CN108159083A - It is a kind of to be used to prevent composition of traumatic brain injury and preparation method thereof - Google Patents
It is a kind of to be used to prevent composition of traumatic brain injury and preparation method thereof Download PDFInfo
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- CN108159083A CN108159083A CN201711500619.9A CN201711500619A CN108159083A CN 108159083 A CN108159083 A CN 108159083A CN 201711500619 A CN201711500619 A CN 201711500619A CN 108159083 A CN108159083 A CN 108159083A
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Abstract
The invention belongs to medical life sciences, are related to application of the clostridium butyricum preparation in prevention traumatic brain injury.The present inventor discloses clostridium butyricum to treating the new application of traumatic brain injury for the first time, and provides the preparation method of clostridium butyricum preparation.The present invention is target spot by enteron aisle, plays neuroprotection in intracerebral by brain intestines axis, improves traumatic brain injury.Clostridium butyricum preparation of the present invention has good effect on treatment traumatic brain injury, has no toxic side effect, and has good economic results in society, is worthy of popularization.
Description
Technical field
The present invention relates to the new applications of clostridium butyricum, and in particular to clostridium butyricum is in traumatic brain injury drug is treated
Using belonging to field of biological pharmacy.
Background technology
Traumatic brain injury (TBI) is the central nervous system disease that all brain structures as caused by being damaged external force and function change
Disease.External force can be the machinery strike of a hard object or bullet or fall down directly touching for rear cranium brain and ground
It hits, is even impacted caused by explosion.Different according to the degree that TBI is damaged, patient can show as abrading, bleeding, brain function
Obstacle or even death.The time of damage may temporarily take several days, it is also possible to through the remaining years of patient.According to documents and materials report
It leads, the whole world has more than 57,000,000 brain injury patients and will appear different degrees of neurological dysfunction.The World Health Organization predicts
To the year two thousand twenty, traumatic brain injury will become lethal one of the most important reason that disables in the whole world.
Traumatic brain injury is a kind of serious central nervous system disease, and cerebral injury while frequently can lead in nerve
The disorder of secretion and the inhibition of vagus reflex, so as to which gastrointestinal tract be made irritability damage, such as gastrointestinal motility disorder, enteron aisle occur
The change of barrier function, the increase of intestinal mucosa permeability, the amount reproduction of harmful intestinal tract flora and transposition, this is further increased
The appearance of the complication such as the risk and pyemia of infection, multiple organ failure.The traumatic brain damage of clinical treatment at present
Wound is thorough due to still lacking to traumatic brain injury pathogenesis mainly using cranial nerve nutrient drug and physiotherapy
The effect of solution, nerve protection medicine, tends not to reach expected.And to gastrointestinal symptom mainly to promote based on gastroenteritic power drug,
Such as Domperidone, metoclopramide etc., but it is often ineffective, and have different degrees of side effect.
TBI mainly includes two stages of primary injury and secondary lesion, and primary injury is that external force directly acts on
The moment on head causes brain tissue that damage and compressing occurs, and the result of denaturation displacement, contusion either bleeding occurs.
On the basis of primary injury, in coming minutes, a few houres or several days secondary lesion occurs for tissue after wound, macroscopic view
On, using intracranial pressure raising and intracranial hematoma for mainly show, it is microcosmic on, appearance of free radicals increases, vasopermeability increases, calcium surpasses
Load, inflammatory reaction, nitric oxide toxicity, nerve excitability toxicity geology a series of physiology and pathology such as generation on change
Become, nerve cell is made denaturation apoptosis occur, and the obstacle of nervous function finally occur.Often there is diffusivity in serious craniocerebral trauma
Axonal injury, and make patient evolution to vegetative state.Research is thought, as liver, lungs, enteron aisle is irritability damage after TBI
One of organ that wound is earliest, most often involves, enteron aisle are also to determine Systemic stress response and the crucial sexual organ of prognosis.There is document report
Road, the mouse of TBI models will appear the gastrointestinal functions such as intestinal tympanites, enteroparalysis, intestinal bleeding, dry and hard excrement and intestinal motive force deficiency
Obstacle shows, and even because of bacterium and endotoxin translocation, leads to SIRS or MODS, is digested with clinically patients with brain injury
The performances such as gastrointestinal hemorrhage, stress ulcer, abdominal distension, vomiting, diarrhea are consistent.After cerebral injury, contacting between brain and enteron aisle will
It is disturbed, neuroendocrine disturbance, immunoregulatory disorders and sympathetic activation occurs, enteron aisle gastrointestinal mucosa ischemic is caused to lack
The appearance that oxygen, gastrointestinal tract dyskinesis and gut barrier function destroy increases plasma endotoxin and D-ALPHA-Hydroxypropionic acid level.
Brain and enteron aisle seem mutual indepedent, and actual relationship is very close, researches show that：Brain-gut axis is between brain and enteron aisle
Two-way traffic access, by some peptides, neurotransmitter, cell factor and enteric microorganism metabolite etc. by brain and stomach
Intestinal tight links together.Probiotics can adjust intestinal flora, and influence host's behavior by regulating and controlling intestines-brain axis.
And clostridium butyricum has been widely used in because of its unique microbiologic properties and physiological function in real life.The hair of TBI
Neurotherapeutic after interpretation of the cause, onset and process of an illness system and neurotrosis is always the research field that current cerebral injury receives much attention, and is also directly related to disease
The progression of the disease of people and prognosis.More and more research confirmations, the mitigation of the intestinal tract injury symptom of nervous system Disease
Its nervous system disease, such as cerebral ischemia re-pouring injured, depression, Parkinson's disease can be improved.
Intestines-brain axis is the two-way exchange system between enteron aisle and brain, by neuroendocrine, immune, vagus nerve approach structure
Into probiotics can adjust intestinal flora, and influence host's behavior by regulating and controlling intestines brain axis.Clostridium butyricum (Clostridium
Butyricum) there is the intestinal mucosa of repairing damage, diminish inflammation, nutrition enteron aisle includes enteric bacteria sense to a variety of causes
Intestines problem caused by dye, chemotherapy of tumors, surgical operation etc. has the effect of good, up to the present, there is not yet clostridium butyricum
Treat the relevant report of traumatic brain injury.
Invention content
The defects of the purpose of the present invention is overcoming the prior art and deficiency, the object of the present invention is to provide one plant of clostridium butyricums
And the application in traumatic brain injury is treated.
Clostridium butyricum (Clostridium butyricum) WZMC1018 of the present invention, existed on October 22nd, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center is referred to as CGMCC (addresses：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is clostridium butyricum
(Clostridium butyricum) WZMC1018, deposit number are CGMCC No.9830.
A kind of clostridium butyricum preparation for treating traumatic brain injury, it is characterised in that the preparation method of clostridium butyricum preparation is such as
Under：
(1) prepared by seed liquor：Picking single bacterium colony clostridium butyricum (Clostridium butyricum) WZMC1018, preservation
Number is CGMCC No.9830, is inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, and Anaerobic culturel is for 24 hours to get to butyric acid
Clostridium WZMC1018 seed liquors；
(2) it ferments：Step (1) is obtained into containing with weight after seed liquor has sterilized by the inoculum concentration access of 1% weight ratio
Measure peptone 1-5%, yeast extract 0.5-5%, glucose 1-10%, oligofructose 0.5-2%, the ammonium sulfate of percentages
0.1%th, potassium dihydrogen phosphate 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.02%, magnesium sulfate 0.02% and chlorine
Change calcium 0.002%, remaining for deionized water fluid nutrient medium in, through 35 DEG C -37 DEG C stand anaerobic fermentations for 24 hours -48h to get
To clostridium butyricum WZMC1018 zymotic fluids；
(3) thalline is collected：The clostridium butyricum zymotic fluid that step (2) culture is obtained, 3,000rpm 5~10min of centrifugation, goes
Fall supernatant, obtain bacterium mud, bacterial suspension is made in 2~3 washings of deionized water, again with 3,000rpm centrifugations 5~
10min removes supernatant fluid and obtains sediment；
(4) degreasing is eluted：By the sediment obtained in step (3), carry out dissolving repeatedly using ethyl alcohol and elute, 10,
000rpm centrifuges 15~20min, recycles sediment, and washing steps are 2~3 times, then with the washing of deionized water 3~5 times,
3000rpm centrifuges 5~10min, obtains sediment；
(5) prepared by bacteria suspension：By the sediment obtained in step (4) in sterile saline or PBS liquid, than turbid, obtain
Viable bacteria bacterium amount a concentration of 1.0 × 10 must be contained9The suspension of CFU/mL is to get to clostridium butyricum preparation.
The present invention is using the clostridium butyricum of effective dose as medicament active composition, according to certain preparation process, is added in
The excipient substances such as conventional excipient, flavoring agent, preservative, lubricant, binder, are made any one and are suitable for clinically making
Dosage form, such as tablet, capsule, granule, powder, oral liquid, enema dosage form.Clostridium butyricum of the present invention exists
When any dosage form is made, it is respectively provided with the effect for the treatment of traumatic brain injury.
Clostridium butyricum composition of the present invention can be that viable bacteria probiotics is made separately as active ingredient in clostridium butyricum
Preparation or clostridium butyricum are combined with other non-probiotic active compositions is made active bacteria formulation, to play synergistic treatment work
With improving the therapeutic effect to traumatic brain injury.
Meaning effective dose of the invention refers to using clostridium butyricum according to described above alone or in combination as active ingredient system
Into total viable count for including of solid live bacteria preparation cannot be below 1 × 109CFU/mL can reach 1 × 1010CFU/mL with
On.
Currently preferred preparation process is first to be inoculated with strain in liquid medium, is cultivated or multistage amplification is trained
It supports, then centrifuges liquid culture, collect bacterium mud, preparation process is not limited to preparation of the present invention, other well known
Technique can be with.
Advantages of the present invention and effect：
The present inventor discloses clostridium butyricum to treating the new application of traumatic brain injury for the first time, and provides clostridium butyricum system
The preparation method of agent.The present invention is target spot by enteron aisle, plays neuroprotection in intracerebral by brain intestines axis, improves traumatic
Cerebral injury.
Specific embodiment
The present invention illustrates the preparation method of probiotics so that oral clostridium butyricum active bacteria powder is specifically prepared as an example, other doses
The preparation method of type is omitted, and is no longer illustrated one by one, and the specific dosage form for preparing is not limited to method described below.If it does not refer in particular to
Conventional means bright, that technological means used in embodiment is well known to those skilled in the art.
The preparation of 1 clostridium butyricum of embodiment (Clostridium butyricum) WZMC1018 preparations
(1) prepared by seed liquor：Picking single bacterium colony clostridium butyricum (Clostridium butyricum) WZMC1018, preservation
Number is CGMCC No.9830, is inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, and Anaerobic culturel is for 24 hours to get to butyric acid
Clostridium WZMC1018 seed liquors；
(2) it ferments：Step (1) is obtained into containing with weight after seed liquor has sterilized by the inoculum concentration access of 1% weight ratio
Measure peptone 5%, yeast extract 2%, glucose 8%, oligofructose 2%, ammonium sulfate 0.1%, the di(2-ethylhexyl)phosphate of percentages
Hydrogen potassium 0.1%, dipotassium hydrogen phosphate 0.1%, manganese sulfate 0.02%, magnesium sulfate 0.02% and calcium chloride 0.002%, remaining for go from
In the fluid nutrient medium of sub- water, anaerobic fermentations are stood for 24 hours to get to clostridium butyricum WZMC1018 zymotic fluids through 37 DEG C；
(3) thalline is collected：The clostridium butyricum zymotic fluid that step (2) culture is obtained, 3,000rpm centrifugation 10min, removes
Supernatant, obtains bacterium mud, and 3 washings of deionized water are made bacterial suspension, centrifuge 10min with 3,000rpm again, remove
Supernatant fluid obtains sediment；
(4) degreasing is eluted：By the sediment obtained in step (3), carry out dissolving repeatedly using ethyl alcohol and elute, 10,
000rpm centrifuges 15min, recycles sediment, and washing steps are 3 times, then with the washing of deionized water 5 times, and 3000rpm centrifugations 5~
10min obtains sediment；
(5) prepared by bacteria suspension：By the sediment obtained in step (4) in sterile saline or PBS liquid, than turbid, obtain
Viable bacteria bacterium amount a concentration of 1.0 × 10 must be contained9The suspension of CFU/mL is to get to clostridium butyricum preparation.
Embodiment 2 treats the zoopery of traumatic brain injury
First, materials and methods：
Traumatic brain injury 1. (TBI) model foundation
This experiment is hit using the weight-drop that Feeney methods are established causes traumatic brain injury model, and self-control freely falling body is hit
Device, specific experiment step are as follows：
(1) experiment mice weight is weighed, after the satisfaction of 3.5% chloraldurate (350mg/kg) intraperitoneal injection of anesthesia, by mouse
It is fixed on strike model experiment platform.
(2) scalp is sterilized using Iodophor cotton balls after hair right over removal cranium brain, along median line longitudinal incision scalp and shelled
From periosteum, right parietal bone is exposed.
(3) 3mm under sagittal suture side 2mm, coronal suture, bores a bone hole, diameter is about 2mm using dental burr.
(4) lance is impacted in 20cm eminences freely falling body using counterweight (weight 20g), impact head end and endocranium are perpendicular
Contact after shock, removes percussion device and is stopped blooding with gelfoam immediately.
(5) after no active bleeding, incision of scalp is sutured, and animal is transferred to 37 DEG C of insulating boxs from experimental bench, revived
The neurologic defect after record mouse of corresponding time point of waking up scores and carries out other experiments.
2. Neuroscore：
This experiment is evaluated using nervous function severity score (neurological severity scores, NSS)
Neurologic impairment situation.NSS scorings include ten different test events, are mainly used to assess the limb motion of mouse, shy
Reflection and harmony are frightened, 1-10 points of scoring range, score is higher, and the neurologic impairment for showing mouse is heavier.
3. the acquisition of zoological specimens
(1) brain tissue Western blot collections of specimens：14d sacrificed by decapitation mouse, pushes rapidly skull aside after cerebral injury, takes
Go out complete brain tissue, acquire the cortex of cerebral injury area and surrounding 2mm tissues, be put into liquid nitrogen, and be transferred to -80 DEG C of ice as early as possible
Case, every group of 6 mouse.
(2) histopathology collection of specimens：14d after cerebral injury, deep anaesthesia mouse take full brain after heart perfusion is fixed
Be placed in 4% paraformaldehyde and fix 72h, per replacing a fixer for 24 hours, be placed in 70% alcohol and continue to fix two days,
Routine paraffin wax embeds, and slice, every group of mouse 6 is made.
4. brain tissue Nissl is dyed and immunohistochemistry：
The making of paraffin pathological section includes materials, fixed, dehydration, waxdip and embedding and slice, in light microscope
Lower observation damage location brain tissue neuronal cell and tigroid body.According to immunofluorescence dyeing step operation, Occludin and ZO-
1 albumen carries out immunofluorescence dyeing, fluorescence microscopy Microscopic observation, and 5 different visuals field of every section collecting are taken pictures, image
Analysis software calculates Occludin, ZO-1 immune fluorescence intensity of damage zone unit area.
The expression of the Occludin and ZO-1 of 5.Western blot methods detection mouse damage stove brain surrounding tissue
6. statistical method
Above experimental result is analyzed, and the data obtained is subjected to statistical analysis, metering by SPSS19.0
Data represents with mean ± standard error, and with one-way analysis of variance (ANOVA) between group, comparing two-by-two between each group is examined using LSD
It tests.It is statistically significant that P ＜ 0.05 represent each group difference.
2nd, result
1. nervous function assesses (NSS) interpretation of result
The NSS scorings of sham-operation group mouse are relatively low always, illustrate to anaesthetize and perform the operation it is smaller on the influence of the nervous function of mouse,
And then all there is apparent neurological dysfunction in TBI groups and 1 clostridium butyricum preparation processing group mouse of embodiment, with the time
Passage, the nervous function of two groups of mouse all make moderate progress, but 1 clostridium butyricum preparation processing group mouse of embodiment restores compared with TBI moulds
Type group mouse is more preferable, and the two is in all significant difference of 4d, 7d, 10d, 14d.NSS scorings are established in traditional neural functional defect
It on the basis of scoring, eliminates that some are cumbersome and be not easy the operation carried out, simplifies the test different for ten, be mainly used for evaluating
Feeling, movement, flat reflective and the balanced capacity of mouse, scoring is higher, and damage is more serious.Continuous nervous function evaluation and test after wound
As a result show that TBI group mouse NSS scorings are significantly raised, and after the 1 clostridium butyricum preparation processing gavage treatment of continuous embodiment compared with
TBI model group mouse NSS scorings reduce, and clostridium butyricum preparation has been prompted, which to have post-traumatic neurological dysfunction, improves work
With.Our pleasantly surprised discoveries, without the clostridium butyricum preparation of elution degreasing step in processing TBI model mices, obtaining effect will
The clostridium butyricum preparation (i.e. 1 clostridium butyricum preparation of embodiment) by eluting degreasing step is markedly less than, absolutely proves elution
Degreasing step is in the importance for the clostridium butyricum preparation for preparing treatment TIB.
1.2 brain tissue Nissl coloration results are analyzed
Nissl dyes the tigroid body that can show neuron.Tigroid body is widely present in the cell space and dendron of neuron,
There is basophilla.Tigroid body is big under normal circumstances and quantity is more；After neuronal damage, quantity can become rareness even
It disappears.14d after causing injury, Sham-operated control group cell arrangement rule, neuron indigo plant dye, cell space form is normal, Nissl body content
It is abundant, depigmentation unobvious, in the coarse intensive particle of navy blue.TBI model groups, 1 clostridium butyricum preparation processing group of embodiment are small
Mouse damages surrounding visible cell irregular arrangement, and neuronal quantity significantly reduces, has and shrinkage deformation, Nissl occur compared with multi-neuron
Corpusculum is rare or disappears, and tigroid body depigmentation is apparent, and the coloring of remaining cell tigroid body is shallower, and 1 clostridium butyricum preparation of embodiment
Processing group mouse can be substantially reduced TBI tigroid body depigmentation quantity.Our pleasantly surprised discoveries, without the butyric acid shuttle of elution degreasing step
Bacteria preparation processing TBI model mices, obtain effect to be markedly less than through elution degreasing step clostridium butyricum preparation (i.e.
1 clostridium butyricum preparation of embodiment), absolutely prove importance of the elution degreasing step in the clostridium butyricum preparation for preparing treatment TIB.
The immunofluorescence dyeing situation of Occludin and ZO-1 albumen around 1.3 brain tissue impairment stoves
In Sham-operated control group, Occludin and ZO-1 albumen is expressed in brain tissue cortex in strong positive, and distribution is wide
It is general；After TBI, the positive expression of Occludin and ZO-1 in the cortex of brain tissue significantly reduces, and damage zone distribution significantly reduces；
Compared to TBI model groups, Occludin and ZO-1 protein expressions increased 1 clostridium butyricum preparation processing group mouse of embodiment.
The expression of Occludin and ZO-1 albumen around 1.4 brain tissue impairment stoves
Compared with Sham-operated control group, the expression quantity of Occludin and ZO-1 albumen is substantially reduced (P ＜ in TBI model groups
0.01).Compared with TBI model groups, the expression quantity of Occludin and ZO-1 albumen is bright in 1 clostridium butyricum preparation group mouse of embodiment
Aobvious raising (P ＜ 0.05).Clostridium butyricum preparation without elution degreasing step is handling TBI model mices, effective, but imitates
Fruit is weaker.This experimental result shows the Blood-Brain Barrier around the cerebral injury stove of 1 clostridium butyricum preparation group mouse of embodiment
GAP-associated protein GAP Occludin and ZO-1 expression are significantly increased compared with TBI groups mouse, illustrate that blood-brain barrier function has obtained significantly changing
It is kind.
Conclusion：
The symptom of the cerebral injury of TBI model mices can arrive improvement after clostridium butyricum formulation application.Clostridium butyricum system of the present invention
Agent has good effect on treatment traumatic brain injury, has no toxic side effect.Auxiliary material is only the preferred embodiments of the invention, and
The non-range for being used for limiting implementation of the invention.
Claims (2)
1. a kind of clostridium butyricum preparation for treating traumatic brain injury, it is characterised in that the preparation method of clostridium butyricum preparation is such as
Under：
(1) prepared by seed liquor：Picking single bacterium colony clostridium butyricum (Clostridium butyricum) WZMC1018, deposit number
It for CGMCC No.9830, is inoculated in sterilized MRS fluid nutrient mediums, 37 DEG C, Anaerobic culturel is for 24 hours to get to clostridium butyricum
WZMC1018 seed liquors；
(2) it ferments：Step (1) is obtained into containing with weight hundred after seed liquor has sterilized by the inoculum concentration access of 1% weight ratio
Divide the peptone 1-5% than meter, yeast extract 0.5-5%, glucose 1-10%, oligofructose 0.5-2%, ammonium sulfate
0.1%th, potassium dihydrogen phosphate 0.05-0.3%, dipotassium hydrogen phosphate 0.05-0.3%, manganese sulfate 0.02%, magnesium sulfate 0.02% and chlorine
Change calcium 0.002%, remaining for deionized water fluid nutrient medium in, through 35 DEG C -37 DEG C stand anaerobic fermentations for 24 hours -48h to get
To clostridium butyricum WZMC1018 zymotic fluids；
(3) thalline is collected：By the obtained clostridium butyricum zymotic fluid of step (2) culture, 3,000rpm 5~10min of centrifugation, remove on
Clear liquid obtains bacterium mud, and 2~3 washings of deionized water are made bacterial suspension, centrifuge 5~10min with 3,000rpm again,
Remove supernatant fluid and obtain sediment；
(4) degreasing is eluted：By the sediment obtained in step (3), carry out dissolving repeatedly using ethyl alcohol and elute, 10,000rpm
15~20min is centrifuged, recycles sediment, washing steps are 2~3 times, then with the washing of deionized water 3~5 times, 3000rpm centrifugations 5
~10min, obtains sediment；
(5) prepared by bacteria suspension：By the sediment obtained in step (4) in sterile saline or PBS liquid, than turbid, contained
Viable bacteria bacterium amount a concentration of 1.0 × 109The suspension of CFU/mL is to get to clostridium butyricum preparation.
2. clostridium butyricum preparation according to claim 1, which is characterized in that total viable count that the clostridium butyricum includes is not
1 × 10 can be less than9CFU/mL。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408561A (en) * | 2019-07-08 | 2019-11-05 | 康美华大基因技术有限公司 | A kind of clostridium butyricum and its application and the composition containing the clostridium butyricum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104997812A (en) * | 2015-08-06 | 2015-10-28 | 温州医科大学附属第二医院 | Drug for resisting cerebral ischemia-reperfusion injury as well as preparation method and application of drug |
| CN105055457A (en) * | 2015-08-06 | 2015-11-18 | 温州医科大学附属第二医院 | Drug for preventing and treating alzheimer's disease as well as preparation method and application of drug |
-
2017
- 2017-12-31 CN CN201711500619.9A patent/CN108159083A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104997812A (en) * | 2015-08-06 | 2015-10-28 | 温州医科大学附属第二医院 | Drug for resisting cerebral ischemia-reperfusion injury as well as preparation method and application of drug |
| CN105055457A (en) * | 2015-08-06 | 2015-11-18 | 温州医科大学附属第二医院 | Drug for preventing and treating alzheimer's disease as well as preparation method and application of drug |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408561A (en) * | 2019-07-08 | 2019-11-05 | 康美华大基因技术有限公司 | A kind of clostridium butyricum and its application and the composition containing the clostridium butyricum |
| CN110408561B (en) * | 2019-07-08 | 2021-08-17 | 康美华大基因技术有限公司 | Clostridium butyricum, application thereof and composition containing clostridium butyricum |
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