Pediococcus pentosaceus strain

Abstract

The present application belongs to the field of life science, and particularly relates to a functional probiotic strain Pediococcus pentosaceus. The Pediococcus pentosaceus WMU002 strain of the present application is derived from human body, and is high in safety after screening, domestication and verification. The Pediococcus pentosaceus WMU002 strain selected by the present application can obviously improve the motor disorder caused by Parkinson's disease and improve the behavior ability, overcome the defects and deficiencies of the prior art, and creatively apply the Pediococcus pentosaceus to the prevention and treatment of Parkinson's disease, and further develop a probiotic preparation for preventing or treating Parkinson's disease. The Pediococcus pentosaceus preparation composition of the present application can be used as a drug for preventing and treating Parkinson's disease, has no toxic side effects, and can be used for the auxiliary treatment of clinical Parkinson's disease patients. The present application has good social and economic benefits, and is worth popularization and application.

Description

Technical Field

[0001] This invention relates to novel uses of Pediococcus pentosaceus, specifically its application in the preparation of compositions for the prevention and treatment of Parkinson's disease, belonging to the field of biopharmaceuticals. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting more than 6.1 million people worldwide. According to the World Health Organization, globally, the rate of disability and death caused by PD is increasing faster than any other neurological disorder. The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the abnormal accumulation of α-synuclein in the neuronal contents. In addition to the main motor features (resting tremor, rigidity, and postural instability), PD is associated with a wide range of nonmotor symptoms, including hyposmia, sleep disturbances, depression, constipation, and other autonomic dysfunction symptoms, all of which negatively impact quality of life. PD is a multifactorial, progressive disease with an unknown cause; risk factors such as age, genetics, and environmental factors all play a role in its development. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that maintains intracellular redox homeostasis and protects neurons from oxidative damage. Mounting evidence suggests that GABA concentrations are reduced in Parkinson's disease, and that GABA levels in the cerebral cortex are negatively correlated with the severity of Parkinson's symptoms (GABAergic changes in the thalamocortical circuit in Parkinson's disease. HumBrain Mapp (2020) 41(4): 1017-29.). Therefore, current treatments only address the symptoms and cannot halt the degenerative process. The probiotic preparation of this invention partially solves this medical problem, effectively preventing and treating Parkinson's disease without side effects, and has significant social implications.

[0003] Probiotics are a class of active beneficial microorganisms that are beneficial to the host, reside in the human gut and other parts of the body, and can produce definite health effects, thereby improving the host's microecological balance and playing a beneficial role. Probiotics are live bacteria that are beneficial to health when ingested in sufficient quantities. Gut microorganisms are believed to play a role in many mental illnesses. There is a two-way communication pathway between the gut and the brain, called the gut-brain axis, which may become a potential therapeutic target. Pediococcus pentosaceus belongs to the genus Pediococcus of the family Streptococcus and is a Gram-positive bacterium. The antibacterial mechanism of Pediococcus pentosaceus mainly includes: (1) secreting bacteriocins to directly kill pathogenic bacteria; (2) secreting organic acids that penetrate into the cell membrane of pathogenic bacteria, lowering the intracellular pH and inhibiting metabolism. At the same time, the decrease in pH can inhibit the expression of pathogenic bacteria virulence factor genes; (3) inhibiting the adhesion of pathogenic bacteria by competing with pathogenic bacteria for intestinal epithelial cell adhesion sites; (4) agglutinating with pathogenic bacteria, preventing them from playing a pathogenic role. Currently, the probiotic function of Pediococcus pentosaceus and its application in the processing of fermented meat products, fermented dairy products, and fermented vegetables are rarely isolated from the human body, and there is no evidence of its application in the prevention and treatment of Parkinson's disease. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects and shortcomings of existing technologies, creatively apply Pediococcus pentosaceus in the prevention and treatment of Parkinson's disease, and further develop probiotic preparations for the prevention and treatment of Parkinson's disease. The objective of this invention is to provide a safe and effective probiotic strain, probiotic preparation, and preparation method for the prevention and treatment of Parkinson's disease.

[0005] This invention provides a Pediococcus pentosaceus strain WMU002 that can prevent and treat Parkinson's disease.

[0006] The Pediococcus pentosaceus strain of this invention is WMU002, which was deposited on May 12, 2022, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101), and classified as Pediococcus pentosaceus, with accession number CGMCC No. 24884.

[0007] The Pediococcus pentosaceus strain WMU002 of this invention has the following biological characteristics:

[0008] (1) Colony morphology: Milky white round colonies with smooth, raised surfaces, neat edges, and opaque;

[0009] (2) Individual morphology: Spherical, mostly in two or four tufts, Gram positive;

[0010] Optimal growth conditions: It grows well under facultative anaerobic conditions, with an optimal growth temperature of 35–40℃; a minimum growth temperature of 25–28℃; a maximum growth temperature of 43–45℃; and an optimal growth pH of 6.5–7.0. It does not grow at pH 4.5–5.0 or 8.0–8.5.

[0011] Another object of the present invention is to provide a new use for Pediococcus pentosaceus strain WMU002, namely, the use of Pediococcus pentosaceus strain WMU002 in the preparation of probiotic formulations for the prevention and treatment of Parkinson's disease, which can be pharmaceuticals, health products and beverages, etc.

[0012] This invention uses an effective dose of Pediococcus pentosaceus strain WMU002 as the active pharmaceutical ingredient, and adds conventional excipients, nutrients and other excipients according to a certain formulation process to make any probiotic preparation suitable for the treatment and prevention of Parkinson's disease. The dosage form used clinically can be granules, powders, oral liquids, enemas, etc.

[0013] The effective dose referred to in this invention means that the total number of live bacteria in a solid live bacterial preparation made with Pediococcus pentosaceus as an active ingredient, alone or in combination as described above, shall not be less than 1 × 10⁻⁶. 7 CFU / g.

[0014] The *Pediococcus pentosaceus* mentioned in this invention refers to the live cell form of *Pediococcus pentosaceus* strain WMU002, and the total number of live bacteria in the probiotic preparation composition shall not be less than 1 × 10⁻⁶. 7 CFU / g is generally around 1×10 9 CFU / g or higher, up to 1×10 12 CFU / g or 1×10 12 CFU / g or higher.

[0015] The preferred formulation process of this invention is as follows: Pediococcus pentosaceus formulation preparation: Pediococcus pentosaceus strain WMU002 seed culture is inoculated at a 1% (by weight) into a sterilized liquid culture medium containing 1% (by weight) peptone, 1% beef extract, 0.5% yeast extract, 2% glucose, 0.2% K₂HPO₄, 0.05% MgSO₄·7H₂O, 0.02% MnSO₄·4H₂O, 0.3% fructooligosaccharides, and 0.2% diammonium citrate. After fermentation at 36℃–38℃, the bacterial cells are separated by centrifugation. The bacterial cells are then added to a freeze-drying protective solution containing 5% (by weight) sodium glutamate and 5% high-fat milk powder. After mixing and freeze-drying, live Pediococcus pentosaceus bacterial powder is obtained. The dried bacterial powder is mixed with a pharmaceutical carrier to obtain the final dosage form probiotic composition. The formulation process is not limited to that described in this invention.

[0016] Since this invention discloses for the first time the application of Pediococcus pentosaceus WMU002 strain as a pharmaceutical active ingredient in the preparation of drugs for the treatment and prevention of Parkinson's disease, a pharmaceutical preparation is made by combining Pediococcus pentosaceus WMU002 strain as an active ingredient with excipients.

[0017] An effective dose refers to a solid live bacterial preparation containing Pediococcus pentosaceus as an active ingredient, alone or in combination as described above, with a total live bacterial count not less than 1 × 10⁻⁶. 7 CFU / g.

[0018] The total viable count of *Pediococcus pentosaceus* in the *Pediococcus pentosaceus* preparation composition of the present invention shall not be less than 1 × 10⁻⁶. 7 CFU / g is generally around 1×10 9 CFU / g or higher, up to 1×10 11 CFU / g or 1×10 11 CFU / g or higher.

[0019] This invention uses Pediococcus pentosaceus strain WMU002 to prevent and treat Parkinson's disease. The effect is significant, and the effect increases with the increase of dosage, and no toxic side effects have been found.

[0020] Advantages and effects of the present invention:

[0021] This invention discloses for the first time the use of Pediococcus pentosaceus strain WMU002 in the prevention and treatment of Parkinson's disease, and provides a method for preparing a Pediococcus pentosaceus preparation composition. The Pediococcus pentosaceus strain WMU002 selected in this invention is derived from humans and has been screened, domesticated, and verified, demonstrating high safety. This invention can significantly improve motor impairments caused by Parkinson's disease and improve behavioral abilities. The metabolites of Pediococcus pentosaceus strain WMU002 include active substances such as γ-aminobutyric acid (GABA), which can exert anti-Parkinson's disease effects in the brain. Therefore, the Pediococcus pentosaceus preparation composition of this invention can be used as a drug for the prevention and treatment of Parkinson's disease, with no toxic side effects, and can be used as an adjunct treatment for clinical Parkinson's disease patients. Attached Figure Description

[0022] Figure 1 The time to cross the balance beam in the balance beam experiment. Time to cross beam(s): time to cross the balance beam; Con: normal control group; MPTP: Parkinson's disease model group; MPTP+PP: Pediococcus pentosaceus WMU002 strain treatment group; ** Compared with the normal control group, P < 0.01; ## Compared with the Parkinson's disease model group, P < 0.01.

[0023] Figure 2 Number of foot slips in the balance beam experiment. Con: normal control group; MPTP: Parkinson's disease model group; MPTP+PP: Pediococcus pentosaceus WMU002 strain treatment group; ** Compared with the normal control group, P < 0.01; ## Compared with the Parkinson's disease model group, P < 0.01.

[0024] Figure 3 Latency time(s): Latency period; Con: Normal control group; MPTP: Parkinson's disease model group; MPTP+PP: Pediococcus pentosaceus WMU002 strain treatment group; * Compared with the normal control group, P < 0.05; ## Compared with the Parkinson's disease model group, P < 0.01.

[0025] Figure 4Score of crawling state in the pole climbing experiment. Con: normal control group; MPTP: Parkinson's disease model group; MPTP+PP: Pediococcus pentosaceus WMU002 strain treatment group; * Compared with the normal control group, P < 0.05; ## Compared with the Parkinson's disease model group, P < 0.01.

[0026] Figure 5 Nissl staining results. Con: normal control group; MPTP: Parkinson's disease model group; MPTP+PP: Pediococcus pentosaceus WMU002 strain treatment group. Detailed Implementation

[0027] The preparation of Pediococcus pentosacchari preparations involves first preparing Pediococcus pentosacchari powder, and then adding appropriate excipients as needed to form the corresponding dosage form. This invention takes the preparation of oral Pediococcus pentosacchari live bacteria powder as an example, and the specific preparation dosage form is not limited to the method described below.

[0028] Example 1: Isolation and Identification of Pediococcus pentosaceus strain WMU002

[0029] Using the feces of a healthy young adult as the isolation sample, the Pediococcus pentosaceus strain WMU002 was obtained by spreading and culturing on modified MRS agar medium at 37°C for 48 hours.

[0030] MRS medium formula: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g K2HPO4, 0.5g MgSO4·7H2O, 0.2g MnSO4·4H2O, 3g fructooligosaccharides, 2g diammonium citrate, 15g agar, 1L distilled water, adjust pH to 7.0, sterilize at 115℃ for 15 minutes.

[0031] The Pediococcus pentosaceus strain WMU002 of this invention has the following microbiological characteristics:

[0032] (1) Colony morphology: Milky white round colonies with smooth, raised surfaces, neat edges, and opaque;

[0033] (2) Individual morphology: Spherical, mostly in two or four tufts, Gram positive;

[0034] Optimal conditions: It grows well under facultative anaerobic conditions, with an optimal growth temperature of 35–40℃; a minimum growth temperature of 25–28℃; a maximum growth temperature of 43–45℃; and an optimal growth pH of 6.5–7.0. It does not grow at pH 4.5–5.0 or 8.0–8.5.

[0035] Table 1. Biochemical reactions of Pediococcus pentosaceus strain WMU002

[0036]

[0037] The concentration of GABA in the culture supernatant of Pediococcus pentosaceus strain WMU002 was determined by ELISA. The results showed that the concentration of GABA in the bacterial culture supernatant reached its peak at 48 h, with a range of 3-4 μmol / L.

[0038] The Pediococcus pentosaceus strain WMU002 of this invention was deposited on May 12, 2022, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), with accession number CGMCCNo. 24884.

[0039] Example 2: Preparation of a Pediococcus pentosaceus WMU002 probiotic formulation composition

[0040] 1. Seed culture preparation: Pick a single colony of Pediococcus pentosaceus WMU002 and inoculate it into 50 mL of MRS liquid medium. The pH is 7.0±0.5. Incubate at 37℃ for 24 h to obtain the seed culture. The formula of MRS medium is as follows: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 5 g sodium acetate, 2 g K2HPO4, 0.5 g MgSO4·7H2O, 0.2 g MnSO4·4H2O, 3 g fructooligosaccharide, 2 g diammonium citrate, 1 L distilled water. Adjust the pH to 7.0 and sterilize at 115℃ for 15 min.

[0041] 2. Fermentation culture: Take the seed liquid from step 1 and inoculate it into a new modified MRS medium at an inoculation rate of 1%. Incubate anaerobically at 37°C for 24 hours. After the culture is completed, remove the fermentation supernatant and centrifuge to separate the bacterial cells.

[0042] 3. Preparation of bacterial powder: The bacterial cells collected in step 2 are mixed with a freeze-drying protective solution containing 5% monosodium glutamate and 5% high-fat milk powder by weight, and then freeze-dried to obtain Pediococcus pentosaceus bacterial powder with a viable count of not less than 1×10⁻⁶.7 CFU / g or 1×10 7 CFU / mL.

[0043] Example 3: Animal experiments on the treatment of Parkinson's disease

[0044] Objective: To observe the neuroprotective effect of the Pentosacchariformis WMU002 preparation of the present invention on a mouse model of Parkinson's disease.

[0045] I. Materials and Methods:

[0046] 1. Drug: Pediococcus pentosaceus WMU002 bacterial powder, containing 1×10⁻⁶ live bacteria. 9 For details on the preparation method of CFU / g, please refer to Example 2.

[0047] 2. Experimental animals: Male C57BL / 6J mice, weighing 18-22 grams.

[0048] 3. Preparation of Parkinson's Disease Mouse Model and Animal Grouping: Mice were intraperitoneally injected with 25 mg / kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) once daily for one week. MPTP+PP group: After MPTP treatment, mice were given 0.2 mL of 1×10⁻⁶ MPTP solution daily. 9 Mice were treated by gavage with a CFU / mL suspension of Pediococcus pentosaceus WMU002 for 4 weeks. Mice in the Con and MPTP groups were treated with the same volume of physiological saline. After the experiment, the mice's motor behavior was tested. Following the behavioral tests, mouse brain tissue was perfused and fixed, and Nissl staining was performed for pathological examination. Separately, brain tissue was collected for ELISA to determine the GABA content.

[0049] 4. Testing of balance and motor coordination

[0050] A) Balance Beam Test: Used to test the balance ability and motor coordination of mice. The balance beam is 0.8m long and 14mm wide. Each mouse is placed at one end of the balance beam and guided to climb into a black box for 2 days. On the third day, the mice are placed horizontally on the beam and are allowed to cross it within 5 minutes. The number of times the feet slip (front paws or hind paws sliding on the smooth surface of the beam) and the time taken for the mouse to cross are recorded.

[0051] B) Rotating bar test: Used to test the coordinated movement ability of mice. Before the experiment, mice were trained once a day for 3 days. On the 4th day, the mice were placed on an accelerating rotating cylinder. The speed of the cylinder was increased slowly to 40 revolutions within 5 minutes. After the test, the mice were rotated 2 times continuously without attempting to walk, and the latency of the mice was measured.

[0052] C) Pole Climbing Test: Used to assess the coordination ability of mice. A pole 40 cm long and 1.5 cm in diameter was fixed to a base and wrapped with non-stick gauze for easy gripping by the mice. Before the test, the mice were trained to ensure they could climb down the pole; at the end of the test, both of the mice's hind legs reached the bottom. The total time to climb down the pole and the mouse's state during the climb were measured. Three consecutive tests were performed on each mouse, and the average value was calculated for statistical analysis. The scoring criteria for the mouse's pole climbing state are as follows: 0: Climbs down the pole smoothly with all four limbs; 0.5: Climbs down in a spiral motion step by step, but the hind legs slip; 1.0: Stops several times to climb down, but grips the pole tightly; 1.5: Slips on the pole and falls; 2.0: Unable to grip the pole and falls directly.

[0053] 5. Nissl staining: Mice were fixed in formaldehyde and embedded in paraffin, then cut into 5μm sections. The sections were dewaxed with xylene and rehydrated with a gradient of ethanol. The sections were then stained with 1% tar violet dye for 30 minutes, washed with distilled water, separated with 70% ethanol, dehydrated with a gradient of ethanol, and finally fixed in xylene and sealed with neutral resin for observation under a microscope.

[0054] 6. ELISA determination of GABA content in brain tissue: The content of GABA in brain tissue was determined by ELISA. Brain tissue and lysis buffer were mixed at a ratio of 1:9, PMSF was added, and the mixture was homogenized. The supernatant was collected. The absorbance (OD value) of each sample at 450 nm was measured by a reader.

[0055] II. Experimental Results:

[0056] 1. Pediococcus pentosaceus WMU002 can inhibit MPTP-induced motor dysfunction.

[0057] In the balance beam experiment, compared with the Con group, the MPTP group had a significantly increased time to cross the balance beam and a significantly increased number of slips, while these conditions were reversed after PP treatment. Figure 1 and Figure 2 In the rotator bar experiment, the latency of the MPTP group was significantly shorter than that of the Con group. Figure 3 The latency period was longer in the MPTP+PP group than in the MPTP group. Figure 3 In the pole climbing experiment, compared with the Con group, the MPTP group showed a significant increase in climbing performance scores, while the MPTP+PP group showed a significant decrease in scores compared with the MPTP group. Figure 4 These results indicate that Pediococcus pentosaceus WMU002 can improve MPTP-induced motor dysfunction.

[0058] 2. Pediococcus pentosaceus WMU002 can improve MPTP-induced neuropathological damage ( Figure 5 ).

[0059] 3. Treatment with Pediococcus pentosaceus WMU002 reversed the MPTP-induced decrease in GABA levels.

[0060] We measured the concentration of GABA in the mouse brain. The results showed that the mean GABA concentration in the Con group was 9.112±0.4820, the mean GABA concentration in the MPTP group was 7.353±0.4839, and the GABA content after treatment was 9.166±0.3098, indicating that Pediococcus pentosaceus WMU002 can alleviate the decrease in GABA in the brain of Parkinson's disease model mice.

[0061] in conclusion:

[0062] This invention creatively targets the gut microbiota as a therapeutic target, pioneering a new model for the prevention and treatment of Parkinson's disease. It has developed a highly effective drug for the prevention and treatment of Parkinson's disease with no toxic side effects. Animal experiments have demonstrated that the Pediococcus pentosaceus WMU002 preparation can significantly improve the motor behavior of Parkinson's disease mice and reduce nerve damage. This invention uses the Pediococcus pentosaceus WMU002 strain to prevent and treat Parkinson's disease, achieving significant efficacy without any toxic side effects. It can be used as a nutritional supplement in conjunction with other nutrients for the adjunctive treatment of clinical patients.

Claims

1. A Pediococcus pentosaceus strain WMU002, preservation number: CGMCCNo.24884.

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