purchased from the institute of biotechnology, of south-kyo, and paraformaldehyde as referred to in the following examples was purchased from the company, inc. Biotechnology, of meku; the Fast DNA Spin Kit for Feces kit referred to in the examples below was purchased from MP Biomedicals company.

The following examples relate to the following media:

MRS solid Medium (g/L): 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 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 1mL/L, agar 20g/L, cysteine amino acid salt 0.5g/L.

MRS liquidVolume medium (g/L): 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 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 1mL/L, cysteine amino acid salt 0.5g/L.

The preparation method of the bifidobacterium longum suspension in the following examples is as follows:

streaking bifidobacterium longum CCFM1029 on MRS solid culture medium (containing 0.05% cysteine), and culturing at 37 ℃ for 48 hours to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium (containing 0.05% cysteine), and is cultured for 24 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium (containing 0.05% cysteine) according to the inoculum size of 2% (v/v), and culturing at 37deg.C for 24 hr to obtain bacterial solution; centrifuging the bacterial liquid for 10min by 8000g to obtain bifidobacterium longum bacterial cells; washing Bifidobacterium longum cells with normal saline, and re-suspending in 200g/L glycerol solution until the bacterial concentration is 1×10 ¹⁰ CFU/mL to obtain bacterial suspension, and preserving the bacterial suspension at-80 ℃ for later use.

Example 1: bifidobacterium longum CCFM1029 alleviates MPTP-induced motor dysfunction in mice

The method comprises the following specific steps:

1. animal experiment group: 50 healthy female C57BL/6 mice with the weight of 18-20_g and the age of 6-8 weeks are randomly divided into 5 groups of 10 mice each, wherein the 5 groups are respectively: blank Control (Control), modeling (MPTP), positive Control (L-DOPA), bifidobacterium longum CCFM1029 intervention, and Bifidobacterium longum Y1 intervention.

1. Blank control group: a 1-7 day adaptation period, free drinking water diet; and 200 mu L of physiological saline is infused into the stomach every day for 8-41 days, and the volume of the physiological saline with the same volume as MPTP in the manufacturing module is 0.1mL/20g of body weight is injected into the abdominal cavity for 33-37 days.

2. And (5) manufacturing a module: a 1-7 day adaptation period, free drinking water diet; 200. Mu.L of physiological saline is infused daily for 8-41 days, 30mg/kg MPTP (dissolved in sterilized physiological saline) is injected intraperitoneally for 33-37 days, and the volume is 0.1ml/20g body weight.

3. Positive control group: a 1-7 day adaptation period, free drinking water diet; 200 mu L of physiological saline is infused every day for 8-33 days, 30mg/kg of MPTP (dissolved in sterilized physiological saline) is injected intraperitoneally for 33-37 days, 100mg/kg of L-DOPA+25mg/kg of Benserazide is infused every 33-41 days, and the volume of each time of gastric lavage is 0.1mL/20g of body weight.

4. Bifidobacterium longum CCFM1029 intervention group: a 1-7 day adaptation period, free drinking water diet; for 8-41 days, 200 μl of 1×10 is infused per day ⁹ CFU/mL bifidobacterium longum CCFM1029 was injected intraperitoneally (in sterile saline) at 30mg/kg MPTP (dissolved in sterile saline) for 33-37 days at a volume of 0.1mL/20g body weight.

5. Bifidobacterium longum Y1 intervention group: a 1-7 day adaptation period, free drinking water diet; for 8-41 days, 200 μl of 1×10 is infused per day ⁹ CFU/mL bifidobacterium longum Y1 was injected intraperitoneally at 30mg/kg MPTP (dissolved in sterile saline) for 33-37 days at a volume of 0.1mL/20g body weight.

2. Behavioural testing: evaluating mice agility and sensory dyskinesia using a pole climbing experiment; evaluating the mouse movement coordination and balance by a balance beam experiment; the rod rotating experiment tests the coordination condition of the limb activities of the mice. All mice received behavior training once daily starting on days 28-30. Behavioral testing was performed 24 hours after the last MPTP injection on day 37, with the following specific methods of behavioral testing:

1. climbing pole experiment

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

2. Balance beam experiment

The experimental device is a wood rod with the length of 50cm, the width of 0.8cm and the height of 0.8cm, the wood rod is arranged on two columns with the height of 50cm from the experimental table top, one end of the wood rod is used as a starting point, and a black box is placed at the other end of the wood rod as an end point. On each training day, mice were placed in a black box for 5min to become familiar with the environment, then placed 5cm from the black box, and trained 3 times to the endpoint, followed by 15cm, 30cm and 50cm balancers. On the day of the test, the time required for the mice to traverse the balance beam from the end of the start point of the stick and climb into the end point black box was recorded. If the mice fall halfway or turn back to the starting point, the test is re-performed. If the mice were unable to reach the endpoint or were continuously dropped from the stick within 60s, they were recorded as 60s. Each mouse was tested 3 times (1 min interval between tests), and the average time of 3 walking the balance beam was used as a statistical index.

3. Rotating rod experiment

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

3. Experimental results

As can be seen from fig. 1A-C, the total drop time of MPTP model mice in the pole-climbing test and the total travel time in the balance beam test were significantly increased, and the total residence time in the rotating rod test was significantly shortened (all p < 0.0001) compared to the blank control group. Compared with the modeling group, the positive control group and the bifidobacterium longum CCFM1029 intervention group obviously relieve the MPTP-induced PD mouse movement dysfunction on a pole climbing (p <0.0001 ), a balance beam (p <0.0001, p < 0.01) and a rod rotating test (p <0.001, p < 0.01), and the effect of the bifidobacterium longum CCFM1029 intervention group is similar to that of the positive control group; bifidobacterium longum Y1 failed to alleviate motor performance in the behavioural test of PD mice, and in the rod climbing test and the balance beam test, bifidobacterium longum Y1 intervention group had a tendency to exacerbate motor dysfunction in PD mice compared to model group mice.

The experimental result shows that bifidobacterium longum CCFM1029 has the function of relieving the MPTP-induced motor dysfunction of PD mice.

Example 2: bifidobacterium longum CCFM1029 increases MPTP-induced mouse monoamine and metabolite levels thereof

The method comprises the following specific steps:

1. animal experiment group: as in example 1.

2. Detection of monoamines and their metabolites: detection by high performance liquid chromatography. The striatal sample of each group of mice was weighed to 10mg or more, 100. Mu.L of 0.1M or 0.2M perchloric acid solution was added to each 10mg of the sample, and after the sample was minced with a 1mL syringe head, the sample was further minced to a homogenized state using an ultrasonic chopper. The homogenized sample was centrifuged at 15000rpm for 15min at 4℃and after centrifugation, the supernatant was filtered through a 0.22 μm aqueous filter to the vial liner.

The high performance liquid chromatograph 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 mu L. The fluorescence detection condition is that the excitation wavelength is 280nm and the absorption wavelength is 320nm.

3. Experimental results

As can be seen from FIGS. 2A-F, the DOPA, DA, 5-HT, DOPAC, 5-HIAA, HVA levels in the striatum of the model mice 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 significantly improves the striatum DOPA, DA, 5-HT, DOPAC, 5-HIAA and HVA levels, and has statistical significance (all p < 0.01); compared with the modeling group, the bifidobacterium longum CCFM1029 intervention group remarkably improves the level 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 level 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 intake of bifidobacterium longum CCFM1029 significantly restores MPTP-induced reduction of DA, 5-HT and its metabolites; intervention by bifidobacterium longum Y1 did not have the effect of alleviating MPTP-induced reduction of mouse striatal neurotransmitters.

Therefore, the experimental result shows that the intervention of bifidobacterium longum CCFM1029 produces neuroprotection on the MPTP-induced mice, and the level of the striatal monoamine and the metabolites thereof of the MPTP-induced mice is obviously improved.

Example 3: bifidobacterium longum CCFM1029 increased brain antioxidant levels in MPTP-induced mice, reducing MPTP-induced oxidative stress

The method comprises the following specific steps:

1. animal experiment group: as in example 1.

2. Detection of brain tissue antioxidants

The levels of superoxide dismutase (Superoxide Dismutase, SOD), catalase (CAT) and reduced Glutathione (GSH) were detected using a detection kit. The brain tissue of the mice was sonicated and lysed in normal saline. After centrifugation, the supernatant was analyzed using the test kit according to the manufacturer's instructions. The antioxidant levels in brain tissue of all mice were estimated from the antioxidant standard activity, then normalized to protein concentration and expressed in enzyme units per mg protein.

3. Experimental results

As can be seen from figures 3A-C, brain CAT, GSH levels were significantly reduced in model mice compared to the placebo group (all p < 0.01); the positive control group and the bifidobacterium longum CCFM1029 intervention group significantly improve the MPTP induction reduction of the content of brain CAT and GSH in mice, and the difference is statistically significant (all p < 0.05) by 2.33 times and 1.99 times, 2.50 times and 2.07 times respectively higher than those of the model group. However, the positive control group and bifidobacterium longum CCFM1029 did not have a significant difference in brain SOD levels in MPTP-induced mice (FIG. 3B), and intervention with bifidobacterium longum Y1 did not have an effect of alleviating the decrease in brain antioxidant levels in MPTP-induced mice.

These results indicate that CCFM1029 enhances MPTP-induced brain antioxidant levels and capacity in mice, and that this capacity may be mediated by CAT and GSH.

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

The method comprises the following specific steps:

1. animal experiment group: as in example 1.

2. Determination of fecal short-chain fatty acid content

Placing the collected mouse feces in liquid nitrogen, transferring to a-80 ℃ box, taking out before short chain fatty acid content detection, performing vacuum freeze drying, accurately weighing 0.05g of freeze-dried feces sample, dissolving in 0.5mL of saturated sodium chloride solution, soaking for 30min, homogenizing by a tissue homogenizer, adding 0.02mL of 10% sulfuric acid, shaking for 30s, accurately adding 0.8mL of diethyl ether solution into the feces solution in a fume hood, shaking for 30s, centrifuging for 15min (8000 g,4 ℃), and transferring the supernatant to a centrifuge tube containing 0.3g of anhydrous sodium sulfate，Shaking uniformly, centrifuging for 15min (8000 g,4 ℃) and taking the supernatant into a gas quality volumetric flask, and detecting the content of short chain fatty acid by GC-MS, wherein the detection result is shown in the figure.

3. Experimental results

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), valeric acid (p < 0.0001) in the colon contents of the model mice showed a significant decrease compared to the blank, and all were statistically significant, which resulted in a significant decrease in the overall SCFAs content of the model (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 content (p < 0.01) in the excrement of the MPTP-induced mice, and has no statistical difference compared with the modeling group although the butyric acid and valeric acid content has an ascending trend; the levels of acetic acid, propionic acid, butyric acid and valeric acid in the mouse faeces of the bifidobacterium longum CCFM1029 intervention group showed significant increases (all p < 0.001), 2.52 times, 2.07 times, 2.76 times and 2.44 times respectively, compared with the control group, the total concentration of SCFAs in the mouse faeces of the CCFM1029 intervention group also increased significantly (p < 0.0001), while the intervention of bifidobacterium longum Y1 only significantly up-regulated the level of acetic acid in the mouse faeces (p < 0.01).

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

The method comprises the following specific steps:

1. animal experiment group: as in example 1.

2. After extracting the metagenome of the fecal bacteria of each group by adopting a Fast DNA Spin Kit for Feces kit, carrying out PCR amplification on the 16s V3-V4 region sequence, and then carrying out intestinal flora diversity in fecal samples by a second-generation sequencer. The results are shown in FIG. 5.

3. Experimental results

As shown in fig. 5A-B, the Shannon and Simpson indices of the mock set were significantly reduced (all P < 0.001) compared to the placebo group; positive control and bifidobacterium longum Y1 intervention groups increased Shannon index and Simpson index compared to the modeling group, but did not have statistical differences compared to the modeling group; the bifidobacterium longum CCFM1029 intervention group significantly reversed MPTP-induced reduction in alpha diversity (all P < 0.0001), 1.40-fold and 1.24-fold increases in Shannon and Simpson indices, respectively, compared to the modeling group.

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

The method comprises the following specific steps:

1. animal experiment group: as in example 1.

2. After extracting the metagenome of the fecal bacteria of each group by adopting a Fast DNA Spin Kit for Feces kit, carrying out PCR amplification on the 16s V3-V4 region sequence, and then forming the intestinal flora in the fecal sample by a second-generation sequencer. The results are shown in FIG. 6.

3. Experimental results

The portal level analysis shows that the intestinal flora of mice mainly consists of Firmicutes, bacteroidetes, proteobacteria, verrucomicrobia, actinobacteria five groups. At the genus level, the flora difference at the genus level was accomplished using a LEfSe assay. Taking LDA score >3.0 and alpha <0.05 as difference analysis threshold values, detecting 15 kinds of the genus having inter-group differences in total; as shown in fig. 6, the abundance of bifidobacteria (Bifidobacterium), lactobacillus (Lactobacillus), bacterioides (Bacteroides) and Shigella (Shigella) changes significantly in chronic stress conditions and can 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 bacterioides 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 indicate that the 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:

the lactobacillus sake CCFM1029 is streaked on MRS solid culture medium and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 ¹⁰ CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus sake CCFM1029 bacterial powder;

the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain 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 products, and the specific preparation process of the capsule products is as follows

The bifidobacterium longum CCFM1029 is streaked on an MRS solid culture medium and cultured for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial liquid for 10min at 6000r/min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to concentration of 1×10 ¹⁰ CFU/mL, obtaining bacterial suspension; the bacterial suspension is added into sodium alginate solution with the concentration of 30g/L to the concentration of 2 multiplied by 10 ⁹ After CFU/mL, fully stirring to uniformly disperse cells of 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; after the formed colloidal particles are stationary and solidified for 30min, 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 capsule product;

the preparation method of the culture medium comprises the following steps: the medium was obtained by dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the medium, and then adjusting the pH to 6.8.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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. Bifidobacterium longumBifidobacterium longum) Use of CCFM1029 in the manufacture of a medicament for preventing parkinson's disease, said use not being for the purpose of treatment of the disease;

the bifidobacterium longum CCFM1029 was deposited at the microorganism strain collection in Guangdong province on 10 th and 11 th 2018 under the accession number GDMCC No:60461, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.

2. The use according to claim 1, wherein the viable count of bifidobacterium longum in the medicament is not less than 5 x 10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

3. Use according to claim 1 or 2, wherein the medicament comprises bifidobacterium longum @Bifidobacterium longum) CCFM1029 and pharmaceutical excipients.

4. Use according to claim 1 or 2, wherein the medicament comprises bifidobacterium longum @Bifidobacterium longum) CCFM1029 and a pharmaceutical carrier.

5. The use according to claim 4, wherein the pharmaceutical carrier is selected from the group consisting of microcapsules, microspheres, nanoparticles and liposomes.

6. Use according to claim 3, wherein the pharmaceutical excipients comprise excipients and/or additives.

7. The use according to claim 6, wherein the excipient comprises binders, fillers, disintegrants and/or lubricants; the additives include solubilizers, co-solvents, and/or preservatives.

8. The use according to claim 3 or 4, wherein the pharmaceutical product is in the form of a powder, granule, capsule, tablet, pill or oral liquid.