CN106974941A - A kind of probiotics preparation method for sterility treatment - Google Patents
A kind of probiotics preparation method for sterility treatment Download PDFInfo
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Abstract
The present invention have studied the physiological action of vagina microorganism and fine intercellular by laboratory facilities, it was found that some vagina microorganisms specific can be combined with sperm, and then cause spermatozoa mnotion to decline even motionless, can also to stimulate sperm to produce the change of protein level and then influence it with ovum binding ability.The beneficial discovery based on more than, present invention determine that promoting by intervening vaginal flora the technology path of sperm motility, will be strong and do not influence the microorganism of sperm basic physiological character to pick out with sperm binding ability, isolation is acted on using its occupy-place to adhere to sperm harmful microbe, so as to lift sperm motility, and then alleviation or therapeutic action are played to infertility.In addition, the probiotic microorganisms in microbial inoculum contribute to the various diseases caused by disturbed vaginal flora for the treatment of, significantly, recurrence probability is low, with good promotion prospect for therapeutic effect.
Description
Technical field
The present invention relates to microbial technology field, further to grinding for vagina microorganism and fine intercellular physiological action
Study carefully, and in particular to a kind of probiotics preparation method for sterility treatment.
Background technology
The female genital tract infection that microecology in vaginas is unbalance to be caused is common gynecological disease.According to statistics because infectious diseases is medical
Account for out-patient more than half, it is vaginitis, cervicitis and pelvic infecton, the only hair of bacterial vaginosis BV every year to come first 3
Patient's number is just millions of, and severe one can cause the diseases such as chronic cervicitis, cervical carcinoma and ovary, fallopian tube cneoplasms, while being also to make
Into the main cause of infertile, premature labor, premature rupture of fetal membranes, infant of low-birth weight's premature labor, recurrent miscarriage, chorioamnionitis etc., serious shadow
The healthy and quality of life of patient is rung, and clinical data shows that incidence of infertility can rise when suffering from colpitis, and study
Prove that appropriate supplement vagina probiotics has mitigation to inflammation, while being also hopeful to be applied in infertility
Tens kinds of microorganisms are may separate out under normal condition, in vagina, including:Lactobacillus, corynebacteria, bifid bar
Bacterium, bacteroid, fusiform bacilarmature, anhemolytic streptococcus, enterococcus, MRSE, EHEC, Gardnerella, disappear
Change streptococcus, and protozoon, virus, mycoplasma and candida albicans bacterium etc., wherein lactobacillus is excellent in vagina normal flora
Gesture bacterium, its separation rate is up to 50%~80%.Constituted between these floras and host, environment mutually restriction, it is mutually coordinated, dynamic
The microecology in vaginas system of balance
Microecology in vaginas system is complex system in the big ecosystem of human body four, its procreation and disease in the mankind
Played an important role in terms of defense mechanism.There are some researches show because part vagina microorganism has direct physiology to spermatid
Influence, therefore the unbalance of vaginal flora may influence sperm to be combined with egg cell, so as to directly result in infertile generation.This
In the case of, it is necessary to clearly different vagina microorganisms and the physiological action of fine intercellular, pass through vagina microorganism on this basis
Regulation promote spermatid in the vigor of intravaginal.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of probiotics system for sterility treatment
Preparation Method, to solve spermatid impacted technical problem of locomitivity in conventional vaginal environment.
Another technical problem to be solved by the present invention is that conventional vaginal environment is also easy to produce Flora Disturbance phenomenon, so as to cause
The generation of gynecological disease.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of probiotics preparation method for sterility treatment, comprises the following steps：
1) separate microorganism from vaginal fluid, culture obtains some single bacterium colonies；
2) strain idenfication is carried out to the microorganism in some single bacterium colonies, obtains some bacterial strains；
3) some bacterial strains are taken, are incubated altogether in liquid environment with spermatid respectively, under microscopic visualization with each bacterium
The adhesive capacity of strain and spermatid is that foundation filters out aimed strain；
4) aimed strain is cultivated, freeze-dried powder microbial inoculum is prepared.
Preferably, step 1) in be used for separate microorganism culture medium include LB basal mediums, MRS solid cultures
Base, BHI solid mediums, the BHI culture mediums containing 5% (g/g) skimmed milk, BSM solid mediums.
Preferably, step 1) described in from vaginal fluid separate microorganism, specifically include following operation：Take vagina
Secretion is added into EP pipes sterile 1.5ml, with sterile PBS buffer according to 101~106Doubling dilution is carried out, respectively with 50
μ L amount is coated in solid medium, cultivates 48h.
Preferably, the vaginal fluid is stored under -80 DEG C of environment and in glycerine environment.
Preferably, the solid medium include LB basal mediums, MRS solid mediums, BHI solid mediums,
BHI culture mediums containing 5% (g/g) skimmed milk, BSM solid mediums；Wherein it is coated on MRS solid mediums, the training of LB bases
The sample supported in base, BSM solid mediums passes through 102~104Dilute again；It is coated on BHI solid mediums and containing 5% (g/g)
Sample in the BHI culture mediums of skimmed milk passes through 103~105Dilute again.
Preferably, the strain idenfication includes：The DNA of microorganism in some single bacterium colonies is extracted respectively, after being expanded through PCR
Sequencing, determines strain classification.
Preferably, step 3) specifically include following operation：
A some bacterial strains) are taken, are inoculated in respectively in the fluid nutrient medium corresponding to it, in CO2In 37 DEG C in incubator
Under the conditions of cultivate to OD values be 0.6；
B) by step A) obtained by some nutrient solutions it is every kind of take 200 μ L, respectively contain the thin of Hela cells with 2mL seminal fluid, 2mL
Born of the same parents' nutrient solution is mixed in 15ml centrifuge tubes, in CO21~1.5h is cultivated in incubator under the conditions of 37 DEG C；
C) separation of solid and liquid takes precipitation, and methanol is added after being cleaned with PBS and is fixed, and Gram's staining, oil mirror observation is each
Experimental group microorganism filters out aimed strain accordingly to the adhesive capacity of spermatid.
Preferably, step C) described in separation of solid and liquid be with the centrifugation of 1000rpm rotating speed；Cleaned with PBS
Number of times be 5 times.
Preferably, the aimed strain include Lactobacillus crispatus, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus vaginalis,
Yue Shi lactobacillus, lactobacillus acidophilus or lactobacillus fermenti.
Preferably, containing the fragment after bacterial secretory product or bacteria breaking in the freeze-dried powder microbial inoculum.
In above technical scheme, the fluid nutrient medium corresponding to it refers to the single bacterium colony of the isolated bacterial strain
The liquid form of residing solid medium；The liquid form is typically the solid medium without things such as agar or gelatin
State during matter.That is, the bacterial strain is isolated from which single bacterium colony, just the solid residing for that single bacterium colony is trained
The liquid form for supporting base is used as the inoculation place of the bacterial strain.
The present invention have studied the physiological action of vagina microorganism and fine intercellular by laboratory facilities, it is found that some vaginas are micro-
Biology specific can be combined with sperm, and then it is even motionless to cause spermatozoa mnotion to decline, and can also stimulate sperm to produce albumen
The change of level and then influence its binding ability with ovum.The beneficial discovery based on more than, intervenes present invention determine that passing through
Vaginal flora promotes the technology path of sperm motility, will be strong and do not influence sperm basic physiological character with sperm binding ability
Microorganism is picked out, and act on isolation using its occupy-place adheres to sperm harmful microbe, so that sperm motility is lifted, and then
Alleviation or therapeutic action are played to infertility.
Microbial bacterial agent provided by the present invention in situ can be combined with sperm, be supported on the basis of Sperm Motility is ensured
The interference of anti-inoculating microbe, contributes to increase to become pregnant probability.In addition, probiotic microorganisms therein contribute to treatment various by vagina
The disease that Flora Disturbance is caused, significantly, recurrence probability is low, with good promotion prospect for therapeutic effect.
Brief description of the drawings
Fig. 1 is the coherent condition microscopy figure of the medial vagina microorganism of the embodiment of the present invention 1 and spermatid.
Embodiment
The embodiment to the present invention is described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.In addition to being defined, institute in following examples
Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.
Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following examples；The experiment
Method, is conventional method unless otherwise specified；Quantitative test in following examples, is respectively provided with three repetition experiments, as a result
Average；% in following examples, is weight/mass percentage composition unless otherwise instructed.
Embodiment 1
A kind of probiotics preparation method for sterility treatment, comprises the following steps：
A, flora culture of isolated
(1) preparation of culture medium：LB basal mediums, MRS solid mediums, BHI solid mediums, BHI+5% degreasings
Newborn culture medium, BSM solid mediums；
(2) the vaginal fluid sample that taking-up is preserved with glycerine from -80 degree refrigerators, it is appropriate in nothing after after its thawing, taking
In bacterium 1.5ml EP pipes, with sterile PBS according to 101—106Doubling dilution is carried out, MRS, LB, BSM press 102、104Dilution BHI,
BHI+5% skimmed milks press 103、105The μ L of dilution 50 be coated in solid medium, cultivate 48h under anaerobism and aerobic condition.
B, bacterial strain identification：
(1) in aseptic superclean bench according to the form of bacterium colony, size, color, neat in edge in flat board whether, protuberance
Whether, choose single bacterium colony and be inoculated in corresponding fluid nutrient medium；
(2) Grain stain observation thalli morphology, tentatively judges gram positive bacteria and Glan negative bacterium；
(3) sample presentation is sequenced after the extraction of genomic DNA, PCR amplification purifications, determines strain.
C, bacterial strain screening：
(1) by the above-mentioned strain inoculation that strain is determined in corresponding fluid nutrient medium, in CO at 37 DEG C2Incubator
In (anaerobic bacteria is filled and sealed) cultivate, make its OD value be equal to 0.6 after terminate cultivate；
(2) the 200 above-mentioned inoculums of μ l are taken, cell culture fluid and 2ml essences that 2ml contains a small amount of Hela is separately added into
In the 15ml centrifuge tubes of daughter cell, 37 DEG C of cell culture incubator cultures；
(3) cultivate, 1000 turns/min, cleaned repeatedly with PBS 5 times, methanol is fixed by l~1.5 hour, leather is blue
Albert'stain Albert, oil mirror observation；
(4) filter out, sperm has the bacterial strain of good adhesion ability.
D, probiotics compounding：
(1) probiotics filtered out from vagina is compounded, makes freeze-dried powder preparation；
(2) after being incubated altogether with spermatid, the extraction of genomic DNA is carried out, which bacterial strain rear sample presentation sequencing determines and fine
Born of the same parents have good adhesive capacity.
E, the further checking of zoopery：
(1) oestrous cycle normal female rats 12 are only randomly divided into normal group by the foundation of rat ovulation rate model
With kidney tonifying Ovarian hyperstimulation group, the daily morning 9:00 takes vaginal fluid to do the inspection of picture row cell detachment, determines the dynamic of rat
The feelings cycle；
(2) normal group and kidney tonifying Ovarian hyperstimulation group metaoestrus, daily the μ g/200g of intraperitoneal injection GnRHa 1.8, continuous 9
My god, HMG70IU/200g is injected simultaneously within the 9th day, HCG injection 180IU/200g after 48 hours, normal group gives isometric 0.9%
Physiological saline is injected intraperitoneally, and 10 points of HCG injection group evening is with male and female mouse 2:1 mates, and the super decorporation group of kidney tonifying is from injecting after GnRHa with benefit
Kidney menstruation regulating side 1ml (200gd) normal group gives Isodose continuous normal saline gavage 11 days
(3) method of external sperm reduction, with different PH (PH6~PH8) nutrient solution culture rat seminal fluid 2 hours, profit
With computer assisted sperm activity analysis instrument (CASA) determine rat spermatozoa motility parameters (sperm motility, average path speed,
Mean linear movement velocity, averaged curve movement velocity), it is determined that making the pH value that sperm is weakened；
(4) experiment packet is observed, and is incubated the spermatid weakened altogether with different vagina microorganism bacterial strains 1 hour, and normal
Group and kidney tonifying Ovarian hyperstimulation group artificial insemination, it is pregnant after become pregnant 10 days after put to death, observe embryo nidation situation, meter record each group implantation embryo
Tire number.
Embodiment 2
A kind of probiotics preparation method for sterility treatment, comprises the following steps：
1) separate microorganism from vaginal fluid, culture obtains some single bacterium colonies；
2) strain idenfication is carried out to the microorganism in some single bacterium colonies, obtains some bacterial strains；
3) some bacterial strains are taken, are incubated altogether in liquid environment with spermatid respectively, under microscopic visualization with each bacterium
The adhesive capacity of strain and spermatid is that foundation filters out aimed strain；
4) aimed strain is cultivated, freeze-dried powder microbial inoculum is prepared.
On the basis of above technical scheme, following condition is met：
Step 1) in be used for separate microorganism culture medium include LB basal mediums, MRS solid mediums, BHI solids
Culture medium, the BHI culture mediums containing 5% (g/g) skimmed milk, BSM solid mediums.
Step 1) described in from vaginal fluid separate microorganism, specifically include following operation：Vaginal fluid is taken to add
Enter into EP pipes sterile 1.5ml, with sterile PBS buffer according to 101~106Doubling dilution is carried out, is applied respectively with 50 μ L amount
It is distributed in solid medium, cultivates 48h.
The vaginal fluid is stored under -80 DEG C of environment and in glycerine environment.
The solid medium includes LB basal mediums, and MRS solid mediums, BHI solid mediums contain 5% (g/
G) the BHI culture mediums of skimmed milk, BSM solid mediums；Wherein it is coated on MRS solid mediums, LB basal mediums, BSM solid
Sample in body culture medium passes through 102~104Dilute again；It is coated on BHI solid mediums and containing 5% (g/g) skimmed milk
Sample in BHI culture mediums passes through 103~105Dilute again.
The strain idenfication includes：The DNA of microorganism in some single bacterium colonies is extracted respectively, is sequenced after being expanded through PCR, it is determined that
Strain classification.
Step 3) specifically include following operation：
A some bacterial strains) are taken, are inoculated in respectively in the fluid nutrient medium corresponding to it, in CO2In 37 DEG C in incubator
Under the conditions of cultivate to OD values be 0.6；
B) by step A) obtained by some nutrient solutions it is every kind of take 200 μ L, respectively contain the thin of Hela cells with 2mL seminal fluid, 2mL
Born of the same parents' nutrient solution is mixed in 15ml centrifuge tubes, in CO2In incubator 1h is cultivated under the conditions of 37 DEG C；
C) separation of solid and liquid takes precipitation, and methanol is added after being cleaned with PBS and is fixed, and Gram's staining, oil mirror observation is each
Experimental group microorganism filters out aimed strain accordingly to the adhesive capacity of spermatid.
Step C) described in separation of solid and liquid be with the centrifugation of 1000rpm rotating speed；The number of times cleaned with PBS is 5
It is secondary.
The aimed strain includes Lactobacillus crispatus, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus vaginalis, Yue Shi breast bars
Bacterium, lactobacillus acidophilus or lactobacillus fermenti.
Contain the fragment after bacterial secretory product or bacteria breaking in the freeze-dried powder microbial inoculum.
Embodiment 3
A kind of probiotics preparation method for sterility treatment, comprises the following steps：
1) separate microorganism from vaginal fluid, culture obtains some single bacterium colonies；
2) strain idenfication is carried out to the microorganism in some single bacterium colonies, obtains some bacterial strains；
3) some bacterial strains are taken, are incubated altogether in liquid environment with spermatid respectively, under microscopic visualization with each bacterium
The adhesive capacity of strain and spermatid is that foundation filters out aimed strain；
4) aimed strain is cultivated, freeze-dried powder microbial inoculum is prepared.
On the basis of above technical scheme, following condition is met：
Step 1) in be used for separate microorganism culture medium include LB basal mediums, MRS solid mediums, BHI solids
Culture medium, the BHI culture mediums containing 5% (g/g) skimmed milk, BSM solid mediums.
Step 1) described in from vaginal fluid separate microorganism, specifically include following operation：Vaginal fluid is taken to add
Enter into EP pipes sterile 1.5ml, with sterile PBS buffer according to 101~106Doubling dilution is carried out, is applied respectively with 50 μ L amount
It is distributed in solid medium, cultivates 48h.
The strain idenfication includes：The DNA of microorganism in some single bacterium colonies is extracted respectively, is sequenced after being expanded through PCR, it is determined that
Strain classification.
Step 3) specifically include following operation：
A some bacterial strains) are taken, are inoculated in respectively in the fluid nutrient medium corresponding to it, in CO2In 37 DEG C in incubator
Under the conditions of cultivate to OD values be 0.6；
B) by step A) obtained by some nutrient solutions it is every kind of take 200 μ L, respectively contain the thin of Hela cells with 2mL seminal fluid, 2mL
Born of the same parents' nutrient solution is mixed in 15ml centrifuge tubes, in CO2In incubator 1.5h is cultivated under the conditions of 37 DEG C；
C) separation of solid and liquid takes precipitation, and methanol is added after being cleaned with PBS and is fixed, and Gram's staining, oil mirror observation is each
Experimental group microorganism filters out aimed strain accordingly to the adhesive capacity of spermatid.
The aimed strain includes Lactobacillus crispatus, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus vaginalis, Yue Shi breast bars
Bacterium, lactobacillus acidophilus or lactobacillus fermenti.
Embodiment 4
A kind of probiotics preparation method for sterility treatment, comprises the following steps：
1) separate microorganism from vaginal fluid, culture obtains some single bacterium colonies；
2) strain idenfication is carried out to the microorganism in some single bacterium colonies, obtains some bacterial strains；
3) some bacterial strains are taken, are incubated altogether in liquid environment with spermatid respectively, under microscopic visualization with each bacterium
The adhesive capacity of strain and spermatid is that foundation filters out aimed strain；
4) aimed strain is cultivated, freeze-dried powder microbial inoculum is prepared.
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All any modifications, equivalent substitutions and improvements done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (10)
1. a kind of probiotics preparation method for sterility treatment, it is characterised in that comprise the following steps：
1) separate microorganism from vaginal fluid, culture obtains some single bacterium colonies；
2) strain idenfication is carried out to the microorganism in some single bacterium colonies, obtains some bacterial strains；
3) some bacterial strains are taken, are incubated altogether in liquid environment with spermatid respectively, under microscopic visualization with each bacterial strain with
The adhesive capacity of spermatid is that foundation filters out aimed strain；
4) aimed strain is cultivated, freeze-dried powder microbial inoculum is prepared.
2. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that step
It is rapid 1) in be used for the culture medium of separate microorganism and include LB basal mediums, MRS solid mediums, BHI solid mediums contain
The BHI culture mediums of 5% (g/g) skimmed milk, BSM solid mediums.
3. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that step
It is rapid 1) described in from vaginal fluid separate microorganism, specifically include following operation：Take vaginal fluid add to 1.5ml without
In the EP pipes of bacterium, with sterile PBS buffer according to 101~106Doubling dilution is carried out, solid training is coated on 50 μ L amount respectively
Support in base, cultivate 48h.
4. a kind of probiotics preparation method for sterility treatment according to claim 3, it is characterised in that institute
Vaginal fluid is stated to be stored under -80 DEG C of environment and in glycerine environment.
5. a kind of probiotics preparation method for sterility treatment according to claim 3, it is characterised in that institute
Stating solid medium includes LB basal mediums, and MRS solid mediums, BHI solid mediums contain 5% (g/g) skimmed milk
BHI culture mediums, BSM solid mediums；Wherein it is coated in MRS solid mediums, LB basal mediums, BSM solid mediums
Sample pass through 102~104Dilute again；It is coated in BHI solid mediums and BHI culture mediums containing 5% (g/g) skimmed milk
Sample pass through 103~105Dilute again.
6. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that institute
Stating strain idenfication includes：The DNA of microorganism in some single bacterium colonies is extracted respectively, is sequenced after being expanded through PCR, is determined strain classification.
7. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that step
It is rapid 3) to specifically include following operation：
A some bacterial strains) are taken, are inoculated in respectively in the fluid nutrient medium corresponding to it, in CO2In 37 DEG C of conditions in incubator
Lower culture to OD values are 0.6；
B) by step A) obtained by some nutrient solutions it is every kind of take 200 μ L, respectively contain with 2mL seminal fluid, 2mL Hela cells cell training
Nutrient solution is mixed in 15ml centrifuge tubes, in CO21~1.5h is cultivated in incubator under the conditions of 37 DEG C；
C) separation of solid and liquid takes precipitation, and methanol is added after being cleaned with PBS and is fixed, Gram's staining, oil mirror observes each experiment
Group microorganism filters out aimed strain accordingly to the adhesive capacity of spermatid.
8. a kind of probiotics preparation method for sterility treatment according to claim 7, it is characterised in that step
Rapid C) described in separation of solid and liquid be rotating speed centrifugation with 1000rpm；The number of times cleaned with PBS is 5 times.
9. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that institute
Stating aimed strain includes Lactobacillus crispatus, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus vaginalis, Yue Shi lactobacillus, acidophilus breast bar
Bacterium or lactobacillus fermenti.
10. a kind of probiotics preparation method for sterility treatment according to claim 1, it is characterised in that institute
State in freeze-dried powder microbial inoculum containing the fragment after bacterial secretory product or bacteria breaking.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110934895A (en) * | 2019-12-26 | 2020-03-31 | 南昌大学 | Application of lactobacillus crispatus in preparing female contraceptive medicament |
| CN113975301A (en) * | 2021-11-24 | 2022-01-28 | 暨南大学 | Probiotic preparation for improving sperm motility and application thereof |
| CN115181712A (en) * | 2022-09-14 | 2022-10-14 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus johnsonii LJM20 and microbial inoculum and application thereof |
| CN115181711A (en) * | 2022-09-14 | 2022-10-14 | 中国农业科学院北京畜牧兽医研究所 | Human milk bacillus LHM11, microbial inoculum and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267319A (en) * | 2016-09-08 | 2017-01-04 | 南昌大学 | A kind of preparation method containing vagina probiotic bacteria sanitary towel |
-
2017
- 2017-03-09 CN CN201710137240.XA patent/CN106974941A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267319A (en) * | 2016-09-08 | 2017-01-04 | 南昌大学 | A kind of preparation method containing vagina probiotic bacteria sanitary towel |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110934895A (en) * | 2019-12-26 | 2020-03-31 | 南昌大学 | Application of lactobacillus crispatus in preparing female contraceptive medicament |
| CN113975301A (en) * | 2021-11-24 | 2022-01-28 | 暨南大学 | Probiotic preparation for improving sperm motility and application thereof |
| CN115181712A (en) * | 2022-09-14 | 2022-10-14 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus johnsonii LJM20 and microbial inoculum and application thereof |
| CN115181711A (en) * | 2022-09-14 | 2022-10-14 | 中国农业科学院北京畜牧兽医研究所 | Human milk bacillus LHM11, microbial inoculum and application |
| CN115181711B (en) * | 2022-09-14 | 2022-12-13 | 中国农业科学院北京畜牧兽医研究所 | Human milk bacillus LHM11, microbial inoculum and application |
| CN115181712B (en) * | 2022-09-14 | 2022-12-13 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus johnsonii LJM20 and microbial inoculum and application thereof |
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Application publication date: 20170725 |