A kind of Lactobacillus rhamnosus and its application for being used to prepare vagina antibacterial medicines
Technical field
The present invention relates to microbial technology field, adjusting and bacterial vaginosis disease further to microecology in vaginas environment
The treatment of disease, and in particular to a kind of Lactobacillus rhamnosus and its application for being used to prepare vagina antibacterial medicines.
Background technology
For healthy women intravaginal there are multiple-microorganism, they constitute mutually restriction, mutually association between host, environment
Adjust, the microecology in vaginas system of dynamic equilibrium.The vaginal flora of healthy women is mainly made of lactobacillus, including plant breast bar
Bacterium, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus rhamnosus etc..Newborn bar under normal circumstances
Bacterium can play a protective role 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
A large amount of numerous plants of his conditionity pathogenic microorganism such as Gardnerella, various anaerobic bacterias, bending vibrios etc., usual BV be actually with
A kind of mixed infection based on bacterium protein of Gardnerella vaginalis.Using antibiosis extract for treating, 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
An important factor for by pathogenic infection, the acid and some antimicrobial agents that in addition lactobacillus metabolism produces can also effectively inhibit
The numerous plant of growth of other bacteriums.
Healthy women intravaginal has individual difference, and anti-pathogenic bacteria energy between each strain of lactobacillus there are a variety of lactobacillus
Force difference is different obvious., it is necessary to consider the species of lactobacillus when selecting 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.It is not actually that China's woman vagina is excellent that market, which has product " Lactobacillus delbrueckii ", at present
Gesture flora, colonization ability is poor, can not maintain stable viable bacteria content, it is impossible to meet the needs of gynecological clinic.
Lactobacillus rhamnosus (Lactobacillus rhamnosus), is lactobacillus, is one of Body normal flora,
It is distributed widely in human body intestinal canal, is also distributed in vagina.Based on current research, Lactobacillus rhamnosus is mainly used for
Adjust intestinal flora, prevention and treatment diarrhea, exclude toxin, pre- anti-caries and anticancer etc..Thus for conventional bacterial strain and
Speech, Lactobacillus rhamnosus does not have specific bacteriostasis in the category of cognition of the prior art.
The content of the invention
The present invention is intended to provide a kind of specific Lactobacillus rhamnosus, to solve conventional Lactobacillus rhamnosus in the prior art
Technical problem without vaginal pathogenic rejection ability.
Another technical problem to be solved by the present invention is that conventional Lactobacillus rhamnosus production hydrogen peroxide manufacture ability is relatively low.
The invention solves another technical problem be conventional Lactobacillus rhamnosus lactic acid production it is relatively low.
The invention solves another technical problem be in the prior art conventional Lactobacillus rhamnosus colonizing in vagina,
Survival benefit is bad.
The invention solves another technical problem be in the prior art be directed to vaginal pathogenic microbial inoculum class antibacterial medicines
Therapeutic effect is bad.
To realize above technical purpose, the present invention uses following technical scheme:
A kind of separated Lactobacillus rhamnosus (Lactobacillus rhamnosus), it is RD- to be numbered in of the invention
0060 (Lactobacillus rhamnosus), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its preservation is compiled
Number it is CGMCC No.14184.
Above-mentioned Lactobacillus rhamnosus (Lactobacillus rhamnosus) RD-0060 is cloudy for the China's Healthy women of child-bearing age
Screening is got in road secretion, has been preserved in on May 24th, 2017 common in China Committee for Culture Collection of Microorganisms
Microorganism center (abbreviation CGMCC), depositary institution address are:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, preservation registration number are CGMCC No.14184, and the Classification And Nomenclature of the bacterial strain is Lactobacillus rhamnosus
(Lactobacillus rhamnosus)。
Above-mentioned separated Lactobacillus rhamnosus RD-0060, is sequenced using 16SrDNA, with mouse in GenBank databases
Lee's sugar lactobacillus base sequence homology score value is more than 98%.
On the basis of above technical scheme, invention further provides above-mentioned Lactobacillus rhamnosus to be used to prepare vagina
The application of pathogenic bacteria antibacterial medicines.
Preferably, the medicine is microbial inoculum, which colonizes and survives on vaginal epithelial cell in vaginal environment.
Preferably, the medicine is microbial inoculum, which 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, escherichia coli, the false list of verdigris
Born of the same parents bacterium or salmonella.
Meanwhile invention further provides above-mentioned Lactobacillus rhamnosus to be used to prepare vaginal disease prevention or medicine
Application.
Preferably, the vaginal disease is vulvovaginal candidiasis, trichomonas vaginitis, senile vagina
Scorching, non-specific vagina infection or Combination vagina infection.
The isolated first one plant of Lactobacillus rhamnosus of the present invention, studies its and is metabolized performance and find that the bacterial strain possesses excellent
Good lactic acid production capacity and production hydrogen peroxide energy, it is very strong to the bacteriostasis of pathogenic bacteria, while there is prominent vagina epithelium
Cell adherence ability.The new property found based on more than, present invention determine that using it the new application of vagina antibacterial medicines is prepared,
Realize the treatment of various bacterial vaginal disease.
The beneficial effect produced using above-mentioned technical proposal is:(1) Lactobacillus rhamnosus RD-0060 bacterial strains of the invention
It can for a long time preserve, and resist bacterial vaginosis BV and various vagina infections, including candida albicans vaginitis, gonorrhoea, disease
Toxicity vaginitis, and urethral infection etc..(2) bacterial strain of the invention is directly collected in healthy human body, has what is actively stablized
Biological characteristics, without domestication and rejuvenation technique, is directly entered preparation process.(3) bacterial strain of the invention, which has, suppresses to add
Moral receives Salmonella, the effect that suppresses Candida albicans, and compared with commercially available control bacterium, advantageous vaginal epithelial cell sticks
Power, has the advantageous ability in the field planting of primate vagina.
Brief description of the drawings
Fig. 1 is the full face of Lactobacillus rhamnosus RD-0060 colonial morphologies in the specific embodiment of the invention;
Fig. 2 is the gram stain microscopy photo of Lactobacillus rhamnosus RD-0060 in the specific embodiment of the invention;
Fig. 3 is the 16SrDNA gene PCR amplified productions of Lactobacillus rhamnosus RD-0060 in the specific embodiment of the invention
Electrophoretogram;
Fig. 4 is the lactate detection collection of illustrative plates of Lactobacillus rhamnosus RD-0060 in the specific embodiment of the invention;
Fig. 5 is Lactobacillus rhamnosus RD-060 hydrogen peroxide colour developing 5min and 10min figures in the specific embodiment of the invention
Piece;
Fig. 6 be in the specific embodiment of the invention Lactobacillus rhamnosus RD-0060 to kanamycins, fleraxacin and A Mo
The antibiotic susceptibility test bacterium colony figure of XiLin/clavulanic acid;
Fig. 7 be in the specific embodiment of the invention Lactobacillus rhamnosus RD-0060 (left side) and Lactobacillus delbrueckii (right side) to the moon
The fungistatic effect photo of road Gardnerella;
Fig. 8 be in the specific embodiment of the invention Lactobacillus rhamnosus RD-0060 (left side) and Lactobacillus delbrueckii (right side) to big
The fungistatic effect photo of the uncommon bacterium of intestines angstrom;
Fig. 9 be in the specific embodiment of the invention Lactobacillus rhamnosus RD-0060 (left side) and Lactobacillus delbrueckii (right side) to gold
The staphylococcic fungistatic effect photo of color;
Figure 10 is that Lactobacillus rhamnosus RD-0060 is colonized machin the moon after machin vagina in the specific embodiment of the invention
The electrophoretogram of road microbiota part bacterial strain 16SrDNA fragment pcr amplification products;
Figure 11 is the scanning electron microscope (SEM) photograph of Lactobacillus rhamnosus RD-0060 of the present invention.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiments to belonging to known structure or function.In addition to being defined, institute in following embodiments
Technical and scientific term has 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 embodiments;The experiment
Method, is conventional method unless otherwise specified;Quantitative test in following embodiments, is respectively provided with three repeated experiments, as a result
It is averaged;% in following embodiments, is mass percentage unless otherwise instructed.
Used Bacteria Culture based component and preparation method are as follows in following embodiments:
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) treat that culture medium temperature is cooled to room temperature and pour into culture dish, it is a according to culture dish size about 10ml/ or 20ml/;
(5) in being operated in super-clean bench.Into agar shape after cooling, mark culture medium title and prepare the date, be put in 4 DEG C of refrigerators
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 stand-by to be put in 4 DEG C of refrigerators.
3rd, hydrogen peroxide (H2O2) identification culture medium preparation:
(1) with meat soup solid medium (MRS) preparation steps (1) to (4);
(2) take out after pot no pressure subject to sterilization, slightly cool down, but still it is (dense eventually in adding TMB in super-clean bench when being 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, it is stand-by to be put in 4 DEG C of refrigerators.
Embodiment 1 (separation of Lactobacillus rhamnosus RD-0060 floras and inoculation, purifying, Zengjing Granule)
1st, the separation and inoculation of Lactobacillus rhamnosus RD-0060 floras:Subject's vagina is gathered with two sterile cotton swabs
Secretion on side wall, 24 it is interior when small be inoculated in the culture dish equipped with prepared MRS culture mediums with various concentrations, and mark letter
Breath, culture dish is placed in anaerobic jar, and be put into CO2Aerogenesis bag, is placed in 37 DEG C of incubators, is incubated more than 48h.
2nd, the purifying of Lactobacillus rhamnosus strain RD-0060, Zengjing Granule:According to bacterium colony different shape (surface, edge
Deng), size count respectively, homomorphosis, it is of the same size be denoted as one kind, a little bacterium in oese picking single bacterium colony, presses
The single bacterium colony that " oblique line method " is seeded to MRS solid mediums to be isolated and purified;On bacterium toothpick picking MRS solid mediums
The a little bacterium of single bacterium colony, is seeded to MRS fluid nutrient mediums, is placed in 37 DEG C of incubators, Anaerobic culturel 24h-48h, filters out new
Bacterial strain.
Embodiment 2 (identification and preservation of Lactobacillus rhamnosus RD-0060 bacterial strains)
1st, cultural character, dyeing microscopic examination and morphological feature:The bacterium colony obtained after culture such as attached drawing 1, bacterium colony is generally in milk
White, circle, neat in edge, surface are smooth, semi-cylindrical hill, opaque;The bacterium pure culture smear is taken to carry out gram dye
Color, as a result such as attached drawing 2, is presented Gram-positive, rod-short, holds blunt circle, in short catenation, the results showed that:Separated bacterial strain
Preliminary judgement is 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 '), carries out PCR
Amplification, takes PCR product to carry out gel electrophoresis, determines 16SrDNA genetic fragments, single clearly PCR is about being obtained at 1500bp
Product band, is shown in attached drawing 3.PCR product 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, it is same with Lactobacillus rhamnosus
Property similarity in source is more than 99%.The category kind finally identified is Lactobacillus rhamnosus.The variable region sequences of its 16SrDNA 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 measure bacterial strain biochemical reactions, acquired results are as shown in table 1:
The physio-biochemical characteristics experimental result of 1 rhamnose lactic acid bacteria RD0060 of table
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 |
Solidification production acid |
Bacterium dynamic test |
- |
+:Represent positive;-:Represent negative
Biochemical identification, mirror are carried out to bacterial strain using the API 50CHL lactobacillus identification systems of French Mei Liai companies production
It is as shown in table 2 to determine result statistics
2 rhamnose lactic acid bacteria RD0060 API 50CH test bar reaction results of table
+:Represent positive;-:Represent negative;d:Represent weakly positive
Thus biochemical collection of illustrative plates judges that bacterial strain biochemical characteristic meets the biochemical characteristic of Lactobacillus rhamnosus.
3rd, the preservation of bacterial strain
Lactobacillus rhamnosus (Lactobacillus rhamnosus) RD-0060 of the present invention is from China's Healthy reproduction age woman
Screening is got in female's vaginal fluid, is preserved in on May 24th, 2017 in China Committee for Culture Collection of Microorganisms
Common micro-organisms center (abbreviation CGMCC), depositary institution address are:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology of institute, preservation registration number are CGMCC No.14184, and the Classification And Nomenclature of the bacterial strain is Lactobacillus rhamnosus
(Lactobacillus rhamnosus)。
Embodiment 3 (Lactobacillus rhamnosus RD-0060 metabolites measure)
1. lactic acid content measures in Lactobacillus rhamnosus RD-0060 metabolites:Using high-efficient liquid phase technique (0.005M sulfuric acid
(0.28ml sulfuric acid (98%) -1000ml water, pH are about that 2.1) lactic acid of this strain culturing 24-48h zymotic fluids is carried out to aqueous solution
Detection, as a result indicate that lactic acid (L/D lactic acid summation) is about 35mg/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 attached drawing 4
2. content of hydrogen peroxide measures in Lactobacillus rhamnosus RD-0060 metabolites:By the peroxidating of Mcgroarty etc.
Thing enzyme process carries out hydrogen peroxide semiquantitative determination, and the Lactobacillus rhamnosus RD-0060 of separated identification is inoculated in H2O2Identification
MRS-TMB tablets, after 37 DEG C of Anaerobic culturel 24h, take out tablet, expose thalline in atmosphere.Produce H2O2Lactobacillus bacterium colony will be changed into
Blueness, without producing H2O2Bacterium colony is non-discolouring, the H according to Coloring Time to generation2O2Sxemiquantitative is carried out, the result is shown in Fig. 5, in figure
Bacterium colony has obvious blue and shows during 5min, and a large amount of bluenesss are obvious during 10min occurs, it is clear that this bacterium is easy to produce peroxidating
Hydrogen, production hydrogen peroxide is very capable, these results suggest that this Lactobacillus rhamnosus RD-0060 can produce lactic acid and hydrogen peroxide,
Help to maintain microecology in vaginas balance.
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 2010, use
AGP test paper disk method measures the sensitiveness of strains, investigated for 0 generation and passes on the Lactobacillus rhamnosus RD-0060 in 30 generations
To the sensitiveness of each antibiotic, strains sensitiveness rank, the measurement result such as institute of table 3 are judged according to the size of inhibition zone
Show:
The antibiotic sensitivity test result of 3 rhamnose lactic acid bacteria RD0060 of table
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 bacitracin, fleraxacin and vancomycin slight sensitive;To oxacillin, to block that mould
Element, Meropenem, gentamicin medium sensitivity;West is drawn to ampicillin, benzyl penicillin, tetracycline, clindamycin, erythromycin, piperazine
Woods, ceftriaxone, amoxicillin/clavulanate, azithromycin and Amoxicillin are sensitive.
Attached drawing 6. wherein is shown in the antibiotic sensitive figure of kanamycins, fleraxacin and amoxicillin/clavulanate
Embodiment 5 (toxicity test)
5 SPF grades of Kunming mouses, every fresh Lactobacillus rhamnosus RD-0060 of mouse peritoneal injection 0.3ml suspend
Bacterium solution (is more than 1 × 109CFU/ mouse).By 2015 editions Chinese Pharmacopoeia requirements, every mouse weight is measured daily, and is observed, remembered
Record the changes such as every front and rear behavior of mouse injection and physiology.All the weight of animals have increase in the results show 7 days, have no bright
Aobvious poisoning symptom, 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 rhamnosus RD-0060 mitotic stabilities)
The present embodiment is from growth characteristics, morphology, Biochemical Characteristics, metabolin component, antibiotic sensitive characteristic, hereditary capacity
And toxotest etc. has carried out this Lactobacillus rhamnosus strain RD-0060 the study on the stability of passage 30 generations (C30).
1st, Lactobacillus rhamnosus RD-0060 isolate and purify, colony morphological observation, dyeing microscopic examination and biochemical characteristic detection method
With the Part I of embodiment 1 and embodiment 2.The result shows that:By passage, colonial morphology is shown in in off-white color, circular, edge
Neatly, surface is smooth, semi-cylindrical hill, opaque, significant changes does not occur, passage is stablized;It is blue that Gram's staining is rendered as leather
Family name's positive bacillus, dyeing microscopic examination photo do not change with 0 generation.
2nd, Genetic Analysis:Part II of the method with embodiment 2.Respectively to the 0th of Lactobacillus rhamnosus RD-0060 the
In generation (C0), the 30th generation (C30) bacterial strain, carry out 16SrDNA fragment PCR amplifications, and pcr amplification product electrophoretic analysis, purpose band is clear
Clear and single, size is about 1500bp, and amplification is correct, and C0, C30 twice PCR amplification are consistent, will measure sequence and use NCBI
In BLAST instruments be compared with the known array in GenBank databases, be Lactobacillus rhamnosus, homology phase
Like degree 100%.
3rd, metabolite measures:Method is each with embodiment 3, Plasma lactate content about 35mg/mL, hydrogen peroxide experiment display
Occurs blueness in 5min for bacterium colony, a large amount of bluenesss are obvious during 10min occurs, it was demonstrated that bacterial strain metabolism produces hydrogen peroxide
Ability and lactic acid producing ability are stablized.
4th, antibiotic sensitivity test:Method is with embodiment 4, using using AGP test paper disk method measure strains
Sensitiveness, judge this lactobacillus rhamnosus strain to bacitracin, fluorine according to the scope of restraining fungi criteria for interpretation of paper disc method
Luo Shaxing and vancomycin slight sensitive, to oxacillin, kanamycins, Meropenem, gentamicin medium sensitivity, to ammonia benzyl
XiLin, benzyl penicillin, tetracycline, clindamycin, erythromycin, Piperacillin, ceftriaxone, amoxicillin/clavulanate, Archie
Mycin and Amoxicillin are sensitive.
5th, toxicity test:Method with embodiment 5, with mouse peritoneal injection to the C0 of this Lactobacillus rhamnosus RD-0060,
C30, for bacterial strain carried out toxotest, wherein, the concentration > 10 of test9CFU/ mouse.As a result it is:Whole test mices 7 days
Poisoning symptom is inside showed no, weight has increase, no animal dead.According to the above results, press《New drug pharmacology, toxicological study skill
Art requires supplementary notes》, which belongs to non-toxic type bacterial strain.
Comprehensive, the present embodiment repeatedly passes on Lactobacillus rhamnosus RD-0060 with MRS medium cultures, from morphology, life
Chemical, metabolin feature and hereditary capacity, susceptibility characteristic, toxicity test etc. have inquired into the numerous plant of passage to Lactobacillus rhamnosus
Influence.The result shows that:With MRS subcultures within 30 generations its morphology, biochemical, hereditary capacity, metabolin and susceptibility
Characteristic is consistent with initial separation bacterial strain.
Embodiment 7 (Lactobacillus rhamnosus RD-0060 bacterial strains pharmacodynamic experiment)
First, Lactobacillus rhamnosus RD-0060 bacterial strains antibacterial experiment in vitro
(1) Lactobacillus rhamnosus RD-0060 and Lactobacillus delbrueckii suppress the experiment of gardnerella vaginalis in vitro:According to 3%
Inoculum density prepares the MRS agar bacteria cakes of Lactobacillus rhamnosus RD-0060, and the Anaerobic culturel 24h at 37 DEG C, prepares commercially available with method
Lactobacillus delbrueckii bacteria cake;100 μ L of gardnerella vaginalis are taken, are inoculated in 10mL BHI solid mediums, are put into rhamnose breast bar
Bacterium RD-0060 and Lactobacillus delbrueckii bacteria cake, 37 DEG C of Anaerobic culturel 48h;Obvious inhibition zone occurs around lactobacillus.The result is shown in figure
7, wherein left circle is Lactobacillus rhamnosus RD-0060 inhibition zone effects, vernier caliper measurement antibacterial circle diameter is 31.3mm, right circle
For Lactobacillus delbrueckii inhibition zone effect, bacteriostatic diameter 26.3mm, conclusion is Lactobacillus rhamnosus RD-0060 to vagina Gardner
The fungistatic effect of bacterium is better than Lactobacillus delbrueckii.
(2) Lactobacillus rhamnosus RD-0060 and Lactobacillus delbrueckii suppress the experiment of pathogenic bacteria in vitro:It is dense according to 3% inoculation
Degree prepares the MRS agar bacteria cakes of Lactobacillus rhamnosus RD-0060, and the Anaerobic culturel 24h at 37 DEG C, commercially available De Shi breasts are prepared with method
Bacillus bacteria cake;By staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and salmonella are seeded in pancreas junket soya peptone
Liquid (TSB) agar medium, is put into Lactobacillus rhamnosus RD-0060 and Lactobacillus delbrueckii bacteria cake.18-24h is cultivated in 33 DEG C,
Inhibition zone is observed, vernier caliper measurement RD-0060 antibacterial circle diameters are about 22mm, and right figure is Lactobacillus delbrueckii inhibition zone effect,
Bacteriostatic diameter is 12mm, and conclusion is false to staphylococcus aureus, escherichia coli, verdigris for Lactobacillus rhamnosus RD-0060
Monad and the fungistatic effect of salmonella are better than Lactobacillus delbrueckii, and representative diagram is shown in attached drawing 8-9
2nd, adhesive forces are tested:According to the lactobacillus number being attached on vaginal epithelial cell monolayer, determine 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 per the density in holes, when 48 is small after VK2/E6E7 form monolayer;
Commercially available lactobacillus (Lactobacillus delbrueckii) and Lactobacillus rhamnosus RD-0060, adhesion 4 are separately added into the CFU of varying number per hole
Hour, gently vibrated on shaking table in adhesion process, each group is respectively equipped with two parallel laboratory tests;After adhesion, 1ml is used
0.05%tritonX-100 cell lysis, is made suspension bacteria liquid, and dilution, takes 100 μ l bacterium solutions to be equably inoculated in MRS fine jades respectively
On fat culture medium flat plate;After when Anaerobic culturel 48 is small, clone's number of each tablet is counted.
The results show:The 4h adherence rates of Lactobacillus rhamnosus RD-0060 are respectively 35.2% and 45.3%, commercially available similar moral
Family name's lactobacillus strain 4h adherence rates are respectively 23.6% and 20.7%, and the adhesive force of Lactobacillus rhamnosus RD-0060 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 weight, are divided into 2 groups, positive controls animal 5, tests
Group 5:
Field planting is prepared with Lactobacillus rhamnosus RD-0060:The freeze-drying bacterium powder of Lactobacillus rhamnosus RD-0060 is weighed,
It is 10 to make field planting amount6.Control group uses listing product (Lactobacillus delbrueckii capsule), each to add the training of MRS liquid under aseptic technique
Base 0.5mL is supported, is uniformly mixed, 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, checks the color of vaginal fluid, character and secretory volume and measure vaginal secretion
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, MRS agar plates of ruling again are with the bacterium colony that is purified and carry out biochemical and molecule and reflect
It is fixed.
Molecular biology method identifies (16SrDNA gene sequencings):The separated bacterial strain being purified into is carried out
Sequence amplification, sequencing and the analysis of 16SrDNA, 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 is greater than or equal to 99% when comparing homology, is then accredited as same species.
2nd, experimental result and analysis
Vagina mucosa and secretion overview:Observed once weekly after implantation, it turns out that all experimental animal vaginas
Mucosal secretions do not find obvious exception.
Vaginal fluid pH value measures:After Lactobacillus rhamnosus RD-0060 implantation, most of test group of animals vagina point
Secretion 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.
4 Lactobacillus rhamnosus RD-0060 of table 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, and cleannes reduce;
And experimental group Lactobacillus rhamnosus RD-0060 implantation after, vaginal fluid cleannes are substantially better than control group, it is seen that quantity
The Gram-positive bacterium vaginae not waited, cleannes are apparently higher than control group.Vaginal fluid cleannes judge the result such as institute of table 5
Show.
5 Lactobacillus rhamnosus RD-0060 of table 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.
Experimental group secretion is carried out amplification to isolate and purify, is analyzed by vaginal flora, from the whole 3 animal the moon of experimental group
The information for examination Lactobacillus rhamnosus RD-0060 for examination Lactobacillus rhamnosus RD-0060, isolated is found in road secretion
As shown in Figure 10.It is seen that found in animal subject vaginal fluid for examination Lactobacillus rhamnosus RD-0060, because
This can be seen that Lactobacillus rhamnosus RD-0060 can effectively be colonized in Chinese machin intravaginal.
Embodiment 8 (Lactobacillus rhamnosus RD-0060 freezes preservation and freeze-dried powder stability)
In order to detect survival rates of the Lactobacillus rhamnosus RD-0060 in the case of fermenting and is lyophilized, by Lactobacillus rhamnosus
RD-0060 is grown in the modified MRS culture medium of pH 6.5, uses the ferment tank of 100 liters of scales.Received in early stage plateau
Collect thalline, its viable count reaches about 3 × 109CFU/ml.Thalline is collected by centrifuging, after being washed with phosphate buffer, with
Freeze drying protectant (including skimmed milk power and sucrose etc.) mixes.Then, mixture is placed in freeze drier and be freeze-dried.Sample
Product freezed at -40 DEG C about 2 it is small when, be then dried in vacuo at -20 DEG C 20-30 it is small when, then 35 DEG C it is dry 3 it is small when.Dry powder
Dispensed in the aluminium foil bag for being put into drier, and be stored in 2-8 DEG C and room temperature (25 DEG C).Surveyed in the 0th, 3, June by plate count
Determine viable count.
Initial every gram of dry powder of Lactobacillus rhamnosus RD-0060 contains up to 82,000,000,000 viable bacterias (8.2 × 1010Cfu/g), exist
There is optimal storage stability at 2-8 DEG C.After being stored 6 months at 2-8 DEG C, retain the 64.6% of initial viable count, be shown in Table 6.
6 Lactobacillus rhamnosus RD-0060 freeze-dried powder microbial inoculum stability test results of table
The embodiment of the present invention is 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 all any modification, equivalent and improvement done in the application range of the present invention etc., should all
Within protection scope of the present invention.
Sequence table
<110>Zhao Xiangjiang
<120>A kind of lactobacillus gasseri and its application for being used to prepare vagina antibacterial medicines
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1480
<212> DNA
<213>Lactobacillus gasseri (Lactobacillus gasseri)
<400> 1
attcctggtc taccttagac ggctgactcc tataaagggt tatcccaccg gctttgggtg 60
ttacagactc tcatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 120
gcgtgctgat ccgcgattac tagcgattcc agcttcgtgt aggcgagttg cagcctacag 180
ttcgaactga gaacggcttt cagagatccg cttaccttcg caggttcgct tctcgttgta 240
ccgtccattg tagcacgtgt gtagcccagg tcataagggg catgatgact tgacgtcatc 300
cccaccttcc tccggtttgt caccggcagt ctcattagag tgcccaactt aatgatggca 360
actaatgaca agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 420
acgacagcca tgcaccacct gtctcagcgt ccccgaaggg aacacctaat ctcttaggtt 480
tgcactggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct 540
ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg tcgtactccc 600
caggcggagt gcttaatgcg ttagctgcag cactgagagg cggaaacctc ccaacactta 660
gcactcatcg tttacggcat ggactaccag ggtatctaat cctgttcgct acccatgctt 720
tcgagcctca gcgtcagttg cagaccagag agccgccttc gccactggtg ttcttccata 780
tatctacgca ttccaccgct acacatggag ttccactctc ctcttctgca ctcaagttca 840
acagtttctg atgcaattct ccggttgagc cgaaggcttt cacatcagac ttattgaacc 900
gcctgcactc gctttacgcc caataaatcc ggacaacgct tgccacctac gtattaccgc 960
ggctgctggc acgtagttag ccgtgacttt ctaagtaatt accgtcaaat aaaggccagt 1020
tactacctct atctttcttc actaccaaca gagctttacg agccgaaacc cttcttcact 1080
cacgcggcgt tgctccatca gacttgcgtc cattgtggaa gattccctac tgctgcctcc 1140
cgtaggagtt tgggccgtgt ctcagtccca atgtggccga tcagtctctc aactcggcta 1200
tgcatcattg ccttggtaag ccgttacctt accaactagc taatgcaccg caggtccatc 1260
caagagtgat agcagaacca tcttttaaac tctagacatg cgtctagtgt tgttatccgg 1320
tattagcatc tgtttccagg tgttatccca gtctcttggg caggttaccc acgtgttact 1380
cacccgtccg ccgctcgctt gtatctagtt tcatttggtg caagcaccaa attcatctag 1440
gcaagctcgc tcgactgcat gtatagcatg ccgccactgg 1480