CN103074270A - Lactobacillus crispatus and application - Google Patents
Lactobacillus crispatus and application Download PDFInfo
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- CN103074270A CN103074270A CN2012104594847A CN201210459484A CN103074270A CN 103074270 A CN103074270 A CN 103074270A CN 2012104594847 A CN2012104594847 A CN 2012104594847A CN 201210459484 A CN201210459484 A CN 201210459484A CN 103074270 A CN103074270 A CN 103074270A
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Abstract
The invention discloses a Lactobacillus crispatus and an application. The Lactobacillus crispatusis is preserved in the China General Microbiological Culture Collection Center (CGMCC) with accession number of CGMCC NO.6406. The Lactobacillus crispatus has high acid resistance, high-yield hydrogen peroxide characteristic and vagina pathogenic bacteria growth inhibition effect, and the invention also relates to an application of the Lactobacillus crispatus LCR15 for adjusting vaginal flora, preventing and treating colpitis.
Description
Technical field
The present invention relates to a kind of new bacterial strain and application thereof of lactobacillus, more precisely, the invention describes lactobacillus crispatus (
Lactobacillus crispatus) a new bacterial strain LCR15, and highly-acidproof, high yield hydrogen peroxide characteristic, to the bacteriostatic action of vaginal pathogenic and prevention with treat colpitic function.
Background technology
There is multiple-microorganism in the healthy women intravaginal, has consisted of mutual restriction between they and host, the environment, mutually coordinates, the microecology in vaginas system of running balance.The vaginal microbial flora of healthy women mainly is made of Bacterium lacticum, comprises lactobacillus crispatus, Lactobacillus Jensenii and lactobacillus gasseri etc.Bacterium lacticum can play a protective role to vagina under normal circumstances, and can cause vaginitis with the disorder of the dominant microecology in vaginas of Bacterium lacticum.
Bacterial vaginitis (BV) is common disease, the frequently-occurring disease of gynaecology.It is because the imbalance of intravaginal microecological balance, anerobe and gardnerella vaginalis are crossed the syndrome that Sheng is grown, the amphimicrobian Bacterium lacticum is suppressed the sex expression of caused a kind of vaginal mucosa inflammation.Use antibiotic therapy, although but the symptom of respite BV also makes this Bacterium lacticum that has reduced further reduce, and increase the weight of the microecology in vaginas imbalance, thereby BV is recurred repeatedly.
Produce H
2O
2Bacterium lacticum is the intravaginal dominant bacteria of healthy women, is the important factor that the protection vagina is avoided pathogenic infection.The other acid that produces of Bacterium lacticum metabolism and some antimicrobial agents growth and breeding that also can effectively suppress other bacteriums.There is the scholar to identify by microorganism culturing and finds, produce H
2O
2The shortage of Bacterium lacticum, can make the increases such as gardnerella vaginalis, anaerobism gram negative bacillus, mycoplasma hominis, chlamydozoan.
Lactobacillus crispatus also can be colonizated in human oral cavity and enteron aisle for mainly being colonizated in a kind of Bacterium lacticum among the human vagina.Lactobacillus crispatus belongs to the main product H in several healthy women vaginas
2O
2One of bacterium.Studies show that much lactobacillus crispatus not only can change vaginal pH (producing lactic acid by fermentation), and can make a response to the vaginal pH variation.Healthy vaginal pH about 4.0, be subject to infected by microbes after, its environment pH value can rise to about neutrality.Lactobacillus crispatus may have following three kinds of physiological actions: one, produce lactic acid, reduce the pH value of intravaginal environment, suppress the generation of non-acidophilia microorganism, stimulate the generation of acidophilia microorganism; Two, produce H
2O
2Regulate the microecology in vaginas balance, prevent the infection that the intrusion of foreign nationality bacterium causes; Three, adherence inhibition, lactobacillus crispatus can adhere to the vaginal epithelial cell surface, suppresses simultaneously streptococcus aureus, intestinal bacteria to the adhesive attraction of vaginal epithelial cell.
Summary of the invention
The purpose of this invention is to provide a kind of lactobacillus crispatus LCR15, have acidproof and high yield hydrogen peroxide characteristic, vaginal pathogenic is had bacteriostatic action, have prevention and treat colpitic function.
The solution that realizes the foregoing invention purpose is:
A kind of lactobacillus crispatus, described lactobacillus crispatus is CGMCC NO.6406 at the deposit number of Chinese common micro-organisms culture presevation administrative center.
Above-mentioned lactobacillus crispatus LE16 directly separates acquisition in the healthy human body enteron aisle, be preserved in Chinese common micro-organisms culture presevation administrative center (abbreviation CGMCC on July 31st, 2012, the address is: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No. 6406.
The application of described lactobacillus crispatus LCR15 in suppressing vaginal pathogenic.
Described vaginal pathogenic refers to subtilis, enterococcus faecalis, micrococcus flavidus, streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa and bacillus cereus.
The application of described lactobacillus crispatus in regulating vaginal microbial flora, prevention and treatment vaginitis.
The form of lactobacillus crispatus LCR15 of the present invention is shaft-like, has the characteristic of strong acid resistance and high yield hydrogen peroxide, and Gram-positive does not form gemma, produces lactic acid, amphimicrobian.
Description of drawings
Fig. 1 is the thalli morphology of lactobacillus crispatus of the present invention under microscope (* 1000 times).
Embodiment
Lactobacillus crispatus LCR15 of the present invention utilizes glucose, fructose, seminose, N-acetyl-glucosamine, polychrom, salicyl alcohol glucoside (salicin), cellobiose, maltose, lactose, melibiose, sucrose, raffinose, starch and glycogen (glycogen) do not utilize glycerine, red tinea alcohol, D-R, L-arabinose, ribose, the D-wood sugar, the L-wood sugar, ribitol, Beta-methyl-D-xyloside, semi-lactosi, sorbose, rhamnosyl, galactitol, inositol, N.F,USP MANNITOL, sorbyl alcohol, Alpha-Methyl-D-MANNOSE glucoside, Alpha-Methyl-D-Glucose glucoside, Vitamin B17, ursin, trehalose (grass sugar), synanthrin, pine three sugar, Xylitol, gentiobiose, the D-turanose, the D-lyxose, D-Tag, D-rock sugar, L-rock sugar, D-R alcohol, the L-arabitol, Sunmorl N 60S, 2-ketone group-Sunmorl N 60S and 5-ketone group-Sunmorl N 60S.
Lactobacillus crispatus LCR15 fermenting process of the present invention: in the MRS liquid nutrient medium, cultivate 24h for 37 ℃.
Embodiment 1: the bacterial strain microscopy
Choose lactobacillus crispatus LCR15 with transfering loop under the aseptic condition, be applied on the slide glass, film-making and gramstaining are examined under a microscope thalli morphology.
The dull and stereotyped purifies and separates of embodiment 2:MRS
Choose lactobacillus crispatus LCR15 with transfering loop under the aseptic condition, rule at the MRS flat board, flat board places 37 ℃ of incubators to cultivate 48h, selects single bacterium colony and carries out microscopy, reaches the bacterial strain Pure strain separation.
Embodiment 3: strain fermentation
With transfering loop lactobacillus crispatus LCR15 picking two rings from the inclined-plane are cultivated 24h for 37 ℃ to being equipped with the 15ml MRS liquid nutrient medium, with in the 1% inoculum size access 15ml MRS liquid nutrient medium, cultivate 24h, namely get lactobacillus crispatus LCR15 fermented liquid for 37 ℃ again.
Embodiment 4: the 16S rDNA partial sequence (seeing sequence table) of lactobacillus crispatus LCR15.
Embodiment 5: lactobacillus crispatus LCR15 acid resistance
Lactobacillus crispatus LCR15 bacterial strain in the MRS liquid nutrient medium 37 ℃ cultivate after 24 hours, centrifugal collection thalline, get the resuspended liquid of 1.0ml cell to the aseptic PBS of the 9.0ml of pH3.0, pH4.0 and pH5.0, process after 0,1,2,3 hour for 37 ℃ and carry out live bacterial count analysis (table 1).
Table 1 is the result show, process 3 hours viable counts under the pH3.0 condition and be declined by less than an order of magnitude, processing 3 hours viable counts under the pH4.0 condition descends less than 0.5 order of magnitude, processing 3 hours viable counts under the pH4.0 condition does not almost change, illustrate that this bacterial strain has preferably acid-fast ability, the healthy women vaginal pH is about 4.0, so this bacterial strain can normal field planting in vagina.
Embodiment 6: lactobacillus crispatus LCR15 bacteriostasis property
Lactobacillus crispatus LCR15 leaves standstill in 37 ℃ of MRS liquid nutrient mediums and cultivates 24h, and this bacterium liquid is put
On the MRS solid plate, parallel three points of every plate, 37 ℃ leave standstill cultivation 24h.
With the indicator liquid (subtilis, enterococcus faecalis, micrococcus flavidus, bacillus pumilus, streptococcus aureus, intestinal bacteria, Klebsiella pneumonia, Pseudomonas aeruginosa Lactobacterium acidophilum, lactobacillus gasseri and bifidobacterium lactis) of various 200ml overnight incubation respectively with 7ml soft agar mixing, being poured on a little has on the MRS of the lactobacillus crispatus LCR15 flat board, 37 ℃ of aerobic or anaerobism (according to indicator) are cultivated 24 h (aerobic) or 48 h (anaerobism), take pictures and measure inhibition zone size (table 2) around the indicator spot.
Annotate: "-" expression lactobacillus crispatus LCR15 does not have restraining effect to indicator.
Table 2 is the result show, lactobacillus crispatus LCR15 all has restraining effect to most of vagina harmful bacteria, wherein the restraining effect to subtilis, enterococcus faecalis, micrococcus flavidus, streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa and bacillus cereus is the strongest, to the restraining effect of bacillus pumilus, Klebsiella pneumonia slightly a little less than, and to other vagina probiotics such as the equal unrestraint effect of Lactobacterium acidophilum, lactobacillus gasseri and bifidobacterium lactis.
Embodiment 7: lactobacillus crispatus LCR15 produces hydrogen peroxide ability determination experiment
Directly use the H of concentration known
2O
2Solution allocation becomes gradient concentration solution, draws reaction absorbancy and H
2O
2Measurer has linear relationship, can get H
2O
2Concentration standard curve.Lactobacillus crispatus LCR15 carries out respectively the nectar degree and measures and bacterium liquid H behind 37 ℃ of lower cultivation 24h of different fermentations condition
2O
2Concentration determination.Bacterium liquid is centrifugal, gets supernatant liquor 0.5ml, adds 0.5ml substrate buffered soln, and is placed on 37 ℃ of insulation 10min.Toward the 1.0mg/ml TMB hydrochloride solution of 37 ℃ of preheatings of centrifuge tube adding 0.1ml, fully mixing.Toward 3.0 * 10-5mg/ml HRP solution of 37 ℃ of preheatings of centrifuge tube adding 0.1ml, fully mixing begins reaction.Accurate response 10min adds the 0.2ml stop buffer, termination reaction in reaction system.With one times of diluted sample, measure absorbancy at the 450nm place.Bacterium liquid H
2O
2Concentration=(measuring pipe OD-blank tube OD)/(standard pipe OD-blank tube OD) * H
2O
2Concentration of standard solution.
1, nectar degree and bacterium liquid H under the different fermentations condition
2O
2Measurement of concetration
Lactobacillus crispatus LCR15 in aerobic, leave standstill (little aerobic) and three kinds of different fermentations conditions of strictly anaerobic lower 37 ℃ cultivate 24h after, measure respectively nectar degree and bacterium liquid H
2O
2Concentration.
As can be seen from Table 3, anaerobism is cultivated cell concentration and is reached maximum, and H
2O
2Concentration is lower, does not almost measure.And although aerobic cultivation group cell concentration is much smaller than other two groups, and H
2O
2Concentration in addition two groups large.Illustrate that lactobacillus crispatus has high yield H leaving standstill under (little aerobic) and the aerobic condition
2O
2Characteristic.10
8
2, ventilation is to plateau nectar degree and bacterium liquid H
2O
2Concentration as influencing factor
Anaerobism is cultured to the bacterium of plateau, moves to the ventilation of vibrating of 37 ℃ of 120rpm shaking tables, carries out nectar degree and bacterium liquid H
2O
2Concentration.Every 30min sampling is surveyed once, until 2h.
As can be seen from the above table, the vibration ventilation illustrates that bacterium has good tolerance to oxygen, the H of generation to the almost not variation of plateau nectar degree
2O
2Concentration maintains about 0.1mM.
Embodiment 8: lactobacillus crispatus LCR15 suppresses mouse vagina bacterium animalbioassay
40 normal female mouse are divided into 4 groups at random: normal group, model group, natural recovering group and lactobacillus crispatus group, 10 every group.Except normal group, other each group is got 50 μ l amoxycilline Trihydrate bp sodium solutions (concentration is 12.5 mg/ml) with micro sample adding appliance and is carried out mouse vagina flushing administration, and every day 1 time, 5d gets Escherichia coli bacteria liquid (10 with micro sample adding appliance afterwards continuously
8CFU/m l) 20 μ l Mice Inoculated intravaginal, continuous flushing 6d, causes the mouse vagina infection model at every day 1 time.Normal group each physiological saline that injects equal volume every day, continuously 11d.
Lactobacillus crispatus LCR15 is seeded in the MRS liquid nutrient medium, and 37 ℃ leave standstill cultivation 24 h, centrifugal, and bacterium mud is resuspended with PBS liquid (pH=7.4), decide concentration 10 with turbidimetry
9CFU/m l.After the lactobacillus crispatus group causes the mouse vagina infection model, give and inject in the mouse vagina lactobacillus crispatus LCR15 bacterium liquid 20 μ l, continuous 6 d, every day 1 time.Natural recovering group continues the physiological saline to equal volume, continuous 6d, every day 1 time.Model group and normal group are no longer done any intervention.
1. the mensuration of mouse vagina flora and analysis
Get 50 μ l physiological saline with micro sample adding appliance, repeatedly wash mouse vagina 8～10 times, the vaginal washing fluid of getting 30 μ l carries out respectively intestinal bacteria and living preparation of lactobacillus counting.
As can be seen from Table 5, the quantity of intestinal bacteria field planting obviously reduces than the quantity of intestinal bacteria field planting in the model group mouse vagina in the mouse vagina of lactobacillus crispatus treatment group, and the escherichia coli field planting quantity in the natural recovering group mouse vagina has also reduced, but compare with model, difference is not obvious.Bacterium lacticum quantity in the model group mouse vagina is starkly lower than the Bacterium lacticum quantity in the Normal group mouse vagina, and Bacterium lacticum quantity can return to more than the normal level in the lactobacillus crispatus bacterium liquid treatment group mouse vagina.
2. the mouse episioitis is observed
Observe and record organizes respectively that the mouse vulva is red and swollen, what situation of vaginal secretions, and get and respectively organize typical mouse and make vaginal washing fluid pathology smear (pap staining).
Table 6 has shown the inflammatory reaction situation that the intestinal bacteria field planting causes at mouse vagina, the inflammatory reactions such as redness that model group mouse vulva occurs are more serious than the mouse of normal group, all have obviously through symptomatic reactions such as redness, the secretory product of mouse vulva after the treatment of lactobacillus crispatus LCR15 bacterium liquid are many to alleviate.
Simultaneously, vaginal washing fluid pathology smear (pap staining) result shows that the mouse vagina epithelial cell of normal group occupies the majority, and the white corpuscle number is less, and the model group mouse vagina epithelial cell of intestinal bacteria field planting is less, the white corpuscle number is more, illustrates that the vaginal mucosa of mouse destroys serious.White corpuscle number through mouse vagina after the treatment of lactobacillus crispatus LCR15 bacterium liquid reduces, and epithelial cell occupies the majority, and illustrates that the breakage of mouse vagina mucous membrane has obtained recovery.
Above result shows, the lactobacillus crispatus LCR15 among the present invention have regulate vaginal microbial flora, suppress vaginal pathogenic growth and field planting effect, can be used for prevention and treatment bacterial vaginitis.
Claims (4)
1. a lactobacillus crispatus is characterized in that, described lactobacillus crispatus is CGMCC NO.6406 at the deposit number of Chinese common micro-organisms culture presevation administrative center.
2. the application of lactobacillus crispatus LCR15 claimed in claim 1 in suppressing vaginal pathogenic.
3. application according to claim 2 is characterized in that, described vaginal pathogenic refers to subtilis, enterococcus faecalis, micrococcus flavidus, streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa and bacillus cereus.
4. the application of lactobacillus crispatus claimed in claim 1 in regulating vaginal microbial flora, prevention and treatment vaginitis.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178437A (en) * | 2013-11-08 | 2014-12-03 | 苏州欧赛微科生物医药科技有限公司 | Lactobacillus crispatus and its application in gynecological diseases |
| CN104519892A (en) * | 2012-07-09 | 2015-04-15 | 普罗比奥诺夫公司 | Use of thiosulphate in order to potentiate anti-pathogen effect of lactobacillus |
| CN104546947A (en) * | 2014-09-30 | 2015-04-29 | 深圳华大基因科技有限公司 | Application of lactobacillus crispatus in treating or preventing rheumatoid arthritis or related diseases |
| WO2015067141A1 (en) * | 2013-11-08 | 2015-05-14 | 苏州欧赛微科生物医药科技有限公司 | Lactobacillus crispatus and application thereof |
| CN106562998A (en) * | 2015-10-12 | 2017-04-19 | 深圳华大基因研究院 | Applications of lactobacillus crispatus in treatment or prevention of uterus myoma related diseases |
| CN107815432A (en) * | 2017-12-07 | 2018-03-20 | 广州柏芳生物科技有限公司 | A kind of people uses inactivation lactobacillus preparation and application thereof |
| CN107937324A (en) * | 2018-01-10 | 2018-04-20 | 中国科学院微生物研究所 | One plant of Lactobacillus crispatus and its application |
| CN109022475A (en) * | 2018-08-02 | 2018-12-18 | 南昌大学 | The building of Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis |
| WO2021027742A1 (en) * | 2019-08-09 | 2021-02-18 | 四川厌氧生物科技有限责任公司 | Lactobacillus crispatus and application thereof |
| CN113278548A (en) * | 2021-05-17 | 2021-08-20 | 南方医科大学珠江医院 | Lactobacillus crispatus and application thereof in producing products for improving human vaginal environment |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104519892A (en) * | 2012-07-09 | 2015-04-15 | 普罗比奥诺夫公司 | Use of thiosulphate in order to potentiate anti-pathogen effect of lactobacillus |
| WO2015067141A1 (en) * | 2013-11-08 | 2015-05-14 | 苏州欧赛微科生物医药科技有限公司 | Lactobacillus crispatus and application thereof |
| EP3040413A4 (en) * | 2013-11-08 | 2016-07-06 | Suzhou Osel Bio Pharm Co Ltd | Lactobacillus crispatus and application thereof |
| JP2017501741A (en) * | 2013-11-08 | 2017-01-19 | スーヂョウ オーセル バイオ−ファーム カンパニー、リミテッド | Lactobacillus crispatus and its applications |
| CN104178437A (en) * | 2013-11-08 | 2014-12-03 | 苏州欧赛微科生物医药科技有限公司 | Lactobacillus crispatus and its application in gynecological diseases |
| CN104546947B (en) * | 2014-09-30 | 2019-01-08 | 深圳华大基因科技有限公司 | Lactobacillus crispatus is treating or preventing the application in rheumatoid arthritis |
| CN104546947A (en) * | 2014-09-30 | 2015-04-29 | 深圳华大基因科技有限公司 | Application of lactobacillus crispatus in treating or preventing rheumatoid arthritis or related diseases |
| CN106562998A (en) * | 2015-10-12 | 2017-04-19 | 深圳华大基因研究院 | Applications of lactobacillus crispatus in treatment or prevention of uterus myoma related diseases |
| CN107815432A (en) * | 2017-12-07 | 2018-03-20 | 广州柏芳生物科技有限公司 | A kind of people uses inactivation lactobacillus preparation and application thereof |
| CN107937324A (en) * | 2018-01-10 | 2018-04-20 | 中国科学院微生物研究所 | One plant of Lactobacillus crispatus and its application |
| CN107937324B (en) * | 2018-01-10 | 2020-01-21 | 中国科学院微生物研究所 | Lactobacillus crispatus and application thereof |
| CN109022475A (en) * | 2018-08-02 | 2018-12-18 | 南昌大学 | The building of Lactobacillus crispatus BT1386 and its application in treatment bacterial vaginitis |
| WO2021027742A1 (en) * | 2019-08-09 | 2021-02-18 | 四川厌氧生物科技有限责任公司 | Lactobacillus crispatus and application thereof |
| CN113278548A (en) * | 2021-05-17 | 2021-08-20 | 南方医科大学珠江医院 | Lactobacillus crispatus and application thereof in producing products for improving human vaginal environment |
| CN113278548B (en) * | 2021-05-17 | 2022-02-22 | 南方医科大学珠江医院 | Lactobacillus crispatus and application thereof in producing products for improving human vaginal environment |
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Application publication date: 20130501 |