Bifidobacterium longum subsp. infantis capable of relieving parkinson's disease and use thereof

Abstract

The present application relates to Bifidobacterium longum subsp. infantis capable of relieving Parkinson's disease and the use thereof. Said strain is named as Bifidobacterium longum subsp. infantis BI03 strain, with an accession number of CGMCC No. 24473, the preservation date thereof being March 07, 2022. Said strain can significantly relieve the symptoms of Parkinson's disease, specifically embodied in: relieving Parkinson's disease related dyskinesia and increased corticosterone levels; alleviating neuroinflammation related to Parkinson's disease; promoting glutathione and reducing brain oxidative stress injuries, etc.

Description

Bifidobacterium longum subspecies infantis for improving Parkinson's disease and application thereof Technical Field

[0001] The present application belongs to the field of microbial culture technology, and relates to a Bifidobacterium longum subsp. infantis strain for improving Parkinson's disease and its application, and specifically to a Bifidobacterium longum subsp. infantis BI03 strain, a probiotic agent containing the same, and its application in the preparation of a medicine or health product with the efficacy of preventing, improving or treating Parkinson's disease. Background Art

[0002] Parkinson's disease is a chronic, progressive neurological disease characterized by hand tremors, muscle stiffness, and bradykinesia, all of which are related to decreased dopamine levels in the brain. Although a variety of drugs have been developed to alleviate Parkinson's disease symptoms, many patients are still not satisfied with existing treatments, and long-term use of certain drugs can lead to side effects and drug resistance. Therefore, it is very important to find new treatment options and prevent the occurrence and progression of Parkinson's disease. In recent years, probiotics have attracted widespread attention in the field of Parkinson's disease as a potential treatment option.

[0003] Some studies suggest that probiotics may have antioxidant and neuroprotective effects, potentially alleviating Parkinson's disease-related symptoms by affecting the brain-gut axis. The gut microbiome is a key factor influencing the brain-gut axis. Probiotics can modulate gut-brain communication by affecting intestinal metabolites, neurotransmitters, and short-chain fatty acids, thereby alleviating Parkinson's disease-related symptoms. Probiotic intake can modulate the composition and balance of the gut microbiome, thereby reducing the damage to the nervous system caused by factors such as chronic low-grade inflammation and oxidative stress. Chronic inflammation is a common feature of many neurodegenerative diseases and is closely associated with the development and progression of Parkinson's disease. Certain strains of probiotics can alleviate inflammation by inhibiting inflammatory responses and regulating the immune system. Furthermore, oxidative stress is a contributing factor to many neurodegenerative diseases, and certain probiotics may protect the nervous system by increasing antioxidant enzyme activity and scavenging free radicals.

[0004] Research on probiotic intervention in Parkinson's disease may provide new inspiration for improving Parkinson's disease-related movement defects, nerve damage, intestinal flora imbalance, etc. Probiotics can be used as a potential treatment or additional treatment alternative.

[0005] Summary of the Invention

[0006] The present application provides a Bifidobacterium longum subsp. infantis strain for improving Parkinson's disease and its application, in particular, provides a Bifidobacterium longum subsp. infantis BI03 strain, a probiotic agent containing the same, and its application in the preparation of a medicine or health product with the efficacy of preventing, improving or treating Parkinson's disease.

[0007] In a first aspect, the present application provides a Bifidobacterium longum subsp. infantis strain for improving Parkinson's disease. The Bifidobacterium longum subsp. infantis strain for improving Parkinson's disease is named Bifidobacterium longum subsp. infantis BI03 strain, with a preservation number of CGMCC No. 24473 and a preservation date of March 7, 2022.

[0008] This application describes the isolation and preservation of a new strain of Bifidobacterium longum subsp. infantis BI03, which can improve Parkinson's disease, from fecal samples of healthy breastfed infants in Dali Bai Autonomous Prefecture, Yunnan Province. The strain is named Bifidobacterium longum subsp. infantis BI03. This strain can significantly improve the symptoms of Parkinson's disease, specifically: (1) significantly improve Parkinson's disease-related movement disorders and elevated corticosterone; (2) reduce Parkinson's disease-related neuroinflammation; and (3) significantly promote glutathione production and reduce brain oxidative stress damage. This discovery provides new ideas for the development of new drugs to prevent or treat Parkinson's disease. In addition, Bifidobacterium longum subsp. infantis is a probiotic that is highly safe and not prone to resistance when used in the preparation of products for the prevention or treatment of Parkinson's disease.

[0009] The screening steps for the Bifidobacterium longum subsp. infantis BI03 strain involved in this application are as follows:

[0010] (1) Fecal samples of healthy breastfed infants were selected from Dali Bai Autonomous Prefecture, Yunnan Province. They were diluted 10-fold using 0.9% saline solution for three times and then spread on MRS solid culture medium (purchased from Haibo Biological). After incubation at 37°C for 48 h, colonies of different morphologies were picked out and purified by streaking on the surface of MRS solid culture medium. Single colonies were picked out and expanded using MRS liquid culture medium at 37°C and then preserved using 30% glycerol.

[0011] (2) In vitro physiological characteristics tests were conducted on multiple preserved single bacterial strains, and a single bacterial strain with the best gastrointestinal fluid tolerance (artificial simulation), HT-29 cell adhesion ability, antibacterial ability, and functional oligosaccharide utilization ability was screened out, identified, and preserved.

[0012] In a second aspect, the present application provides a bacterial agent for preventing, improving or treating Parkinson's disease, wherein the strain in the bacterial agent includes the Bifidobacterium longum subsp. infantis BI03 strain described in the first aspect.

[0013] The Bifidobacterium longum subsp. infantis BI03 strain involved in the present application can be used alone in relevant products, or in combination with other strains in relevant products.

[0014] Preferably, in the bacterial agent, the viable bacterial count of Bifidobacterium longum subsp. infantis BI03 strain is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 3×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), or 1×10 13 CFU / g (CFU / mL), etc.

[0015] Preferably, the strain in the bacterial agent further includes Bifidobacterium longum BL21 strain, with a deposit number of CGMCC No. 10452 and a deposit date of January 27, 2015.

[0016] The present applicants also creatively discovered that the BI03 strain can be used in combination with the Bifidobacterium longum BL21 strain for the prevention, alleviation, or treatment of Parkinson's disease, demonstrating significantly superior efficacy compared to single bacterial agents or other combination methods. This suggests that the BI03 strain and the BL21 strain have a synergistic effect in achieving the aforementioned therapeutic effects.

[0017] Preferably, the ratio of the viable cell count of the Bifidobacterium longum subsp. infantis BI03 strain to the Bifidobacterium longum BL21 strain is (2-4):1, such as 2:1, 5:2, 3:1, 7:2, or 4:1.

[0018] Based on the potential interaction between the BI03 strain and the BL21 strain in the above-mentioned efficacy, the two strains have a better synergistic effect when the above-mentioned specific mass ratio relationship is met.

[0019] Preferably, the dosage form of the bacterial agent includes freeze-dried powder, capsule, tablet or granule.

[0020] Preferably, the bacterial agent further comprises a protective agent and / or auxiliary additives.

[0021] Preferably, the protective agent is selected from any one or a combination of at least two of skim milk, gelatin, maltodextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0022] Preferably, the auxiliary additive is selected from any one or a combination of at least two of resistant dextrin, fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactalbumin or lactoferrin.

[0023] In a third aspect, the present application provides a use of the Bifidobacterium longum subsp. infantis BI03 strain according to the first aspect or the bacterial agent according to the second aspect in the preparation of a medicine or health product having the efficacy of preventing, improving or treating Parkinson's disease.

[0024] In a fourth aspect, the present application provides use of the Bifidobacterium longum subsp. infantis BI03 strain according to the first aspect or the bacterial agent according to the second aspect in the preparation of a medicament for preventing, improving or treating motor deficits associated with Parkinson's disease.

[0025] In a fifth aspect, the present application provides use of the Bifidobacterium longum subsp. infantis BI03 strain according to the first aspect or the bacterial agent according to the second aspect in the preparation of a drug for preventing, improving or treating Parkinson's disease-related neuroinflammatory damage.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] This application describes the isolation and preservation of a new strain of Bifidobacterium longum subsp. infantis BI03, which can improve Parkinson's disease, from fecal samples of healthy breastfed infants in Dali Bai Autonomous Prefecture, Yunnan Province. The strain is named Bifidobacterium longum subsp. infantis BI03. This strain can significantly improve the symptoms of Parkinson's disease, specifically: (1) significantly improve Parkinson's disease-related movement disorders and elevated corticosterone; (2) reduce Parkinson's disease-related neuroinflammation; and (3) significantly promote glutathione production and reduce brain oxidative stress damage. This discovery provides new ideas for the development of new drugs to prevent or treat Parkinson's disease. In addition, Bifidobacterium longum subsp. infantis is a probiotic that is highly safe and not prone to resistance when used in the preparation of products for the prevention or treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a graph showing the statistical results of the time required for each group of mice to turn their heads to the upside-down position (T-turn).

[0029] FIG2 is a graph showing the statistical results of the time required for each group of mice to descend to the floor (T-total).

[0030] FIG3 is a graph showing the statistical results of the time required for each group of mice to cross the beam and reach the end point.

[0031] FIG4 is a graph showing the statistical results of the latency time to fall of mice in each group.

[0032] FIG5 is a graph showing the statistical results of serum corticosterone levels in mice of each group.

[0033] FIG6 is a graph showing the statistical results of the serum tumor necrosis factor TNF-α levels in each group of mice.

[0034] FIG7 is a graph showing the statistical results of the serum tumor necrosis factor IL-6 levels in each group of mice.

[0035] FIG8 is a graph showing the statistical results of the serum tumor necrosis factor IL-1β levels in each group of mice.

[0036] FIG9 is a graph showing the statistical results of the levels of superoxide dismutase (SOD), a marker of oxidative stress in the brains of mice in each group.

[0037] FIG10 is a graph showing the statistical results of the glutathione (GSH) level, a marker of brain oxidative stress, in each group of mice.

[0038] FIG11 is a diagram showing the statistical results of dopamine levels in the striatum of mice in each group.

[0039] FIG12 is a graph showing the statistical results of 5-HT levels in the striatum of mice in each group.

[0040] FIG13 is a diagram showing the statistical results of dopamine levels in the hippocampus of mice in each group.

[0041] FIG14 is a graph showing the statistical results of 5-HT levels in the hippocampus of mice in each group.

[0042] The data in Figures 1-14 were statistically analyzed using the ggplot2 function of R language (R 4.2.2). Compared with the model group and the MC group, "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001. DETAILED DESCRIPTION

[0043] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0044] The Bifidobacterium longum subsp. infantis BI03 strain involved below is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 24473, the deposit date being March 7, 2022, and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] The Bifidobacterium longum BL21 strain involved below is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 10452 and the deposit date of January 27, 2015 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] The following MRS liquid culture medium includes, by concentration, the following: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.5 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, polysorbate 80 1 mL / L, and cysteine ​​hydrochloride 0.5 g / L. Dissolve in deionized water to a total volume of 1 L, sterilize, and cool for later use.

[0047] The following method for preparing a bacterial suspension involves: inoculating the desired strain into MRS liquid culture medium, culturing at 37°C for 18 hours for activation, and continuously activating twice to obtain an activated solution; inoculating the activated solution into MRS liquid culture medium at an inoculum rate of 5% (v / v), culturing at 37°C for 24 hours to obtain a bacterial solution; centrifuging the bacterial solution at 8000g for 15 minutes, and resuspending the bacteria in PBS to obtain the obtained product.

[0048] Example 1

[0049] In this example, a strain of Bifidobacterium longum subsp. infantis was screened for improving Parkinson's disease, and the steps were as follows:

[0050] (1) Fecal samples of healthy breastfed infants were selected from Dali Bai Autonomous Prefecture, Yunnan Province. They were diluted 10-fold using 0.9% saline solution for three times and then spread on MRS solid culture medium (purchased from Haibo Biological). After incubation at 37°C for 48 h, colonies of different morphologies were picked out and purified by streaking on the surface of MRS solid culture medium. Single colonies were picked out and expanded using MRS liquid culture medium at 37°C and then preserved using 30% glycerol.

[0051] (2) In vitro physiological property tests were conducted on multiple preserved strains of bacteria, and a strain with the best gastrointestinal fluid tolerance (artificial simulation), HT-29 cell adhesion, antibacterial ability, and functional oligosaccharide utilization ability was selected, as follows:

[0052] (2.1) Gastrointestinal fluid tolerance test:

[0053] Artificial simulated gastric fluid: prepare 0.5% NaCl solution, add 0.3% pepsin, adjust the pH to 2.5 with 1 mol / L HCl, then fully dissolve and filter with a 0.22 μm microporous membrane for sterilization.

[0054] Artificial simulated intestinal fluid: prepare 0.5% NaCl solution, add 0.1% trypsin, adjust the pH to 8.0 with 0.1 mol / L NaOH, then fully dissolve and filter with a 0.22 μm microporous filter membrane for sterilization.

[0055] The selected strains were cultured anaerobically in artificial gastric or intestinal fluid for 3 hours. The digestive fluids collected at 0 and 3 hours were then assayed for viable cell counts and their survival rates were calculated. Strain survival rate (%) = B / A × 100%, where A represents the viable cell count (CFU / mL) at 0 hours and B represents the viable cell count (CFU / mL) at 3 hours.

[0056] (2.2) HT-29 cell adhesion ability test:

[0057] The concentration of digested HT-29 cells was adjusted to 1×105 / mL, 1mL was taken from each well and inoculated into a 12-well cell culture plate, incubated in a 5% CO2 incubator until the cells grew into a monolayer, washed twice with sterile PBS, one well was trypsinized, and the cells were counted using a hemocytometer; 1mL of the selected strain suspension was added to the other wells (the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL), incubated at 37°C in a 5% CO2 incubator for 2 h, washed the cells 5 times with sterile PBS to remove the non-adherent bacterial suspension, added 0.2 mL of trypsin EDTA buffer to each well to digest the cells for 5 min, and after digestion, added 0.8 mL of PBS to pipette evenly, and the bacterial solution was diluted to count the viable bacteria.

[0058] (2.3) Antibacterial ability test:

[0059] The ability to inhibit pathogenic bacteria was tested using the Oxford cup method: the pathogenic strain Escherichia coli was inoculated into a liquid beef extract peptone medium and cultured overnight at 37°C and 250 rpm in a constant temperature shaker to prepare a pathogen suspension. The MRS solid medium was cooled to about 55°C and mixed with the pathogen suspension in a certain proportion to ensure that the number of viable pathogens in the system was 10 6 CFU / mL, then quickly pour it into a plate with an Oxford cup placed in advance. After the culture medium cools and solidifies, remove the Oxford cup and inject 200μL of the test strain solution into each well. Cover the plate lightly and place it upright in a 37℃ constant temperature incubator. After incubation for an appropriate time, observe and measure the diameter of the inhibition zone with a vernier caliper. The larger the inhibition zone, the stronger the antibacterial ability.

[0060] (2.4) Functional oligosaccharide utilization ability test:

[0061] Sugar-free culture medium: 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% diammonium hydrogen citrate, 0.2% potassium phosphate (K₂HPO₄), 0.058% magnesium sulfate (MgSO₄), 0.019% manganese sulfate (MnSO₄), 0.1% polysorbate 80 (Tween-80), and 0.1% L-cysteine ​​hydrochloride. Xylo-oligosaccharides, inulin, fructo-oligosaccharides, and galacto-oligosaccharides were purchased from Quantum Hi-Tech (China) Biotechnology Co., Ltd., and xylo-oligosaccharides were purchased from Shandong Longli Biotechnology Co., Ltd.

[0062] Add 2% xylooligosaccharides, inulin, fructo-oligosaccharides, and galacto-oligosaccharides to the sugar-free medium to obtain a medium containing oligosaccharides as a carbon source. Inoculate the selected strain into the oligosaccharide-containing medium and incubate at 37°C for 12 hours. Measure the OD600 absorbance to determine the strain's ability to utilize functional oligosaccharides.

[0063] Example 2

[0064] In this example, the strains screened in Example 1 were subjected to cell morphology and physicochemical experiments as well as molecular biological identification, as follows:

[0065] (1) The cell morphology and physicochemical test results are shown in Table 1.

[0066] Table 1

[0067] (2) The results of 16S rRNA gene sequence determination are shown in SEQ ID No: 1:

[0068] SEQ ID No: 1:

[0069] (3) The results of tuf gene sequence determination are shown in SEQ ID No: 2:

[0070] SEQ ID No: 2:

[0071] The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the results showed that the strain was indeed Bifidobacterium longum subsp. infantis.

[0072] Example 3

[0073] This example verifies the effects of Bifidobacterium longum subsp. infantis BI03 on serum corticosterone, brain oxidative stress markers, motor behavior, brain neurotransmitter levels, and neuroinflammation in Parkinson's disease model mice. The steps are as follows:

[0074] (1) Experimental animals: 48 16-week-old adult male SAMP8 mice (22±2 g) were adaptively raised with normal feed in an animal room (room temperature: 23-25°C, humidity: 50-70%, lighting 10±0.5 h) for one week (with free access to food and water).

[0075] (2) Grouping: After adaptive feeding, the mice were randomly divided into 6 groups: control group (CTL group), model group (MC group), BI03 group, BL21 group, BI03 and BL21 combination group (combination group 1), and ATCC 15697 and BL21 combination group (combination group 2), with 8 mice in each group.

[0076] (3) Intervention methods:

[0077] Except for the control group, the mice in other groups were given MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 30 mg / kg, Sigma-Aldrich) by gavage for 4 consecutive weeks; the probiotic intervention groups (BI03 group, BL21 group, combination group 1, and combination group 2) were given 1×109 The cells were gavaged with 100 μL of bacterial solution (the ratio of viable bacteria counts of BI03 and BL21 in combination group 1 was 3:1, and the ratio of viable bacteria counts of ATCC 15697 and BL21 in combination group 2 was 3:1). The control group was gavaged with 200 μL of normal saline per day for 4 consecutive weeks.

[0078] (4) Impact on motor behavior ability:

[0079] After the intervention, the mice's motor ability was assessed by three behavioral tests: the pole test (PT), the narrow-beam test (NBT), and the rotarod test (RTR). All mice received behavioral training once a day from day 20 to day 22, and behavioral testing was performed on day 29 (24 hours after the last MPTP injection). Both PT and NBT were video recorded, and the video file names were converted to random digital codes and analyzed by three researchers who were blind to the treatment status. The specific operations were carried out using conventional experimental methods in this field:

[0080] (4.1) Pole test (for assessing agility and sensorimotor impairment): Prepare a vertically placed metal pole, approximately 50 cm long and 1 cm in diameter. On each training day, the mouse was first placed in a pole cage to familiarize itself with the environment. The mouse was then placed head-up on a pole 15 cm from the cage bottom three times, followed by three training sessions on poles 30 cm and 50 cm above the cage floor. On the test day, each mouse was placed head-up on the top of the pole, and the time required to turn its head to the head-down position (T-turn) and the time required to descend to the floor (T-total) were recorded. If the mouse fell or jumped off the pole within 60 seconds, the test was repeated. If the mouse could not turn its head down or kept falling off the pole, the T-turn and T-total times were recorded as 60 seconds. Each mouse was tested three times (with a 1-minute interval between tests), and the average time was taken. The results are shown in Figures 1 and 2.

[0081] (4.2) Narrow beam test (assessment of motor coordination and balance): Prepare a thin wooden bar, 50 cm long and 0.8 cm wide. The beam is mounted on two poles and is 50 cm away from the table. A black box is placed at one end of the beam as the end point. Mouse chow is placed in the black box to attract mice. On each training day, the mouse is placed in the black box for 5 minutes to familiarize itself with the environment, and then placed on a beam 5 cm away from the box beam and trained to walk towards the box three times. Subsequently, 15 cm, 30 cm and 50 cm balance beam walking training is performed. On the test day, each mouse is tested three times (with a 1 min interval between tests). The time required to cross the beam and enter the end point is recorded until completion or a maximum of 60 seconds. If the mouse falls halfway or turns back to the starting point, the test is repeated. If the mouse cannot reach the end point within 60 seconds or keeps falling off the beam, the total time is recorded as 60 seconds. The average time of the three trials is taken. The results are shown in Figure 3.

[0082] (4.3) Rotarod test (assessment of motor coordination): An RT-01 mouse rotating rod with an automatic timer and a fall sensor was used during training and formal testing. All mice were first trained at a rotation speed of 10 rpm for 180 s × 3 times on the 20th day, followed by training at a rotation speed of 20 rpm on the 21st day and training at a rotation speed of 30 rpm on the 22nd day. On the test day, each mouse was tested at a rotation speed of 30 rpm for 180 s × 3 times (with a test interval of 1 min), the latency time to fall was recorded, and the average time was taken. The results are shown in Figure 4.

[0083] As shown in Figures 1, 2, 3, and 4, compared to the CTL group, mice in the MC group showed significantly increased reversal and total descent time on the PT, indicating that Parkinson's mice exhibit motor impairments and balance problems, resulting in a prolonged time spent descending the pole. Total walking time on the NBT was significantly increased, indicating that Parkinson's mice exhibit an unstable and uncoordinated gait and are prone to errors. Furthermore, dwell time on the RTR was significantly decreased, indicating that Parkinson's mice exhibit balance impairments and lack of coordination, making it difficult for them to maintain balance on the rotating cylinder. Treatment with the BI03 strain significantly reduced motor deficits on the PT, NBT, and RTR in Parkinson's mice, indicating that BI03 treatment can alleviate motor deficits in Parkinson's mice. The effects were particularly pronounced when the probiotic BI03 was combined with BL21 (Note: ***p<0.001, **p<0.01, *p<0.05 compared with the model group).

[0084] After behavioral testing on day 29, all mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate (2 mL / kg) and subjected to cervical dislocation, and their brains and blood were collected.

[0085] (5) Serum corticosterone test:

[0086] The serum corticosterone concentration of mice in each group was measured using a commercial CORT EIAkit (Cat. No.: K014-H5W, Arbor Assays) and analyzed according to the manufacturer's instructions. The results are shown in Figure 5. As can be seen from the figure, compared with the CTL group, the serum corticosterone content of mice in the MC group was significantly increased, which may be related to the inflammatory response of Parkinson's disease. Inflammation is an important feature of the Parkinson's disease process and may be related to the damage of nerve cells and the release of inflammatory mediators. These inflammatory mediators can stimulate the adrenal glands to secrete more corticosterone. Under the intervention of probiotics, the serum corticosterone content of each group decreased, among which the reversal degree was the most obvious when BI03 and BL21 acted together (Note: Compared with the model group, ***p<0.001, **p<0.01, *p<0.05).

[0087] (6) Detection of serum tumor necrosis factor TNF-α, IL-1β and IL-6:

[0088] ELISA kits (Shanghai ELISA Biotechnology Co., Ltd.) were used to measure serum tumor necrosis factor (TNF)-α, IL-1β, and IL-6 levels in each group of mice. The results are shown in Figures 6, 7, and 8. As shown, compared with the CTL group, Parkinson's mice had increased levels of TNF-α, IL-1β, and IL-6 in the striatum. Administration of the BI03 strain successfully alleviated the elevated levels of TNF-α, IL-1β, and IL-6 in the striatum of Parkinson's mice. The combined effect of BI03 and BL21 was even more pronounced. This suggests that probiotics may help improve symptoms in Parkinson's mice by reducing proinflammatory cytokine levels in the striatum and alleviating neuronal damage and degeneration caused by inflammation (Note: ***p<0.001, **p<0.01, *p<0.05 compared with the model group).

[0089] (7) Detection of brain oxidative stress markers:

[0090] Based on antioxidant activity, superoxide dismutase (SOD) and glutathione (GSH) levels were measured using a test kit. Brain tissue (striatum) from each group of mice was ultrasonically treated in PBS buffer. After centrifugation, the supernatant was collected and analyzed using a test kit according to the kit's instructions. The antioxidant levels in the mouse brain tissue were estimated based on standard antioxidant activity. The results are shown in Figures 9 and 10. As can be seen from the figures, by measuring the antioxidant activity levels in the striatum of mice, we found that the levels and activity of SOD and GSH in the striatum of mice in the MC group were significantly reduced compared to those in the CTL group. SOD and GSH are important antioxidant substances in cells that play a key role in protecting cells from oxidative stress. Neuronal cells in the striatum of Parkinson's disease mice may be damaged, leading to cell dysfunction and death, thereby triggering an inflammatory response and promoting the release of inflammatory mediators. The inflammatory response itself can lead to increased consumption of SOD and GSH, resulting in a decrease in their levels. However, probiotic intervention can effectively reverse this trend, especially in the BI03 group. The BI03 combined with BL21 group showed the most significant reversal (Note: compared with the model group, ***p<0.001, **p<0.01, *p<0.05).

[0091] (8) Detection of brain neurotransmitter levels:

[0092] Striatal and hippocampal samples were collected from both sides of each mouse group, homogenized, and centrifuged at 12,000 g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm membrane, and the filtrate was used for HPLC-MS analysis. For HPLC analysis, a Thermo Vanquish ultra-high performance liquid chromatography system from Thermo Fisher Scientific was used, along with a Waters ACQUITY UPLC HSST3 column (2.1 × 150 mm, 1.8 μm). The flow rate was set at 0.25 mL / min, the column temperature was 40°C, and the injection volume was 2 μL. For mass spectrometry analysis, a Thermo Q Exactive Focus mass spectrometer from Thermo Fisher Scientific was used, and data were acquired using an electrospray ionization (ESI) source in both positive and negative ion modes.

[0093] Parkinson's disease is a neurodegenerative disorder characterized by decreased levels of dopamine and 5-HT in the bilateral striatum and hippocampus. Figures 11, 12, 13, and 14 show that compared with the CTL group, the MC group showed decreased levels of 5-HT and dopamine in the bilateral striatum and hippocampus. 5-HT is a neurotransmitter that regulates mood and cognitive function. Parkinson's disease is often accompanied by decreased 5-HT levels, leading to emotional and cognitive problems. Dopamine is an important neurotransmitter crucial for motor regulation. Loss of dopamine neurons in Parkinson's mice leads to decreased dopamine levels, which in turn causes symptoms such as movement disorders. Probiotic treatment restored neurotransmitter levels. The increase in dopamine and 5-HT levels was more significant in the BI03 and BL21 combination group (Note: ***p<0.001, **p<0.01, *p<0.05 compared with the model group).

[0094] The applicant declares that this application uses the above-mentioned examples to illustrate the present application's Bifidobacterium longum subsp. infantis for ameliorating Parkinson's disease and its uses, but this application is not limited to the above-mentioned examples, that is, it does not mean that this application must rely on the above-mentioned examples for implementation. Those skilled in the art should understand that any improvements to this application, equivalent replacements for various raw materials in the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of this application.

[0095] The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

[0096] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

Claims

1. A Bifidobacterium longum subsp. infantis strain for improving Parkinson's disease, named as Bifidobacterium longum subsp. infantis BI03 strain, with a preservation number of CGMCC No. 24473 and a preservation date of March 7, 2022.

2. A bacterial agent for preventing, improving or treating Parkinson's disease, wherein: The strain in the bacterial agent includes the Bifidobacterium longum subsp. infantis BI03 strain according to claim 1.

3. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 2, wherein: The viable count of the Bifidobacterium longum subsp. infantis BI03 strain is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

4. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 2, wherein: The strains also include Bifidobacterium longum BL21 strain, with a preservation number of CGMCC No.10452 and a preservation date of January 27, 2015.

5. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 4, wherein The ratio of the number of live bacteria of the Bifidobacterium longum subsp. infantis BI03 strain to the number of live bacteria of the Bifidobacterium longum BL21 strain is (2-4):

1.

6. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 2, wherein: The dosage form of the bacterial agent includes freeze-dried powder, capsule, tablet or granule.

7. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 2, wherein: The bacterial agent also includes a protective agent and / or an auxiliary additive.

8. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 7, wherein: The protective agent is selected from any one of skim milk, gelatin, maltodextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol, or a combination of at least two thereof.

9. The bacterial agent for preventing, improving or treating Parkinson's disease according to claim 7, wherein: The auxiliary additive is selected from any one of resistant dextrin, fructooligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, α-lactalbumin or lactoferrin, or a combination of at least two thereof.

10. Use of the Bifidobacterium longum subsp. infantis BI03 strain according to claim 1 or the bacterial agent according to any one of claims 2 to 9 in the preparation of a medicine or health product having the efficacy of preventing, improving or treating Parkinson's disease.

Images