CN107267415A - A kind of lactobacillus reuteri and its application for preparing vagina antibacterial medicines - Google Patents
A kind of lactobacillus reuteri and its application for preparing vagina antibacterial medicines Download PDFInfo
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Abstract
A kind of application the invention provides lactobacillus reuteri and its for preparing vagina antibacterial medicines, the lactobacillus reuteri is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its deposit number is CGMCC No.14110.Study it and be metabolized performance discovery, relatively common lactobacillus reuteri, 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 reuteri 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 reuteri, Lactobacillus Jensenii, lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus rhamnosus etc..Just
Lactobacillus can play a protective role to vagina in the case of often, when the lactobacillus disorder of microecology in vaginas can cause pathogenic bacteria to enter
Invade and produce vaginitis.
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.
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.
Lactobacillus reuteri (Lactobacillus reuteri), is lactobacillus, is one of Body normal flora, extensively
It is general to be distributed in human body intestinal canal, it is also distributed in vagina.For the bacterial strain being conventionally found in vagina, existing
Its in the category of cognition of technology does not simultaneously have specific bacteriostasis.Prior art research shows that lactobacillus reuteri can
Animal alimentary canal environment is preferably resistant to, and can be colonized in humans and animals alimentary canal, regulation gut flora is played, prevents and control
Treat the effect such as diarrhoea, pre- anti-caries.In addition, the Extracellular metabolism of lactobacillus reuteri mainly includes lactic acid, acetic acid and ethanol
Deng, and energy metabolism glycerine produces a kind of special ablastins --- Luo Yishi elements, its Main Ingredients and Appearance is 3-HPA (3-HPA)
Monomer, hydrate and Cyclodimerization body.
The content of the invention
The present invention is intended to provide a kind of specific lactobacillus reuteri, to solve conventional lactobacillus reuteri 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 reuteri production hydrogen peroxide manufacture ability is relatively low.
The invention solves the problems that another technical problem be conventional lactobacillus reuteri lactic acid production it is relatively low.
The invention solves the problems that another technical problem be in the prior art conventional lactobacillus reuteri colonizing in vagina,
Survival benefit 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 reuteri of separation (Lactobacillus reuteri), the present invention
0047 (lactobacillus reuteri), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its preservation is compiled
Number be CGMCC No.14110.
Above-mentioned lactobacillus reuteri (Lactobacillus reuteri) RD-0047 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.14110, and the Classification And Nomenclature of the bacterial strain is lactobacillus reuteri
(Lactobacillus reuteri)。
The lactobacillus reuteri RD-0047 of above-mentioned separation, is sequenced using 16SrDNA, with sieve in GenBank databases
Yi Shi lactobacillus base sequence highest homologies score value is more than 98%.
On the basis of above technical scheme, it is used to prepare vagina invention further provides above-mentioned lactobacillus reuteri
The application of pathogenic bacteria 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 reuteri
Application.
Preferably, the vaginal disease is VVC, trichomonas vaginitis, senile vagina
Scorching, non-specific vagina infection or Combination vagina infection.
The isolated first one plant of lactobacillus reuteri of the present invention, studies its and is metabolized performance and find, 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 having prominent vagina epithelium
Cell adherence ability.The new property found based on more than, present invention determine that new application of vagina antibacterial medicines is prepared using it,
Realize the treatment of various bacterial vaginal disease.
The beneficial effect produced using above-mentioned technical proposal is：(1) lactobacillus reuteri RD-0047 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, with active stabilization
Biological characteristics, without domestication and rejuvenation technique, can directly preparation use.(3) there is bacterial strain of the invention suppression vagina to add moral
Receive the inhibitory action of the pathogenic bacteria such as Salmonella, compared with commercially available control bacterium, advantageous vaginal epithelial cell adhesive force, have
The advantageous ability being colonized in primate vagina.
Brief description of the drawings
Accompanying drawing 1 is the full face of the lactobacillus reuteri RD-0047 colonial morphologies of the present invention；
Accompanying drawing 2 is the lactobacillus reuteri RD-0047 of present invention gram stain microscopy photo；
Accompanying drawing 3 is the electrophoretogram of the lactobacillus reuteri RD-0047 of present invention 16SrDNA gene PCR amplified productions；
Accompanying drawing 4 is the lactobacillus reuteri RD-0047 of present invention lactate detection collection of illustrative plates；
Accompanying drawing 5 is the lactobacillus reuteri RD-0047 hydrogen peroxide colour developing 5min and 10min pictures of the present invention；
Accompanying drawing 6 be the present invention lactobacillus reuteri RD-0047 to gentamicin, kanamycins, tetracycline antibiotic
Sensitivity tests bacterium colony figure；
Accompanying drawing 7 is the lactobacillus reuteri RD-0047 (left side) and Lactobacillus delbrueckii (right side) of the present invention to gardnerella vaginalis
Fungistatic effect photo；
Accompanying drawing 8 is the lactobacillus reuteri RD-0047 (left side) and Lactobacillus delbrueckii (right side) of the present invention to EHEC
Fungistatic effect photo；
Accompanying drawing 9 is the lactobacillus reuteri RD-0047 (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 reuteri RD-0047 of the present invention is colonized machin vagina microorganism area after machin vagina
The electrophoretogram of part 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 reuteri RD-0047 floras, purifying, Zengjing Granule)
1st, the separation of lactobacillus reuteri RD-0047 floras：Gathered with two sterile cotton swabs on subject's vaginal sidewall
Secretion, be inoculated in various concentrations equipped with the culture dish of MRS culture mediums prepared, and label information, culture dish put
In anaerobic jar, and it is put into CO2Aerogenesis bag, is placed in 37 DEG C of incubators, is incubated more than 48h.
2nd, L. reuteri strain RD-0047 purifying, Zengjing Granule：According to bacterium colony different shape (surface, edge
Deng), size count respectively, homomorphosis, it is of the same size be designated as in one kind, oese picking single bacterium colony a little bacterium, press
The single bacterium colony that " oblique line method " is seeded to MRS solid mediums to be isolated and purified；On sterile toothpick picking MRS solid mediums
The a little bacterium of single bacterium colony, be seeded to MRS fluid nutrient mediums, be placed in 37 DEG C of incubators, Anaerobic culturel 24h-48h is filtered out new
Bacterial strain.
Embodiment 2 (identification and preservation of lactobacillus reuteri RD-0047 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 irregular
Type, circular protrusions, smooth, neat in edge；On blood plate, bacteria colony white or milky；The bacterium pure culture smear is taken to carry out
Gram's staining, as a result such as accompanying drawing 2, is presented Gram-positive, shape is in the slight irregular, campylobacter of circular distal, greatly
Small is 0.7-1.0 × 2.0-5.0 μm, is generally existed in single, paired, tuftlet.As a result show：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 ') 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 same with lactobacillus reuteri
Property similarity in source is more than 99%.The category kind finally identified is lactobacillus reuteri.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 reuteri RD0047 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 | - |
| Bacterium dynamic test | - |
+：Represent positive；-：Represent negative
Utilization ability of the RD-0047 bacterium to following carbon source is determined using conventional sugar fermenting experiment, the biochemical reaction is with sugar, alcohol
Based on class fermentation basal medium (peptone 1g, sodium chloride 0.5g, purified water 100mL, pH7.2-7.4), add following
Sugar is as sole carbon source, and addition concentration is 1%, and acquired results are as shown in table 2：
The lactobacillus reuteri RD0047 of table 2 investigates result of the test to different carbon source Utilization ability
| Carbon source kind | As a result | Carbon source kind | As a result |
| Arabinose | + | Cellobiose | - |
| Fructose | + | Galactolipin | + |
| Glucose | + | Lactose | + |
| Maltose | + | Mannitol | - |
| Mannose | - | Melezitose | - |
| Melibiose | + | Gossypose | + |
| Rhamnose | - | Ribose | + |
| Sorbierite | - | Sucrose | + |
| Trehalose | - | Xylose | - |
+：Represent positive；-：Represent negative
Thus biochemical collection of illustrative plates judges that bacterial strain biochemical characteristic meets the biochemical characteristic of lactobacillus reuteri.
3rd, the preservation of bacterial strain
Lactobacillus reuteri (Lactobacillus reuteri) RD-0047 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.14110, the Classification And Nomenclature of the bacterial strain is lactobacillus reuteri (Lactobacillus reuteri).
Embodiment 3 (lactobacillus reuteri RD-0047 metabolites measure)
1. lactic acid content is determined in lactobacillus reuteri RD-0047 metabolites：Using high-efficient liquid phase technique (0.005M sulfuric acid
(0.28ml sulfuric acid (98%) -1000ml water, pH is about that 2.1) lactic acid of this strain culturing 24-48h zymotic fluids is carried out to the aqueous solution
Detection, as a result indicate that lactic acid (L/D lactic acid summation) is about 28mg/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 reuteri RD-0047 metabolites：By Mcgroarty etc. peroxidating
Thing enzyme process carries out hydrogen peroxide semiquantitative determination, and the lactobacillus reuteri RD-0047 of separated identification is inoculated in into H2O2Identification
After MRS-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
Blueness, without producing H2O2Bacterium colony nondiscolouring, according to H of the Coloring Time to generation2O2Sxemiquantitative is carried out, is as a result seen in Fig. 5, figure
The existing obvious blue of bacterium colony shows during 5min, and a large amount of bluenesss substantially occur during 10min, it is clear that this bacterium is easy to produce peroxidating
Hydrogen, production hydrogen peroxide is very capable, these results suggest that this lactobacillus reuteri RD-0047 can significantly produce lactic acid and peroxidating
Hydrogen, helps 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 2015, use
AGP test paper disk method determines the sensitiveness of strains, investigates the lactobacillus reuteri RD-0047 in 0 generation and 30 generations of passage
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 lactobacillus reuteri RD0047 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 OXA, fleraxacin slight sensitive, big to Meropenem, ampicillin, celebrating
The medium sensitivities such as mycin, clindamycin, ceftriaxone, to benzyl penicillin, kanamycins, tetracycline, erythromycin, Piperacillin, ten thousand
Ancient mycin, amoxicillin/clavulanate, azithromycin, Amoxicillin, bacitracin are sensitive.
Accompanying drawing 6. wherein is shown in the antibiotic sensitive figure of gentamicin, kanamycins, tetracycline
Embodiment 5 (toxicity test)
5 SPF grades of Kunming mouses, every fresh lactobacillus reuteri RD-0047 of mouse vagina injection 0.5ml suspends
Bacterium solution (is more than 1 × 109CFU/ mouse).By 2015 editions Chinese Pharmacopoeia requirements, every mouse weight is measured daily, and observe, remember
The changes such as behavior and physiology before and after every mouse injection of record.As a result show that all the weight of animals have increase in 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 reuteri RD-0047 mitotic stabilities)
The present embodiment is from growth characteristics, morphology, Biochemical Characteristics, metabolin composition, antibiotic sensitive characteristic, hereditary capacity
And to this L. reuteri strain RD-0047 pass on the study on the stability in 30 generations (C30) in terms of toxotest.
1st, lactobacillus reuteri RD-0047 isolate and purify, colony morphological observation, dyeing microscopic examination and biochemical characteristic detection method
The Part I of be the same as Example 1 and embodiment 2.As a result show：By passage, colonial morphology is shown in for milky circular colonies, greatly
Small about 0.5~2mm, intermediate projections, neat in edge, surface is smooth, and significant changes do not occur, and passage is stable；Gram's staining is in
It is now 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 the 0th of lactobacillus reuteri RD-0047 the
In generation (C0), the 30th generation (C30) bacterial strain, carry out 16SrDNA fragments PCR amplifications, and by 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 reuteri, homology phase
Like degree 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, according to the scope of restraining fungi criteria for interpretation of paper disc method judge this lactobacillus reuteri strain to erythromycin, fluorine
Luo Sha star slight sensitives, to Meropenem and gentamicin medium sensitivity, to ampicillin, OXA, benzyl penicillin, block that
Mycin, tetracycline, clindamycin, erythromycin, Piperacillin, ceftriaxone, fleraxacin, vancomycin, Amoxicillin/carat
Tie up acid, azithromycin, Amoxicillin, bacitracin sensitivity.
5th, toxicity test：Method be the same as Example 5, with mouse vagina note passage 30 generations (C30) lactobacillus reuteri RD-0047
Bacterium solution has carried out toxotest, wherein, the concentration ＞ 10 of test9CFU/ mouse.As a result it is：In whole test mices 7 days not
See poisoning symptom, body weight has increase, no animal dead.According to the above results, press《New drug pharmacology, toxicological study technical requirements
Supplementary notes》, the bacterial strain belongs to non-toxic type bacterial strain.
Comprehensive, the present embodiment repeatedly passes on lactobacillus reuteri RD-0047 with MRS medium cultures, from morphology, life
The numerous plant of passage has been inquired into terms of chemistry, metabolin feature and hereditary capacity, susceptibility characteristic, toxicity test to lactobacillus reuteri
Influence.As a result show：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 reuteri RD-0047 bacterial strains pharmacodynamic experiment)
First, lactobacillus reuteri RD-0047 bacterial strains antibacterial experiment in vitro
(1) lactobacillus reuteri RD-0047 and Lactobacillus delbrueckii suppress the experiment of gardnerella vaginalis in vitro：According to 3%
Inoculum density prepares lactobacillus reuteri RD-0047 MRS agar bacteria cakes, in Anaerobic culturel 24h at 37 DEG C, is prepared with method commercially available
Lactobacillus delbrueckii bacteria cake；The μ L of gardnerella vaginalis 100 are taken, are inoculated in 10mL BHI solid mediums, the newborn bars of Luo Yishi are put into
Bacterium RD-0047 and Lactobacillus delbrueckii bacteria cake, 37 DEG C of Anaerobic culturel 48h；Obvious inhibition zone occurs around lactobacillus.As a result figure is seen
7, wherein left figure is lactobacillus reuteri RD-0047 inhibition zone effects, and vernier caliper measurement antibacterial circle diameter is 32.3mm, right figure
For Lactobacillus delbrueckii inhibition zone effect, bacteriostatic diameter is 21.5mm, and conclusion is lactobacillus reuteri RD-0047 to vagina Gardner
The fungistatic effect of bacterium is better than Lactobacillus delbrueckii.
(2) lactobacillus reuteri RD-0047 and Lactobacillus delbrueckii suppress the experiment of gardnerella vaginalis in vitro：According to 3%
Inoculum density prepares lactobacillus reuteri RD-0047 MRS agar bacteria cakes, in Anaerobic culturel 24h at 37 DEG C, is prepared with method commercially available
Lactobacillus delbrueckii bacteria cake；By staphylococcus aureus, ETEC, pseudomonas aeruginosa and salmonella are seeded in pancreas junket
Soya peptone liquid (TSB) agar medium, is put into lactobacillus reuteri RD-0047 and Lactobacillus delbrueckii bacteria cake.In 33 DEG C of cultures
18-24h, observes inhibition zone, and vernier caliper measurement RD-0047 antibacterial circle diameters are about 22mm, and right figure is that Lactobacillus delbrueckii is antibacterial
Effect is enclosed, bacteriostatic diameter is 12mm, conclusion is lactobacillus reuteri RD-0047 to staphylococcus aureus, ETEC,
The fungistatic effect of pseudomonas aeruginosa and salmonella is better than Lactobacillus delbrueckii, and 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 reuteri RD-0047, 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 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 reuteri RD-0047 4h adherence rates are respectively 45.8% and 52.8%, commercially available similar moral
Family name's lactobacillus strain 4h adherence rates are respectively 23.6% and 20.7%, and lactobacillus reuteri RD-0047 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 reuteri RD-0047：Lactobacillus reuteri RD-0047 freeze-drying bacterium powder is weighed,
It is 10 to make field planting amount6.Control group is used under listing product (Lactobacillus delbrueckii capsule), aseptic technique, each to add the training of MRS liquid
Base 0.5mL is supported, is well 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, 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 is 99% when comparing homology, 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 reuteri RD-0047 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.
The lactobacillus reuteri RD0047 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 reuteri RD-0047 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.The vaginal fluid cleannes result of determination such as institute of table 5
Show.
The lactobacillus reuteri RD0047 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
Found in road secretion for examination lactobacillus reuteri RD-0047, the information for examination lactobacillus reuteri RD-0047 isolated
As shown in Figure 10.It is seen that found in animal subject vaginal fluid for examination lactobacillus reuteri RD-0047, because
This can be seen that lactobacillus reuteri RD-0047 can effectively be colonized in Chinese machin intravaginal.
Embodiment 8 (lactobacillus reuteri RD-0047 is lyophilized to be preserved and freeze-dried powder stability)
In order to detect survival rates of the lactobacillus reuteri RD-0047 in the case of fermenting and be lyophilized, by lactobacillus reuteri
RD-0047 grows in pH 6.5 modified MRS culture medium, 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.) mixing.Then, mixture is placed in freeze drier and be freeze-dried.Sample
Product are freezed about 2 hours at -40 DEG C, are then dried in vacuo 20-30 hours at -20 DEG C, then dry 3-5 hours at 30 DEG C.It is dry
Powder is dispensed in the aluminium foil bag for being put into drier, and is stored in 4 DEG C and room temperature (25 DEG C).Pass through flat board in the 0th, 1,3,6,9, December
Count and determine viable count.
Initial every gram of dry powder of lactobacillus reuteri RD-0047 contains up to 30,000,000,000 viable bacterias (3 × 1010Cfu/g), at 4 DEG C
There is down optimal storage stability.After being stored 6 months at 4 DEG C, retain the 63.3% of initial viable count, be shown in Table 6.
The lactobacillus reuteri RD0047 freeze-dried powder microbial inoculum stability test results of table 6
| Condition | Viable count/gram dry powder | Viable detection/% |
| 0 month | 3*1010 | —— |
| 1 month, 4 DEG C | 2.6*1010 | 86.7 |
| 1 month room temperature | 2.3*1010 | 76.7 |
| 3 months, 4 DEG C | 2.4*1010 | 80 |
| 3 months room temperatures | 1.5*1010 | 50 |
| 6 months, 4 DEG C | 1.9*1010 | 63.3 |
| 6 months room temperatures | 9.1*109 | 30.3 |
| 9 months, 4 DEG C | 1.6*1010 | 53.3 |
| 9 months room temperatures | 5.3*109 | 17.7 |
| 12 months, 4 DEG C | 1.2*1010 | 40 |
| 12 months room temperatures | 9.8*108 | 3.2 |
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 Long Chuanji pharmaceutcal corporation, Ltds；Guangdong Qiangji Pharmaceutical Co., Ltd.；Zhao Xiangjiang
<120>A kind of lactobacillus reuteri and its application for preparing vagina antibacterial medicines
<160> 1
<210> 1
<211> 1475
<212> DNA
<213>Artificial sequence
<400> 1
gggttccggccttaggcggctccctcctaaaggttaggcc 40
accgactttgggcgttacaaactcccatggtgtgacgggc 80
GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGC 120
TGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGA 160
GTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTAAGAGA 200
TTAGCTTACTCTCGCGAGTTTGCGACTCGTTGTACCGTCC 240
ATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGAT 280
GATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGG 320
CAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGT 360
AACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT 400
CACGACACGAGCTGACGACGACCATGCACCACCTGTCATT 440
GCGTCCCCGAAGGGAACGCCTTATCTCTAAGGTTAGCGCA 480
AGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAA 520
TTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAA 560
TTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCG 600
GAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAA 640
CCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTA 680
CCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGC 720
CTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACT 760
GGTGTTCTTCCATATATCTACGCATTCCACCGCTACACAT 800
GGAGTTCCACTGTCCTCTTCTGCACTCAAGTCGCCCGGTT 840
TCCGATGCACTTCTTCGGTTAAGCCGAAGGCTTTCACATC 880
AGACCTAAGCAACCGCCTGCGCTCGCTTTACGCCCAATAA 920
ATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGC 960
TGGCACGTAGTTAGCCGTGACTTTCTGGTTGGATACCGTC 1000
ACTGCGTGAACAGTTACTCTCACGCACGTTCTTCTCCAAC 1040
AACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCG 1080
GTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCC 1120
CTACTGCTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGT 1160
TCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATC 1200
ATCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGC 1240
ACCGCAGGTCCATCCCAGAGTGATAGCCAAAGCCATCTTT 1280
CAAACAAAAGCCATGCGGCTTTTGTTGTTATGCGGTATTA 1320
GCATCTGTTTCCAAATGTTATCCCCCGCTCCGGGGCAGGT 1360
TACCTACGTGTTACTCACCCGTCCGCCACTCACTGgtgat 1400
ccatcgtcaatcaggtgcaagcaccatcaatcagttgggc 1440
cagtgcgtacgactgcatgtataggcacccccgcc 1475
Claims (9)
1. a kind of lactobacillus reuteri, its deposit number is CGMCC No.14110.
2. a kind of DNA molecular, it is characterised in that the DNA molecular extracts from lactobacillus reuteri described in claim 1, using its as
Masterplate is expanded using 16SrDNA universal primers, amplified production sequence and lactobacillus reuteri base sequence in GenBank databases
The two homology similarity is more than 98%.
3. lactobacillus reuteri 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 reuteri 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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| CN112940970A (en) * | 2021-02-09 | 2021-06-11 | 中国科学院近代物理研究所 | Intestinal probiotics and application thereof in treating tumors and replacing antibiotics |
| CN114891705A (en) * | 2022-07-11 | 2022-08-12 | 东北农业大学 | Acid-producing Min pig source lactobacillus reuteri and culture method and application thereof |
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| CN109385387A (en) * | 2018-12-28 | 2019-02-26 | 上海源耀生物股份有限公司 | The lactobacillus reuteri of anti-TGEV a kind of and its application |
| 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 |
| CN112940970A (en) * | 2021-02-09 | 2021-06-11 | 中国科学院近代物理研究所 | Intestinal probiotics and application thereof in treating tumors and replacing antibiotics |
| CN112940970B (en) * | 2021-02-09 | 2023-01-24 | 中国科学院近代物理研究所 | Intestinal probiotics and application thereof in treating tumors and replacing antibiotics |
| CN114990011A (en) * | 2022-05-25 | 2022-09-02 | 江西仁仁健康产业有限公司 | Lactobacillus reuteri capable of reducing cholesterol and inhibiting gardnerella and application |
| CN114990011B (en) * | 2022-05-25 | 2023-04-14 | 江西仁仁健康微生态科技有限公司 | Lactobacillus reuteri capable of reducing cholesterol and inhibiting gardnerella and application |
| CN114891705A (en) * | 2022-07-11 | 2022-08-12 | 东北农业大学 | Acid-producing Min pig source lactobacillus reuteri and culture method and application thereof |
| CN114891705B (en) * | 2022-07-11 | 2022-09-16 | 东北农业大学 | Acid-producing Min pig source lactobacillus reuteri and culture method and application thereof |
| CN116751705A (en) * | 2023-05-17 | 2023-09-15 | 上海华朴生命健康科技有限公司 | Lactobacillus reuteri with effect of improving colpitis and application thereof |
| CN116751705B (en) * | 2023-05-17 | 2024-03-15 | 上海华朴生命健康科技有限公司 | Lactobacillus reuteri with effect of improving colpitis and application thereof |
| CN116622570A (en) * | 2023-05-22 | 2023-08-22 | 微康益生菌(苏州)股份有限公司 | Lactobacillus reuteri LR08, and application, product and method thereof in preparation of medicines for preventing and treating vaginitis |
| CN116622570B (en) * | 2023-05-22 | 2023-11-03 | 微康益生菌(苏州)股份有限公司 | Lactobacillus reuteri LR08, and application, product and method thereof in preparation of medicines for preventing and treating vaginitis |
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