CN116083284A - Probiotics Pediococcus pentosaceus strain - Google Patents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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Abstract
The invention belongs to the field of life science, and particularly relates to a functional probiotic bacterial strain pediococcus pentosaceus. The pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain is derived from a human body, and has high safety through screening, domestication and verification. The pediococcus pentosaceus WMU002 strain selected by the invention can obviously improve dyskinesia caused by parkinsonism, improve behavior ability, overcome the defects and the shortcomings of the prior art, creatively apply the pediococcus pentosaceus in preventing and treating parkinsonism, and further develop a probiotic preparation for preventing or treating parkinsonism; the pediococcus pentosaceus preparation composition can be used as a medicament for preventing and treating parkinsonism, has no toxic or side effect, and can be used for adjuvant therapy of patients suffering from clinical parkinsonism. The invention has good social and economic benefits and is worthy of popularization and application.
Description
Technical Field
The invention relates to a new application of Pediococcus pentosaceus, in particular to an application of Pediococcus pentosaceus in preparing a composition for preventing and treating Parkinson's disease, and belongs to the field of biological pharmacy.
Background
Parkinson's disease is the second most common neurodegenerative disease affecting more than ten thousand people worldwide, and according to information from the world health organization, the rate of increase of disability and death caused by parkinson's disease is faster than any other neurological disease worldwide. The pathological features of parkinson's disease are loss of dopaminergic substantia nigra neurons and abnormal accumulation of alpha-synuclein in the neuronal content. In addition to the major motor features (resting tremor, stiffness, and postural instability), parkinson's disease is associated with a wide range of non-motor symptoms, including hyposmia, sleep disorders, depression, constipation, and other autonomic dysfunction symptoms, all of which negatively impact quality of life. Parkinson's disease is a multifactorial progressive disease with unknown etiology; risk factors such as age, genetic and environmental factors all play a role in the pathogenesis. Gamma aminobutyric acid (GABA) is an inhibitory neurotransmitter that maintains intracellular redox homeostasis, protecting neurons from oxidative damage. There is growing evidence that in parkinson's disease, GABA concentrations decrease and GABA levels in the cerebral cortex are inversely related to the severity of parkinsonian symptoms (GABAergic changes in the thalamocortical circuit in Parkinson's disease. Hum Brain map (2020) 41 (4): 1017-29.). Thus, current symptomatic treatments alone do not prevent the progression of degeneration. The probiotic preparation of the invention partially solves the medical problem, can effectively prevent and treat the parkinsonism, has no side effect and has great social significance.
Probiotics are a general term for active beneficial microorganisms which are beneficial to a host, colonize the intestinal tract of a human body and the like, and can generate definite health effects so as to improve the micro-ecological balance of the host and exert beneficial effects. Probiotics are living bacteria that when ingested in sufficient amounts would be beneficial to health. Intestinal microorganisms are thought to play a role in many mental diseases, there is a bi-directional communication pathway between the intestine and brain, termed the gut-brain axis, and may be potential therapeutic targets. Pediococcus pentosaceus belongs to Pediococcus of Streptococcaceae, and is gram positive bacteria, and the antibacterial action mechanism of Pediococcus pentosaceus mainly comprises: (1) secretion of bacteriocin to directly kill pathogenic bacteria; (2) The secreted organic acid permeates the pathogenic bacteria cell membrane to reduce intracellular pH to inhibit metabolism, and the pH reduction can inhibit the expression of pathogenic bacteria virulence factor genes; (3) Inhibiting pathogenic bacteria adhesion by competing with pathogenic bacteria for intestinal epithelial cell adhesion sites; (4) By agglomerating with the pathogen, it is rendered incapable of pathogenic action. At present, the probiotic function of Pediococcus pentosaceus and the application thereof in the processing of fermented meat products, fermented dairy products, fermented vegetables and other foods are rarely separated from human bodies, and the application of Pediococcus pentosaceus in preventing and treating Parkinson's disease is not seen.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art, creatively applies Pediococcus pentosaceus in preventing and treating Parkinson's disease, and further develops a probiotic preparation for preventing and treating Parkinson's disease. The invention aims to provide a safe and effective probiotic bacterial strain for preventing and treating parkinsonism, a probiotic preparation and a preparation method.
The invention provides a WMU002 strain with Pediococcus pentosaceus (Pediococcus pentosaceus) for preventing and treating Parkinson's disease.
The pediococcus pentosaceus is a pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain which has been preserved in China general microbiological culture Collection center (CGMCC) (address: north Xielu No. 1, 3, china academy of sciences microbiological study, post code 100101) for 5 months 12 days 2022, and the classification name is pediococcus pentosaceus (Pediococcus pentosaceus), and the preservation number is CGMCC No.24884.
The pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain of the invention has the following biological characteristics:
(1) Colony morphology: the milky white circular colony has smooth and convex surface, neat edge and opaqueness;
(2) Individual morphology: spherical, most of which are in a duplex or quadruple shape and are gram positive;
optimal culture conditions: the growth is good under facultative anaerobic condition, and the optimal growth temperature is 35-40 ℃; the minimum growth temperature is 25-28 ℃; 43-45 ℃ at the highest; the optimal pH for growth is 6.5-7.0; the pH is 4.5-5.0 or 8.0-8.5 without growth.
Another object of the present invention is to provide a new use of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain, namely, an application of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain in preparing probiotic preparation composition for preventing and treating parkinson's disease, wherein the composition can be medicines, health products, drinks, etc.
The invention takes an effective dose of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain as a medicine active ingredient, and adds conventional excipients, nutritional agents and other auxiliary materials according to a certain preparation process to prepare any one of probiotic preparations suitable for treating and preventing the Parkinson disease, and clinically used dosage forms can be granules, powder, oral liquid, enemas and the like.
The effective dose refers to that the solid viable bacteria preparation prepared by taking Pediococcus pentosaceus alone or in combination as an active ingredient contains no less than 1X 10 total viable bacteria 7 CFU/g。
The pediococcus pentosaceus refers to the form of living cells of a WMU002 strain of pediococcus pentosaceus (Pediococcus pentosaceus), and the total number of living bacteria contained in the probiotic preparation composition cannot be less than 1 multiplied by 10 7 CFU/g, typically 1X 10 9 CFU/g, the maximum can reach 1×10 12 CFU/g or 1X 10 12 CFU/g or more.
The preparation process of the invention comprises the following steps of: inoculating pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain seed solution with 1% by weight of inoculating amount containing 1% by weight of peptone, 1% by weight of beef extract, O.5% by weight of yeast extract, 2% by weight of glucose, and K 2 HPO 4 0.2％，MgSO 4 ·7H 2 O 0.05％，MnSO 4 ·4H 2 O0.02%, fructo-oligosaccharide 0.3%, diammonium citrate 0.2% liquid culture medium, fermenting at 36-38deg.C, centrifuging to separate thallus, adding thallus into lyophilized protective liquid containing sodium glutamate 5% and milk powder 5% by weight, mixing, lyophilizing to obtain live Pediococcus pentosaceus bacterial powder, mixing the dried bacterial powder with medicinal carrier to obtain the final probiotic composition. The formulation process is not limited to the present invention.
The invention discloses an application of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain serving as a medicine active ingredient in preparing medicines for treating and preventing parkinsonism for the first time, so that the pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain serving as an active ingredient is combined with auxiliary materials to prepare a medicament.
An effective dose means that the solid viable bacteria preparation prepared by taking Pediococcus pentosaceus alone or in combination as an active ingredient contains a total viable bacteria count of not less than 1X 10 7 CFU/g。
The Pediococcus pentosaceus preparation composition of the invention contains Pediococcus pentosaceus with total viable count not less than 1X 10 7 CFU/g, typically 1X 10 9 CFU/g, the maximum can reach 1×10 11 CFU/g or 1X 10 11 CFU/g or more.
The invention selects pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain to prevent and treat the parkinsonism, has obvious curative effect, enhanced curative effect along with the increase of dosage, and does not find any toxic or side effect.
The invention has the advantages and effects that:
the invention discloses the application of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain in preventing and treating parkinsonism for the first time and provides a preparation method of a pediococcus pentosaceus preparation composition. The pediococcus pentosaceus WMU002 strain selected by the invention is derived from human bodies, and has high safety through screening, domestication and verification. The invention can obviously improve dyskinesia caused by parkinsonism and improve behavioural ability. The metabolite of the pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain has gamma aminobutyric acid and other active matters, and can play the role of resisting parkinsonism in brain. Therefore, the pediococcus pentosaceus preparation composition can be used as a medicament for preventing and treating the parkinsonism, has no toxic or side effect, and can be used for the adjuvant therapy of patients with the clinical parkinsonism.
Drawings
Fig. 1. Time for the balance beam to traverse the balance beam for the balance beam experiment. Time to cross beam(s): time for crossing balance beam; con: normal control group; MPTP: a parkinson's disease model group; mptp+pp: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain treatment group; ** : comparison with the Normal control groupP＜0.01； ## : p < 0.01 compared with the Parkinson disease model group.
Figure 2 number of foot slips for the balance beam experiment. Number of foot slips: number of foot slips; con: normal control group; MPTP: a parkinson's disease model group; mptp+pp: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain treatment group; ** : p < 0.01 compared with the normal control group; ## : p < 0.01 compared with the Parkinson disease model group.
Fig. 3 incubation period of the rotarod experiment. Latex time(s): incubation period; con: normal control group; MPTP: a parkinson's disease model group; mptp+pp: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain treatment group; * : p < 0.05 compared with the normal control group; ## : p < 0.01 compared with the Parkinson disease model group. .
Fig. 4. Crawling state score of the pole climbing experiment. Score of crawling state: crawling state scores; con: normal control group; MPTP: a parkinson's disease model group; mptp+pp: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain treatment group; * : p < 0.05 compared with the normal control group; ## : p < 0.01 compared with the Parkinson disease model group. .
FIG. 5 Nile stain results. Con: normal control group; MPTP: a parkinson's disease model group; mptp+pp: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain in the treatment group.
Detailed Description
The preparation method of the pediococcus pentosaceus preparation composition is that pediococcus pentosaceus bacterial powder is firstly prepared, and then corresponding auxiliary materials are added according to the requirement to prepare corresponding dosage forms.
EXAMPLE 1 isolation and identification of Pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 Strain
The method comprises the steps of taking the excrement of a healthy young person as a separation sample, and performing coating separation culture on an improved MRS agar medium at 37 ℃ for 48 hours to obtain the pediococcus pentosaceus which is a pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain.
Formulation of MRS medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose and K 2 HPO 4 2g，MgSO 4 ·7H 2 O 0.5g，MnSO 4 ·4H 2 O0.2 g, fructo-oligosaccharide 3g, diammonium citrate 2g, agar 15g, distilled water 1L, adjusting pH to 7.0, sterilizing at 115 ℃ for 15 minutes.
The pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain of the invention has the following microbiological characteristics:
(1) Colony morphology: the milky white circular colony has smooth and convex surface, neat edge and opaqueness;
(2) Individual morphology: spherical, most of which are in a duplex or quadruple shape and are gram positive;
optimum conditions: the growth is good under facultative anaerobic condition, and the optimal growth temperature is 35-40 ℃; the minimum growth temperature is 25-28 ℃; 43-45 ℃ at the highest; the optimal pH for growth is 6.5-7.0; the pH is 4.5-5.0 or 8.0-8.5 without growth.
TABLE 1 Biochemical reaction of Pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 Strain
The concentration of the culture supernatant of Pediococcus pentosaceus WMU002 strain was determined by ELISA. The results showed that the concentration of GABA in the bacterial culture supernatant peaked at 48h with GABA ranging from 3-4. Mu. Mol/L.
The pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain is preserved in China general microbiological culture collection center (CGMCC) (address: north Chen West Lu No. 1, 3 of the Korean area of Beijing, china academy of sciences microbiological study, post code 100101) at day 5 and 12 of 2022, with the preservation number of CGMCC No.24884.
Example 2 preparation of Pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 probiotic formulation composition
1. Seed liquid preparation: selecting single colony of pediococcus pentosaceus (Pediococcus pentosaceus) WMU002, inoculating into 50mL MRS liquid culture medium, and pHCulturing at 37deg.C for 24 hr at 7.0+ -0.5 to obtain seed solution; formulation of MRS medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate and K 2 HPO 4 2g，MgSO 4 ·7H 2 O 0.5g，MnSO 4 ·4H 2 0.2g of O, 3g of fructo-oligosaccharide, 2g of diammonium citrate, 1L of distilled water, adjusting the pH to 7.0 and sterilizing for 15min at 115 ℃.
2. Fermentation culture: inoculating the seed solution obtained in the step 1 into a new and improved MRS culture medium with an inoculum size of 1%, performing anaerobic culture at 37 ℃ for 24 hours, ending the culture, removing fermentation supernatant, and centrifuging to obtain thalli;
3. preparing bacterial powder: adding the thallus collected in the step 2 into freeze-drying protection liquid containing 5% of sodium glutamate and 5% of multi-fat milk powder by weight, mixing, freeze-drying to obtain Pediococcus pentosaceus bacterial powder with viable count not less than 1×10 7 CFU/g or 1X 10 7 CFU/mL。
Example 3 experiments on animals treated with Parkinson's disease
The purpose is as follows: the neuroprotection of the pediococcus pentosaceus WMU002 preparation of the present invention on mice model for Parkinson's disease was observed.
1. Materials and methods:
1. medicament: pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 bacteria powder with viable count of 1×10 9 CFU/g, specific preparation method, see example 2.
2. Experimental animals: c57BL/6J mice were male and had a weight of 18-22 g.
3. Preparation of a parkinson's disease mouse model and animal grouping: mice were intraperitoneally injected with 25mg/kg MPTP (1-methyl-4-phenyl 1,2,3, 6-tetrahydropyridine, MPTP) once daily for 1 week. Mptp+pp group: mice were given 0.2mL of 1X 10 daily following MPTP treatment 9 Pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 suspension in CFU/mL was gastric lavage for 4 weeks, and Con and MPTP mice were treated with the same dose of physiological saline. Testing the motor behavior capability of the mice after the experiment is finished, and performing perfusion fixation on brain tissues of the mice after the behavioral test is finished, and performing Nissl staining pathological detection; the GABA content in the brain tissue was measured by ELISA using the brain tissue.
4. Testing of balance and exercise coordination ability
A) Balance beam experiment: used to test the balance ability and motor coordination of mice. The balance beam is 0.8m long and 14mm wide. Each mouse was placed at one end of the balance beam and led to climb into a black box for 2d, at 3d the mouse was placed horizontally on the beam and traversed within 5min, and the number of foot slips (front or rear paws slid over the smooth surface of the beam) and the time of the mouse traversal were recorded.
B) Rotating rod experiment: used to test the ability of mice to coordinate locomotor activity. Prior to the experiment, mice were trained once daily for 3d. And 4d, placing the mice on an acceleration rotation cylinder, slowly increasing the rotation speed to 40 rotations within 5 minutes, continuously rotating for 2 circles under the condition of not attempting walking after the test is finished, and measuring the incubation period of the mice.
C) Pole climbing experiment: used to assess the coordination ability of mice. A rod with the length of 40em and the diameter of 1.5cm is fixed on the base and is wrapped by non-sticky gauze, so that the mouse is convenient to clamp. Prior to testing, the mice were trained to ensure that the poles could be climbed down; at the end of the experiment, both hind limbs of the mice reached the bottom. The total time to climb down from the pole and the status of the mice during the climb down were measured. Each mouse was subjected to 3 consecutive experiments and the average value was calculated for statistical analysis. The scoring criteria for the status of the mice climbing pole are as follows. 0: smoothly climbing down the rod by limbs; 0.5: spiral crawling downwards step by step, but the rear legs have sliding behaviors; 1.0: stopping climbing down for several times, but tightly holding the rod; 1.5: sliding on the rod and falling off; 2.0: the rod cannot be grasped and directly falls down.
Nissl staining: mice were fixed in formaldehyde and embedded in paraffin, cut to 5 μ; the sections were dewaxed with xylene and rehydrated with gradient ethanol. The sections were then stained with 1% tar violet dye for 30 minutes, washed with distilled water, separated with 70% ethanol, dehydrated with gradient ethanol, and finally fixed in xylene and sealed with neutral resin and observed under microscope.
ELISA determination of GABA content in brain tissue: measuring GABA content in brain tissue by ELISA method, mixing brain tissue and lysate according to a ratio of 1:9, adding PMSF, homogenizing by a homogenizer, and taking supernatant; the absorbance (OD value) of each sample was measured at 450nm by a reader.
2. Experimental results:
1. pediococcus pentosaceus WMU002 can inhibit MPTP induced dyskinesia
In the balance beam experiments, the time to traverse the balance beam and the number of foot slips were significantly increased in the MPTP group compared to the Con group, and these conditions were reversed after PP treatment (fig. 1 and 2). In the rod-rotating experiments, the latency was significantly shorter in the MPTP group than in the Con group (fig. 3), while the latency in the mptp+pp group was longer than in the MPTP group (fig. 3). In the pole-climbing experiments, the score of the crawling state of the MPTP group was significantly increased compared to the Con group, while the score of the mptp+pp group was significantly decreased compared to the MPTP group (fig. 4), indicating that pediococcus pentosaceus WMU002 can improve MPTP-induced dyskinesia.
2. Pediococcus pentosaceus WMU002 was able to ameliorate MPTP-induced neuropathological lesions (FIG. 5).
3. Pediococcus pentosaceus WMU002 treatment reversed MPTP-induced GABA level decline
We measured the concentration of GABA in the mouse brain. The results showed that the average concentration of GABA in the Con group was 9.112 ±0.4820, the average GABA level in mptp group mice was 7.353 ± 0.4839, and the GABA content 9.166 ± 0.3098 after treatment, indicating that pediococcus pentosaceus WMU002 could alleviate GABA reduction in brains of parkinson's disease model mice.
Conclusion:
the invention creatively takes intestinal flora as a treatment target point, creates a new mode for preventing and treating the parkinsonism, develops a medicament for preventing and treating the parkinsonism, has good curative effect and no toxic or side effect, and is proved by animal experiment researches, the pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 preparation can obviously improve the motor behavior ability of parkinsonism mice and relieve nerve injury. The invention selects pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain to prevent and treat the parkinsonism, has obvious curative effect, does not find any toxic or side effect, and can be used as a nutritional preparation and other nutrients for auxiliary treatment of clinical patients.
Claims (1)
1. Pediococcus pentosaceus (Pediococcus pentosaceus) WMU002 strain, accession number: CGMCC No.24884.
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