CN107299065A - A kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines - Google Patents
A kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines Download PDFInfo
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- CN107299065A CN107299065A CN201710469896.1A CN201710469896A CN107299065A CN 107299065 A CN107299065 A CN 107299065A CN 201710469896 A CN201710469896 A CN 201710469896A CN 107299065 A CN107299065 A CN 107299065A
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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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/25—Lactobacillus plantarum
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
A kind of application the invention provides Lactobacillus plantarum and its for preparing vagina antibacterial medicines, the Lactobacillus plantarum is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is CGMCC No.14111.Study it and be metabolized performance discovery, relatively common Lactobacillus plantarum, the bacterial strain possesses stronger lactic acid production capacity, and production hydrogen peroxide ability is also very strong, very strong to the bacteriostasis of pathogenic bacteria, while having prominent vaginal epithelial cell adhesive capacity.The new property found based on more than, present invention determine that preparing using it new application of vagina antibacterial medicines, can be achieved the treatment of various bacterial vaginal disease.It is notable to bacterial vaginosis effect using microbial inoculum of the present invention, and safety non-toxic, stability are good, can preserve for a long time, further relate to its purposes in preventing and/or treating the medicine of gynecological disease.
Description
Technical field
The present invention relates to microbial technology field, regulation and bacterial vaginosis disease further to microecology in vaginas environment
The treatment of disease, and in particular to a kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines.
Background technology
There is multiple-microorganism in healthy women intravaginal, they constitute mutually restriction, mutually association between host, environment
Tune, the microecology in vaginas system of dynamic equilibrium.The vaginal flora of healthy women is mainly made up of lactobacillus, including the newborn bar of plant
Bacterium, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus rhamnosus etc..Newborn bar under normal circumstances
Bacterium can be played a protective role to vagina, and vagina is produced when the lactobacillus disorder of microecology in vaginas can cause pathogenic bacteria to be invaded
It is scorching.
Bacterial vaginosis BV (BV) is due to vaginal dysbacteriosis, and the lactobacillus of host itself reduces and causes it
His conditionity pathogenic microorganism such as Gardnerella, various anaerobic bacterias, bending vibrios etc. it is a large amount of it is numerous plant, usual BV be actually with
A kind of mixed infection based on bacterium protein of Gardnerella vaginalis.Using antibiotic therapy, can respite BV symptom, but also reduced this
Lactobacillus further reduce, aggravate microecology in vaginas imbalance so that BV recurs repeatedly.How Control in recurring, thoroughly radical cure
Bacterial vaginosis BV is the thorny problem of ob-gyn's urgent need to resolve.
Research shows：Produce H2O2Lactobacillus with lactic acid is the dominant bacteria of healthy women intravaginal, is that protection vagina is exempted from
By the key factor of pathogenic infection, the acid and some antimicrobial agents that lactobacillus metabolism is produced in addition also can effectively suppress
The growth of other bacteriums is numerous to plant.
There are a variety of lactobacillus in healthy women intravaginal, with anti-pathogenic bacteria energy between individual difference, and each strain of lactobacillus
Power difference is obvious., it is necessary to consider the species of lactobacillus during selection lactobacillus probiotics, it produces acid, production H2O2Ability, and with
The ability of vaginal epithelial cell adhesion, can wherein lactobacillus successfully be colonized in vagina, be the basis of lactobacillus continuous action,
It is the key factor that lactobacillus plays curative effect.Lactobacillus plantarum (Lactobacillusplantarum), is lactobacillus, is people
One of body normal flora, is distributed widely in human body intestinal canal, is also distributed in vagina.For conventional bacterial strain,
Its in the category of cognition of prior art does not simultaneously have specific bacteriostasis.
The actual not China's woman vagina dominant microflora of the existing product " Lactobacillus delbrueckii " in current market, colonization ability compared with
Difference, can not maintain stable viable bacteria content, it is impossible to meet the demand of gynecological clinic.
The content of the invention
The present invention is intended to provide a kind of specific Lactobacillus plantarum, does not have to solve conventional plant lactobacillus in the prior art
There is the technical problem of vaginal pathogenic rejection ability.
Another technical problem to be solved by the present invention is that conventional plant lactobacillus production hydrogen peroxide manufacture ability is relatively low.
The invention solves the problems that another technical problem be conventional plant lactobacillus lactic acid production it is relatively low.
The invention solves the problems that another technical problem be conventional plant lactobacillus colonizing, depositing in vagina in the prior art
Active fruit is not good.
The invention solves the problems that another technical problem be in the prior art be directed to vaginal pathogenic microbial inoculum class antibacterial medicines
Therapeutic effect is not good.
To realize above technical purpose, the present invention uses following technical scheme：
It is RD- to be numbered in a kind of Lactobacillus plantarum of separation (Lactobacillus plantarum), the present invention
0025 (Lactobacillus plantarum), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number
For CGMCC No.14111.
Above-mentioned Lactobacillus plantarum (Lactobacillus plantarum) RD-0025 is China's Healthy women of child-bearing age's vagina
Screening is got in secretion, is preserved in on May 10th, 2017 commonly micro- in China Committee for Culture Collection of Microorganisms
Bio-Centers (abbreviation CGMCC), depositary institution address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences is micro-
Biological study institute, preservation registration number is CGMCC No.14111, and the Classification And Nomenclature of the bacterial strain is Lactobacillus plantarum
(Lactobacillus plantarum)。
The Lactobacillus plantarum RD-0025 of above-mentioned separation, is sequenced using 16SrDNA, with plant in GenBank databases
Lactobacillus base sequence highest homology score value is more than 98%.
On the basis of above technical scheme, it is used to prepare vagina cause invention further provides above-mentioned Lactobacillus plantarum
The application of germ antibacterial medicines.
Preferably, the medicine is microbial inoculum, the microbial inoculum is colonized and survived on vaginal epithelial cell in vaginal environment.
Preferably, the medicine is microbial inoculum, the microbial inoculum is metabolized production H in vaginal environment2O2。
Preferably, the pathogenic bacteria are Gardnerella vaginalis.
Preferably, the pathogenic bacteria are atropic ripple bacterium.
Preferably, the pathogenic bacteria are Candida albicans, staphylococcus aureus, ETEC, the false list of verdigris
Born of the same parents bacterium or salmonella.
Meanwhile, it is used to prepare vaginal disease prevention or medicine invention further provides above-mentioned Lactobacillus plantarum
Using.
Preferably, the vaginal disease is VVC, trichomonas vaginitis, senile vagina
Scorching, non-specific vagina infection or Combination vagina infection.
The isolated first lactobacillus plantarum of the present invention, studies its and is metabolized performance and find, the bacterial strain possesses excellent
Lactic acid production capacity and production hydrogen peroxide energy, it is very strong to the bacteriostasis of pathogenic bacteria, while having prominent vagina epithelium thin
Born of the same parents' adhesive capacity.The new property found based on more than, present invention determine that new application of vagina antibacterial medicines is prepared using it, it is real
The treatment of various bacterial vaginal disease is showed.
The beneficial effect produced using above-mentioned technical proposal is：(1) Lactobacillus plantarum RD-0025 bacterial strains of the invention can
To preserve for a long time, and resist bacterial vaginosis BV and various vagina infections, including candida albicans vaginitis, gonorrhoea, virus
Property vaginitis, and urethral infection etc..(2) bacterial strain of the invention is directly collected in healthy human body, the life with active stabilization
Thing characteristic, without domestication and rejuvenation technique, can directly preparation use.(3) bacterial strain of the invention, which has, suppresses vagina Gardner
Salmonella etc. suppress pathogenic bacteria effect, with it is commercially available control bacterium compared with, advantageous vaginal epithelial cell adhesive force, with
The advantageous ability of primate vagina field planting.
Brief description of the drawings
Accompanying drawing 1 is the full face of the Lactobacillus plantarum RD-0025 colonial morphologies of the present invention；
Accompanying drawing 2 is the Lactobacillus plantarum RD-0025 of present invention gram stain microscopy photo；
Accompanying drawing 3 is the electrophoretogram of the Lactobacillus plantarum RD-0025 of present invention 16SrDNA gene PCR amplified productions；
Accompanying drawing 4 is the Lactobacillus plantarum RD-0025 of present invention lactate detection collection of illustrative plates；
Accompanying drawing 5 is the Lactobacillus plantarum RD-0025 hydrogen peroxide colour developing 5min and 10min pictures of the present invention；
Accompanying drawing 6 is that the Lactobacillus plantarum RD-0025 of the present invention is quick to the antibiotic of vancomycin, gentamicin and erythromycin
Perception experiment bacterium colony figure；
Accompanying drawing 7 is the Lactobacillus plantarum RD-0025 (left side) and Lactobacillus delbrueckii (right side) of the present invention to gardnerella vaginalis
Fungistatic effect photo；
Accompanying drawing 8 is the suppression of the Lactobacillus plantarum RD-0025 (left side) and Lactobacillus delbrueckii (right side) of the present invention to EHEC
Bacterium effect photo；
Accompanying drawing 9 is the Lactobacillus plantarum RD-0025 (left side) and Lactobacillus delbrueckii (right side) of the present invention to gold-coloured staphylococci
Fungistatic effect photo；
Accompanying drawing 10 is that the Lactobacillus plantarum RD-0025 of the present invention is colonized portion of machin vagina microorganism area after machin vagina
Divide the electrophoretogram of bacterial strain 16SrDNA fragment pcr amplification products.
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.
Used Bacteria Culture based component and compound method are as follows in following examples：
1st, meat soup solid medium (MRS) is prepared：
(1) by agar powder wiring solution-forming, 1.5g/100ml deionized waters；
(2) MRS culture medium 17.91g/100ml agar solutions are added, are mixed；
(3) autoclave, 1.0MPa steam sterilizings 20 minutes are put into；
(4) culture dish is poured into after culture medium temperature is down to room temperature, it is individual according to culture dish size about 10ml/ or 20ml/；
(5) in operation in super-clean bench.Into agar shape after cooling, mark culture medium title and preparation date, 4 DEG C of refrigerators are put in
It is stand-by.
2nd, meat soup fluid nutrient medium (MRS) is prepared：
(1) MRS culture mediums are added into deionized water, ratio is 17.9l g/100ml；
(2) autoclave, 1.0MPa steam sterilizings 20 minutes are put into；
(3) take out, dispense into EP pipes, each 1.0ml after pot no pressure subject to sterilization.Mark culture medium title and prepare day
Phase, it is put in 4 DEG C of refrigerators stand-by.
3rd, hydrogen peroxide (H2O2) identification culture medium preparation：
(1) with meat soup solid medium (MRS) preparation steps (1) to (4)；
(2) take out, slightly cool down after pot no pressure subject to sterilization, but still it is (dense eventually in adding TMB in super-clean bench during for liquid condition
Spend 0.25mg/m1), HRP (final concentration 0.01mg/m1), mix；
(3) pour into culture dish after culture medium temperature is down to about 45 DEG C, into agar shape after cooling, mark culture medium title and
The date is prepared, 4 DEG C of refrigerators are put in stand-by.
Embodiment 1 (separation of Lactobacillus plantarum RD-0025 floras and inoculation, purifying, Zengjing Granule)
1st, the separation and inoculation of Lactobacillus plantarum RD-0025 floras：Subject's vaginal side is gathered with two sterile cotton swabs
Secretion on wall, is inoculated in the culture dish equipped with the MRS culture mediums prepared, and label information with various concentrations, will cultivate
Ware is placed in anaerobic jar, and is put into CO2Aerogenesis bag, is placed in 37 DEG C of incubators, is incubated more than 48h.
2nd, lactobacillus plantarum strain RD-0025 purifying, Zengjing Granule：According to bacterium colony different shape (surface, edge etc.),
Size is counted respectively, homomorphosis, it is of the same size be designated as in one kind, oese picking single bacterium colony a little bacterium, by " oblique line
The single bacterium colony that method " is seeded to MRS solid mediums to be isolated and purified；List on sterile toothpick picking MRS solid mediums
The a little bacterium of bacterium colony, is seeded to MRS fluid nutrient mediums, is placed in 37 DEG C of incubators, and Anaerobic culturel 24h-48h filters out new bacterium
Strain.
Embodiment 2 (identification and preservation of Lactobacillus plantarum RD-0025 bacterial strains)
1st, cultural character, dyeing microscopic examination and morphological feature：The bacterium colony obtained after culture such as accompanying drawing 1, bacterium colony is typically in white
Color is circular, and diameter about 3mm, intermediate projections, surface is smooth, fine and closely woven；The bacterium pure culture smear is taken to carry out Gram's staining, as a result
Such as accompanying drawing 2, Gram-positive is presented, as a result nose circle straight-bar, single, paired or short chainlike distribution shows：Separated bacterial strain is preliminary
It is determined as lactobacillus.
2nd, 16SrDNA gene orders are identified：DNA extractions are carried out with bacterial genomes DNA extraction kit, and use primer
To 27F (5 '-AGAGTTTGATCMTGGCTCAG-3 '), 1492R (5 '-TACGGYTACCTTGTTACGACTT-3 ') enters performing PCR
Amplification, takes PCR primer to carry out gel electrophoresis, determines 16SrDNA genetic fragments, single clearly PCR is obtained at about 1500bp
Product band, is shown in accompanying drawing 3.PCR primer is purified and determined dna sequence, using Sanger PCR sequencing PCRs, sequencing primer pair
For 27F/1492R, instrument ABI3730XL is sequenced, sequencing result is compared with GenBank databases, and it is homologous with Lactobacillus plantarum
Property similarity be more than 99%.The category kind finally identified is Lactobacillus plantarum.Its 16SrDNA variable region sequences are shown in sequence table SEQ
ID NO:1。
3rd, physiological and biochemical property：Pass through aesculin hydrolysis experiment, methyl red test (MR experiments), voges-Proskauer test
(VP experiments), indole experiment, triple sugar iron test, kirschner disaccharide iron tests, urease test, phenylalanine deaminase experiment,
Amino acid decarboxylase enzyme test, gelatin liquefaction test, sodium malonate experiment, citrate experiment (citrate experiment), nitrate
Reduction test, litmus milk experiment, bacterium dynamic test determine bacterial strain biochemical reactions, and acquired results are as shown in table 1：
The lactobacillus plantarum RD0025 of table 1 physio-biochemical characteristics experimental result
| Physiology and biochemistry project | As a result |
| Aesculin hydrolysis experiment | + |
| Methyl red test (MR experiments) | + |
| Voges-Proskauer test (VP experiments) | - |
| Indole is tested | - |
| Triple sugar iron test | - |
| Kirschner disaccharide iron tests | - |
| Urease test | - |
| Phenylalanine deaminase is tested | - |
| Amino acid decarboxylase enzyme test | - |
| Gelatin liquefaction test | - |
| Sodium malonate is tested | - |
| Citrate tests (citrate experiment) | - |
| Nitrate reduction test | - |
| Litmus milk is tested | - |
| Bacterium dynamic test | - |
+：Represent positive；-：Represent negative
The API 50CHL lactobacillus identification systems produced using French Mei Liai companies carry out biochemical identification, mirror to bacterial strain
Determine result statistics as shown in table 2
The lactobacillus plantarum RD0025API 50CH test bar reaction results of table 2
+：Represent positive；-：Represent negative
Thus biochemical collection of illustrative plates judges that bacterial strain biochemical characteristic meets the biochemical characteristic of Lactobacillus plantarum.
3rd, the preservation of bacterial strain
Lactobacillus plantarum (Lactobacillus plantarum) RD-0025 of the present invention is from Chinese healthy women of reproductive age
Screening is got in vaginal fluid, is preserved in on May 10th, 2017 general in China Committee for Culture Collection of Microorganisms
Logical microorganism center (abbreviation CGMCC), depositary institution address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, preservation registration number is CGMCC No.14111, and the Classification And Nomenclature of the bacterial strain is Lactobacillus plantarum
(Lactobacillus plantarum)。
Embodiment 3 (Lactobacillus plantarum RD-0025 metabolites measure)
1. lactic acid content is determined in Lactobacillus plantarum RD-0025 metabolites：Using high-efficient liquid phase technique (0.005M sulfuric acid water
(0.28ml sulfuric acid (98%) -1000ml water, pH is about that 2.1) lactic acid of this strain culturing 24-48h zymotic fluids is examined to solution
Survey, as a result indicate that lactic acid (L/D lactic acid summation) is about 30mg/mL, far above Lactobacillus delbrueckii (commercially available) lactic acid content (about
15mg/mL), lactate detection collection of illustrative plates of the invention is referring to accompanying drawing 4
2. content of hydrogen peroxide is determined in Lactobacillus plantarum RD-0025 metabolites：By Mcgroarty etc. peroxide
Enzyme process carries out hydrogen peroxide semiquantitative determination, and the Lactobacillus plantarum RD-0025 of separated identification is inoculated in into H2O2Identification of M RS-
After TMB flat boards, 37 DEG C of Anaerobic culturel 24h, flat board is taken out, thalline is exposed in atmosphere.Produce H2O2Lactobacillus bacterium colony will be changed into blue
Color, without producing H2O2Bacterium colony nondiscolouring, according to H of the Coloring Time to generation2O2Sxemiquantitative is carried out, 5min in Fig. 5, figure is as a result seen
When the existing obvious blue of bacterium colony show, a large amount of bluenesss substantially occur during 10min, it is clear that this bacterium is easy to produce hydrogen peroxide, production
Hydrogen peroxide is very capable, these results suggest that this Lactobacillus plantarum RD-0025 can significantly produce lactic acid and hydrogen peroxide, helps
Balanced in maintaining microecology in vaginas.
Embodiment 4 (antibiotic sensitivity test)
According to the requirement of antibiotic susceptibility test in the 3rd Tiny ecosystem viable bacteria product introduction of version pharmacopeia in 2015, use
AGP test paper disk method determines the sensitiveness of strains, investigates 0 generation (C0) and passes on the Lactobacillus plantarum in 30 (C30) generations
RD-0025 judges strains sensitiveness rank, measurement result to the sensitiveness of each antibiotic according to the size of inhibition zone
As shown in table 3：
The lactobacillus plantarum RD0025 of table 3 antibiotic sensitivity test result
Inhibition zone is determined as slight sensitive less than 10mm, is medium sensitivity in 10-20mm, is sensitivity more than 20mm.
Experimental data shows this bacterium to erythromycin, fleraxacin slight sensitive, quick to Meropenem and gentamicin moderate
Sense, to ampicillin, OXA, benzyl penicillin, kanamycins, tetracycline, clindamycin, erythromycin, Piperacillin, cephalo
Qusong, fleraxacin, vancomycin, amoxicillin/clavulanate, azithromycin, Amoxicillin, bacitracin are sensitive.
Accompanying drawing 6. wherein is shown in the antibiotic sensitive figure of vancomycin, gentamicin and erythromycin
Embodiment 5 (toxicity test)
5 SPF grades of Kunming mouses, every mouse vagina injection 0.5ml Lactobacillus plantarum RD-0025 suspension bacteria liquid is (big
In 1 × 109CFU/ mouse).By 2015 editions Chinese Pharmacopoeia requirements, every mouse weight is measured daily, and observe, record every small
The changes such as behavior and physiology before and after mouse injection.As a result show that all the weight of animals have increase in 7 days, have no obvious nosotoxicosis
Shape, crawler behavior is without exception, no animal dead, it is believed that the bacterial strain belongs to non-toxic type bacterial strain.
Embodiment 6 (test of Lactobacillus plantarum RD-0025 mitotic stabilities)
The present embodiment is from growth characteristics, morphology, Biochemical Characteristics, metabolin composition, antibiotic sensitive characteristic, hereditary capacity
And to this lactobacillus plantarum strain RD-0025 pass on the study on the stability in 30 generations (C30) in terms of toxotest.
1st, Lactobacillus plantarum RD-0025 isolate and purify, colony morphological observation, dyeing microscopic examination and biochemical characteristic detection method it is same
The Part I of embodiment 1 and embodiment 2.As a result show：By passage, colonial morphology is shown in that for white circular colonies, size is about
3mm, intermediate projections, surface is smooth, and significant changes do not occur, and passage is stable；Gram's staining is rendered as Gram-positive bacillus,
Dyeing microscopic examination photo does not change with 0 generation.
2nd, Genetic Analysis：The Part II of method be the same as Example 2.Respectively to Lactobacillus plantarum RD-0025 the 0th generation
(C0), the 30th generation (C30) bacterial strain carries out 16SrDNA fragments PCR amplifications, and by pcr amplification product electrophoretic analysis, purpose band is clear
And it is single, size is about 1500bp, and amplification is correct, and C0, C30 twice PCR amplification are consistent, will measure sequence and uses in NCBI
BLAST instruments be compared with the known array in GenBank databases, be Lactobacillus plantarum, homology similarity
100%.
3rd, metabolite is determined：Method be the same as Example 3, Plasma lactate content about 30mg/mL, hydrogen peroxide experiment display is each
Occurs blueness in 5min for bacterium colony, a large amount of bluenesss substantially occur during 10min, it was demonstrated that bacterial strain metabolism produces hydrogen peroxide
Ability and lactic acid producing ability are stable.
4th, antibiotic sensitivity test：Method be the same as Example 4, strains are determined using using AGP test paper disk method
Sensitiveness, judge this lactobacillus plantarum strain to erythromycin, fluorine sieve according to the scope of restraining fungi criteria for interpretation of paper disc method
Husky star slight sensitive, to Meropenem and gentamicin medium sensitivity, to ampicillin, OXA, benzyl penicillin, to block that mould
Element, tetracycline, clindamycin, erythromycin, Piperacillin, ceftriaxone, fleraxacin, vancomycin, Amoxicillin/carat dimension
Acid, azithromycin, Amoxicillin, bacitracin are sensitive.
5th, toxicity test：Method be the same as Example 5, passage 30 generations (C30) Lactobacillus plantarum RD- is injected with mouse vagina
0025 bacterium solution has carried out toxotest, wherein the concentration ＞ 10 tested9CFU/ mouse.As a result it is：In whole test mices 7 days
Poisoning symptom is had no, body weight has increase, no animal dead.According to the above results, press《New drug pharmacology, toxicological study technology will
Seek supplementary notes》, the bacterial strain belongs to non-toxic type bacterial strain.
Comprehensive, the present embodiment repeatedly passes on Lactobacillus plantarum RD-0025 with MRS medium cultures, from morphology, biochemistry
The numerous shadow planted to Lactobacillus plantarum of passage has been inquired into terms of, metabolin feature and hereditary capacity, susceptibility characteristic, toxicity test
Ring.As a result show：With MRS subcultures within 30 generations its morphology, biochemical, hereditary capacity, metabolin and susceptibility characteristic
It is consistent with initial separation bacterial strain.
Embodiment 7 (Lactobacillus plantarum RD-0025 bacterial strains pharmacodynamic experiment)
First, Lactobacillus plantarum RD-0025 bacterial strains antibacterial experiment in vitro
(1) Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii suppress the experiment of gardnerella vaginalis in vitro：Connect according to 3%
The MRS agar bacteria cakes that concentration prepares Lactobacillus plantarum RD-0025 are planted, in Anaerobic culturel 24h at 37 DEG C, commercially available De Shi is prepared with method
Lactobacillus bacteria cake；The μ L of gardnerella vaginalis 100 are taken, is inoculated in 10mL BHI solid mediums, is put into Lactobacillus plantarum RD-
0025 and Lactobacillus delbrueckii bacteria cake, 37 DEG C of Anaerobic culturel 48h；Obvious inhibition zone occurs around lactobacillus.As a result Fig. 7 is seen, its
Middle left figure is Lactobacillus plantarum RD-0025 inhibition zone effects, and vernier caliper measurement antibacterial circle diameter is 32.3mm, and right figure is De Shi
Lactobacillus inhibition zone effect, bacteriostatic diameter is 21.5mm, and conclusion is Lactobacillus plantarum RD-0025 to the antibacterial of gardnerella vaginalis
Effect is better than Lactobacillus delbrueckii.
(2) Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii suppress staphylococcus aureus in vitro, ETEC,
Pseudomonas aeruginosa and the experiment of salmonella：Lactobacillus plantarum RD-0025 MRS agar bacterium are prepared according to 3% inoculum density
Cake, in Anaerobic culturel 24h at 37 DEG C, commercially available Lactobacillus delbrueckii bacteria cake is prepared with method；By staphylococcus aureus, E
Bacterium, pseudomonas aeruginosa and salmonella are seeded in pancreas junket soya peptone liquid (TSB) agar medium, are put into Lactobacillus plantarum
RD-0025 and Lactobacillus delbrueckii bacteria cake.18-24h is cultivated in 33 DEG C, inhibition zone, vernier caliper measurement RD-0025 inhibition zones is observed
Diameter is about 22mm, and right figure is Lactobacillus delbrueckii inhibition zone effect, and bacteriostatic diameter is 12mm, and conclusion is Lactobacillus plantarum RD-
The fungistatic effect of 0025 pair of staphylococcus aureus, ETEC, pseudomonas aeruginosa and salmonella is better than De Shi breasts
Bacillus, representative diagram is shown in accompanying drawing 8-9
2nd, adhesive forces are tested：According to the lactobacillus number being attached on vaginal epithelial cell monolayer, it is determined that not
With the Adhesion property of lactobacillus.Method is as follows：Take human vagina epithelial cell Vk2/E6E7 and human cervical cancer epithelial cell
Hela, cell is inoculated in 12 orifice plates with 500,000 density per hole, after VK2/E6E7 formation monolayer after 48 hours；
Commercially available lactobacillus (Lactobacillus delbrueckii) and Lactobacillus plantarum RD-0025 are separately added into the CFU of varying number per hole, adhesion 4 is small
When, gently vibrated on shaking table in adhesion process, each group is respectively equipped with two parallel laboratory tests；After adhesion terminates, 1ml is used
0.05%tritonX-100 cell lysis, is made suspension bacteria liquid, dilution, takes 100ul bacterium solutions to be equably inoculated in MRS fine jades respectively
On fat culture medium flat plate；After Anaerobic culturel 48 hours, clone's number of each flat board is counted.
As a result show：Lactobacillus plantarum RD-0025 4h adherence rates are respectively 35.2% and 45.3%, commercially available similar De Shi
Lactobacillus strain 4h adherence rates are respectively 23.6% and 20.7%, and Lactobacillus plantarum RD-0025 adhesive force is higher than commercially available similar
Lactobacillus Lactobacillus delbrueckii bacterial strain.
3rd, rabbit vagina field planting experiment
1st, experimental method
10 healthy animals of selection carry out stratified random packet by body weight, are divided into 2 groups, positive controls animal 5, experiment
Group 5：
Field planting is prepared with Lactobacillus plantarum RD-0025：Lactobacillus plantarum RD-0025 freeze-drying bacterium powder is weighed, it is fixed to make
Plant amount is 106.Control group is used under listing product (Lactobacillus delbrueckii capsule), aseptic technique, each to add MRS fluid nutrient mediums
0.5mL, is well mixed, and vagina is implanted into after all being drawn with vaginal administration device.
It is colonized modeling and sampling：After the monkey menstruation of normal menstrual cycle can be observed, continuous 7 days implantation modeling bacterium.
The vagina of animal is once observed weekly, the color of vaginal fluid, character and secretory volume and measure vaginal secretion is checked
Thing pH, takes 2 aseptic cotton carrier samplings, wherein a cotton swab is used for vaginal fluid cleannes microscopy, another cotton swab is used for bacterium
Cluster analysis.
The separation and purifying culture of vagina bacterium：By the vaginal fluid of collection, vibrated in 2mL D-Hanks buffer solutions,
Gradient dilution is done with phosphate buffer, is respectively coated on MRS agar plates and at 37 DEG C, under anaerobic condition cultivate 24~
48h.The information such as record colonial morphology, the bacterium colony that MRS agar plates are rule again to be purified simultaneously carries out biochemical and molecule mirror
It is fixed.
Molecular biology method identifies (16SrDNA gene sequencings)：The separated bacterial strain being purified into is carried out
16SrDNA sequence amplification, sequencing and analysis, gel extraction method pair is used by the pcr amplification product for being accredited as 16SrDNA fragments
Purpose fragment is sequenced after purification.The 16SrDNA gene orders measured are used into the BLAST instruments and GenBank in NCBI
Known array in database is compared, and homology is 99%, is accredited as same species.
2nd, experimental result and analysis
Vagina mucosa and secretion overview：Observed weekly once after implantation, as a result find all experimental animal vaginas
Mucosal secretions do not find obvious exception.
Vaginal fluid pH value is determined：After Lactobacillus plantarum RD-0025 implantation, most of test group of animals vaginal secretion
Thing pH value is significantly lower than listing product control group, and relative to being decreased obviously before implantation, pH value measurement result is as shown in table 4.
The lactobacillus plantarum RD0025 of table 4 influences result of the test to experimental animal vaginal fluid pH
Vaginal fluid cleannes：Compared before and after implantation, control group vagina miscellaneous bacteria quantity substantially increases, cleannes reduction；
And experimental group Lactobacillus plantarum RD-0025 implantation after, vaginal fluid cleannes are substantially better than control group, it is seen that quantity is not
Deng Gram-positive bacterium vaginae, cleannes are apparently higher than control group.The vaginal fluid cleannes result of determination such as institute of table 5
Show.
The lactobacillus plantarum RD0025 of table 5 influences result of the test to experimental animal vaginal fluid cleannes
I~III is clean-up performance, and III represents that cleanliness factor is minimum.
The progress amplification of experimental group secretion is isolated and purified, analyzed by vaginal flora, from the whole 3 animal the moon of experimental group
Test plant lactobacillus RD-0025 is found in road secretion, the test plant lactobacillus RD-0025 isolated information is as schemed
Shown in 10.It is seen that find test plant lactobacillus RD-0025 in animal subject vaginal fluid, therefore can be with
Find out that Lactobacillus plantarum RD-0025 can effectively be colonized in Chinese machin intravaginal.
Embodiment 8 (Lactobacillus plantarum RD-0025 is lyophilized to be preserved and freeze-dried powder stability)
In order to detect survival rates of the Lactobacillus plantarum RD-0025 in the case of fermenting and be lyophilized, by Lactobacillus plantarum RD-
0025 grows in pH 6.5 modified MRS culture medium, uses the ferment tank of 100 liters of scales.In plateau harvested earlier
Thalline, its viable count reaches about 3 × 109CFU/ml.Thalline is collected by centrifuging, after being washed with phosphate buffer, with jelly
Dry protective agent (including skimmed milk power and sucrose etc.) mixing.Then, mixture is placed in freeze drier and be freeze-dried.Sample
Freeze about 2 hours, be then dried in vacuo 20-30 hours at -20 DEG C at -40 DEG C, then it is dry 3-5 hours at 30 DEG C.Dry powder
Dispensed with aluminium foil bag, and be stored in 4 DEG C and room temperature (25 DEG C).Viable bacteria is determined by plate count in the 0th, 1,3,6,9, December
Number.
Initial every gram of dry powder of Lactobacillus plantarum RD-0025 contains up to 50,000,000,000 viable bacterias (5 × 1010Cfu/g), at 4 DEG C
With optimal storage stability.After being stored 6 months at 4 DEG C, retain the 80% of initial viable count, be shown in Table 6.
The lactobacillus plantarum RD0025 freeze-dried powder microbial inoculum stability test results of table 6
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.
SEQUENCE LISTING
<110>Guangdong Qiangji Pharmaceutical Co., Ltd.；Guangdong Long Chuanji pharmaceutcal corporation, Ltds；Zhao Xiangjiang
<120>A kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines
<160> 1
<210> 1
<211> 1471
<212> DNA
<213>Artificial sequence
<400> 1
aatctctggtccacttaggcggctggttctaaaaggttac 40
cccaccgactttgggtgttacaaactctcatggtgtgacg 80
GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCA 120
TGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGG 160
CGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAG 200
AGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCA 240
TCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCAT 280
GATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCAC 320
CGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACT 360
GATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA 400
TCTCACGACACGAGCTGACGACAACCATGCACCACCTGTA 440
TCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGC 480
ATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTC 520
GAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT 560
CAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAG 600
GCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGG 640
AAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGA 680
CTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCG 720
AGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCC 760
ACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACA 800
CATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCA 840
GTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCAC 880
ATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAA 920
TAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 960
TGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACC 1000
GTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTT 1040
AACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCAC 1080
GCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGAT 1120
TCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTC 1160
AGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGT 1200
ATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAA 1240
TACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATC 1280
TTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTA 1320
TTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCA 1360
GGTTTCCCACGTGTTACTCACCAGTTCGCCActcactcaa 1400
atgtaaatcatgatgcaagcaccaatcaataccagagttc 1440
gttcgactgcatgtatagcacgccgcacctc 1471
Claims (9)
1. a kind of Lactobacillus plantarum, its deposit number is CGMCC No.14111.
2. a kind of DNA molecular, it is characterised in that the DNA molecular extracts from Lactobacillus plantarum described in claim 1, using it as mould
Version utilizes the amplification of 16SrDNA universal primers, amplified production sequence and both Lactobacillus plantarum base sequences in GenBank databases
Homology similarity is more than 98%.
3. Lactobacillus plantarum described in claim 1 is used for the application for preparing vaginal pathogenic antibacterial medicines.
4. application according to claim 3, it is characterised in that the medicine is microbial inoculum, the microbial inoculum is colonized in vaginal environment
And survive on vaginal epithelial cell.
5. application according to claim 3, it is characterised in that the medicine is microbial inoculum, the microbial inoculum is metabolized in vaginal environment
Produce H2O2。
6. application according to claim 3, it is characterised in that the pathogenic bacteria are Gardnerella vaginalis or atropic ripple bacterium.
7. application according to claim 3, it is characterised in that the pathogenic bacteria are Candida albicans, Staphylococcus aureus
Bacterium, ETEC, pseudomonas aeruginosa or salmonella.
8. Lactobacillus plantarum described in claim 1 is used to prepare vaginal disease prevention or the application of medicine.
9. application according to claim 8, it is characterised in that the vaginal disease is VVC, drop
Worm property vaginitis, senile vahinitis, non-specific vagina infection or Combination vagina infection.
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| CN108295096A (en) * | 2017-12-21 | 2018-07-20 | 天津科睿恒生物科技有限公司 | Application of the bacterial strain in prevention and treatment mycotic, bacterial vaginitis preparation |
| CN109439592A (en) * | 2018-12-06 | 2019-03-08 | 青岛东海药业有限公司 | Lactobacillus plantarum preparation and its application |
| CN110016442A (en) * | 2019-02-21 | 2019-07-16 | 河北一然生物科技有限公司 | The application of Lactobacillus rhamnosus and its composite bacterium powder in prevention and treatment vaginitis product |
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| CN108295096A (en) * | 2017-12-21 | 2018-07-20 | 天津科睿恒生物科技有限公司 | Application of the bacterial strain in prevention and treatment mycotic, bacterial vaginitis preparation |
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| CN112313325B (en) * | 2018-10-12 | 2024-03-08 | 株式会社Atogen | Novel lactobacillus plantarum strain and composition for preventing or treating vaginitis comprising the same |
| CN109439592A (en) * | 2018-12-06 | 2019-03-08 | 青岛东海药业有限公司 | Lactobacillus plantarum preparation and its application |
| CN110016442A (en) * | 2019-02-21 | 2019-07-16 | 河北一然生物科技有限公司 | The application of Lactobacillus rhamnosus and its composite bacterium powder in prevention and treatment vaginitis product |
| CN110777087A (en) * | 2019-08-09 | 2020-02-11 | 四川厌氧生物科技有限责任公司 | Lactobacillus johnsonii and application thereof |
| CN112342154A (en) * | 2020-09-03 | 2021-02-09 | 上海上药信谊药厂有限公司 | Probiotics for preventing and treating female genital tract inflammation |
| CN112342154B (en) * | 2020-09-03 | 2022-08-05 | 上海上药信谊药厂有限公司 | Probiotics for preventing and treating female genital tract inflammation |
| CN113699071A (en) * | 2021-08-30 | 2021-11-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicine for treating infectious vaginosis |
| CN114869917A (en) * | 2022-07-07 | 2022-08-09 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus plantarum N13 in preparation of medicine for preventing and treating vaginitis or inhibiting pathogenic bacteria |
| WO2024032452A1 (en) * | 2022-08-09 | 2024-02-15 | 成都倍特生物制药有限公司 | Lactiplantibacillus plantarum strain and use thereof |
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