CN104673702B - One plant of lactobacillus curvatus（Lactobacillus crispatus）And its pharmaceutical applications - Google Patents
One plant of lactobacillus curvatus（Lactobacillus crispatus）And its pharmaceutical applications Download PDFInfo
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Abstract
The invention discloses one plant of lactobacillus curvatus (Lactobacillus.crispatus) and its pharmaceutical applications.Lactobacillus curvatus provided by the present invention are specially lactobacillus curvatus IMC 79, and lactobacillus curvatus IMC 79 of the invention has stronger generation H2O2Ability, can effectively suppress the growth of external pathogen and other conditions pathogenic bacteria；There is the stronger adhesive attraction to vaginal epithelial cell again simultaneously, ensure that field planting of the bacterial strain in vagina epithelium.Lactobacillus curvatus IMC 79 provided by the present invention has certain application prospect in terms of bacterial vaginosis BV preventing and treating.
Description
Technical field
The invention belongs to biological technical field, more particularly to one plant be isolated from human body can be used for prevent and treat bacterial vaginosis BV
Lactobacillus curvatus (Lactobacillus.crispatus)
Background technology
Microorganism inhabites that almost each position, such as skin, oral cavity, enteron aisle, each position of genital tract are all present in human body
Substantial amounts of bacterium, including a variety of different types of bacteriums, " beneficial bacterium " can be roughly classified into and " conditioned pathogen " two is big
Class, stable poised state is kept in the quantity and ratio of healthy human body these bacteriums.Various bacteria may be present in woman vagina,
Wherein, Bacillus acidi lactici is to maintain the most common and most important beneficial bacterium of vaginal health micro-ecological environment, and Bacillus acidi lactici accounts for healthy woman
More than the 90% of cysthus road bacteria total amount.They are mainly caused a disease by producing sour, hydrogen peroxide to suppress pathogen and other conditions
The growth of bacterium.If some factors cause intravaginal lactobacillus quantity to reduce, or even missing completely, then intravaginal can be caused many
Condition pathogen abnormality proliferation, there is the bacterial vaginosis BV using obvious microecology in vaginas environment disturbance as principal character.
Bacterial vaginosis BV is that Women of Childbearing Age is most commonly seen but be not affected by the frequently-occurring disease of attention, in healthy women
Illness rate is about 20%, and pregnant woman is about 15%.Bacterial vaginosis BV is " small disease, burden ", under patients ' life quality is obvious
Drop, mental burden are very heavy；And the risk of the venereal disease such as aids infection, gonorrhoea, NUG substantially increases；It is ill pregnant
Woman may occur in which a series of complications of pregnancy, such as premature labor and chorioamnionitis.At present, the treatment of bacterial vaginosis BV mainly according to
By antibiotic such as metronidazole or lincomycins, but therapeutic effect is bad, and cure rate is less than 50%, and the ratio recurred is high.One
Even if half patient widely applies antibiotic also to cure, clinical symptoms are persistently present, and patient, which enjoys, to be decocted, and doctor suffers from not having good
Plan.How Control in recurring, thoroughly radical cure bacterial vaginosis BV be ob-gyn's urgent need to resolve thorny problem.
In order to improve the therapeutic effect of bacterial vaginosis BV, clinically it is badly in need of new therapeutic scheme.Due to antibiosis extract for treating
The toxic side effect such as liver, renal damage caused by bacterial vaginosis BV less effective and antibiosis extract for treating, has been working hard visit both at home and abroad
Tiny ecosystem therapy of the rope based on probiotic Lactobacillus preparation.Microecologic cure is by kill microorganism as main treatment method
It is changed into increasing the novel therapeutic theory for the purpose of probiotics, the recovery normal micro-ecological environment of vagina.Removed using antibiotic
After pathogenic bacteria, directly supplement makes its Colonization get off, promotes its day from the living preparation of lactobacillus preparation of healthy women vagina separation
The ability of the pathogenic bacteria growing of right resistance.
There are a variety of lactobacillus in healthy women intravaginal, have individual difference, and anti-cause between each strain of lactobacillus curvatus
Germ capacity variance is obvious., it is necessary to consider the species of lactobacillus when selecting lactobacillus probiotics, it produces acid, production H2O2Energy
Power, and the ability with vaginal epithelial cell adhesion, can wherein Bacillus acidi lactici successfully be colonized in vagina, be lactobacillus continuous action
Basis, and lactobacillus play curative effect key factor.
Because different lactobacillus strains has different adhesion affinity, the strong bacterial strain field planting of affinity to vaginal epithelial cell
Ability is strong, can just play a part of suppression and kill multiple pathogenic microorganisms.If living preparation of lactobacillus preparation can not be colonized in vagina epithelium
Cell, in experiment in vitro suppression strong again kill the effects of multiple pathogenic microorganisms and all do not know where to begin, be changed into bad check.
One is improved and effective probiotics strain, and itself must at least possess following standards：(1) must be applied for host
Position is normally colonized bacterium；(2) it can survive and grow in host's application site；(3) it can provide host beneficial effect；(4) it is related
Product must can maintain activity and survival ability during being made and storing.
At present, the agent of state's per vaginam probiotics bacterial only has one kind, and its main component is Lactobacillus delbrueckii viable bacteria.Although it be from
A kind of lactobacillus that healthy women vagina is separated, but be not the main beneficial bacterium in China's woman vagina flora, China is good for
Health woman vagina bacterium is based on lactobacillus curvatus.
The content of the invention
Blank of the present inventor based on above-mentioned field, a kind of lactobacillus curvatus are separated out of China healthy women body
(Lactobacillus.crispatus) bacterial strain, the bacterial strain was in preservation on July 16 Chinese microorganism strain preservation management in 2014
Committee's common micro-organisms center, deposit number are CGMCC No.9468.Inventors have also demonstrated that the bacterial strain is to vagina epithelium
Cell has stronger adhesion, while also demonstrates that the bacterial strain has stronger generation H2O2Ability, microbial inoculum made of the bacterium
Higher activity and survival ability are able to maintain that during storage, illustrates to can be used for preventing and treating bacterial vaginosis BV.
The claimed technical scheme of the present invention is as follows：
One plant of lactobacillus curvatus bacterial strain IMC-79 (Lactobacillus.crispatus), its preserving number are CGMCC
No.9468。
For preventing and/or treating the product of bacterial vaginosis BV, it is characterised in that：The active component of the product includes
Above-mentioned lactobacillus curvatus bacterial strain IMC-79.
For preventing and/or treating the product of bacterial vaginosis BV, it is characterised in that：On the active component of the product is
The lactobacillus curvatus bacterial strain IMC-79 stated.
The product, it is characterised in that：Also containing the customary adjuvant composition for preparing microbial inoculum.
For suppressing the product of pathogenic bacteria, it is characterised in that：The above-mentioned bending lactic acid of the active component of the product
Bacillus strain IMC-79.
For suppressing the product of pathogenic bacteria, it is characterised in that：The active component of the product is above-mentioned bending breast
Acidfast bacilli bacterial strain IMC-79.
Described product, it is characterised in that：Also containing the customary adjuvant composition for preparing microbial inoculum.
Above-mentioned lactobacillus curvatus bacterial strain IMC-79 is in the product for preparing prevention and/or treatment bacterial vaginosis BV
Purposes, it is characterised in that the active component of the product includes above-mentioned lactobacillus curvatus bacterial strain IMC-79.
Above-mentioned lactobacillus curvatus bacterial strain IMC-79 is in the product for preparing prevention and/or treatment bacterial vaginosis BV
Purposes, it is characterised in that：The active component of the product is above-mentioned lactobacillus curvatus bacterial strain IMC-79.
The present invention has separated intravaginal dominant bacteria according to the recognised standard of probiotics strain out of China healthy women body
Group-lactobacillus curvatus, and therefrom filtered out one plant there is higher production H2O2Ability and fixed with vaginal epithelial cell adhesion
The strong probiotic strain of plant ability-lactobacillus curvatus IMC-79.It is demonstrated experimentally that it is demonstrated experimentally that by the lactobacillus curvatus
(Lactobacillusjensenii) IMC-79, according to 106Cfu/ml density is inoculated in MRS fluid nutrient mediums, in anaerobism ring
Under border, after 37 DEG C of quiescent culture 21h, shaken cultivation 3h, rotating speed 220rpm are converted to, H in final gained nutrient solution supernatant2O2
At concentrations up to 365.5 μM.Hydrogen peroxide is efficiently produced, can effectively suppress the life of external pathogen and other conditions pathogenic bacteria
It is long；Meanwhile the lactobacillus curvatus reach 113 to each vaginal epithelial cell average adhesion quantity, this also confirms that the bacterial strain pair
Vaginal epithelial cell has stronger adhesive attraction, ensure that field planting of the bacterial strain in vagina epithelium.
The bacterial strain has sent preservation, the entitled IMC-79 of preservation, and preserving number is CGMCC No.9468, and size about 1-2 is in the least
Rice, opaque, projection milky bacterium colony, colony edge is neat, relatively moistens, and size and form is stable (Fig. 1).In logarithmic growth
The bacterial strain IMC-79 of phase, through smear, Grain stain, the form of optical microphotograph Microscopic observation thalline is：Grain stain is positive,
Bactacin, atrichia.Lactobacillus curvatus (Lactobacillus jensenii) IMC-79 amphimicrobians, growth are most suitable
37-42 DEG C of temperature, optimal pH 5.8-6.2；Can a variety of sugar such as glucose fermentation, fructose, sucrose, trehalose, maltose.
The production H of the bacterial strain confirmed according to experiment2O2Ability and the adhesive capacity to vaginal epithelial cell, the present invention please also
Seek the product that is used to prevent and/or treat bacterial vaginosis BV of the protection using the bacterium as activity.Preparation method is art technology
Conventional meanses known to personnel, such as, but not limited to such as《A kind of probiotics of stable safety and its production and use》
(CN1663573B),《Lactobacillus micro-ecological preparation of inactivation and preparation method thereof》(CN101612169B),《A kind of Bacillus acidi lactici
The preparation method of probiotics》(CN101530160A),《Lactic acid as the probiotic microorganisms in human vagina produces thin
Bacterium》(CN1308436) the disclosed preparation method or operating procedure in the prior art such as, in the market public offering use
The preparation technology of " lactobacillus vaginal capsule ", " vagina with live lactobacillus capsule (determine monarch give birth to) ", with the lactobacillus curvatus
IMC-79 is active component, passes through the preparation method or operating procedure disclosed in the conventional meanses and/or prior art literature
Or the preparation technology for the product that public offering uses in the market, it can obtain for preventing and/or treating bacterial vaginosis
The product of disease, or for suppressing the product of pathogenic bacteria.
The formulation of the claimed product of the present invention is preferably but not limited to：Capsule.
In some embodiments of the present invention, the product for suppressing pathogenic bacteria, the activity of these products are additionally provided
The lactobacillus curvatus IMC-79 that composition is or identified including present invention separation.These products can be used for the other positions of human body
Pathogenic bacteria infection.
Present invention also offers the external method for suppressing pathogenic bacteria, as suppressed pathogenic bacteria in specific environment
Purposes, this purposes are unrelated with disease treatment.
Preservation information
Culture presevation title：IMC-79
Preserving number：CGMCC No.9468
Latin name：Lactobacillus.crispatus
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On July 16th, 2014
Brief description of the drawings
Fig. 1 is the lactobacillus curvatus IMC-79 bacterium colonies from Healthy People guiding separation.
Fig. 2 is adhesive attraction schematic diagrames of the lactobacillus curvatus IMC-79 to vaginal epithelial cell.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The separation and identification of embodiment 1, lactobacillus curvatus bacterial strain
First, the separation of lactobacillus curvatus bacterial strain
MRS culture mediums：Peptone 10.0g；Beef extract 10.0g；Yeast extract 5.0g；Diammonium hydrogen citrate 2.0g；Glucose
20.0g；Tween 80,1.0mL；Sodium acetate (CH3COONa·3H2O)5.0g；Dipotassium hydrogen phosphate (K2HPO4·3H2O)2.0g；Sulphur
Sour magnesium (MgSO4·7H2O)0.58g；Manganese sulfate (MnSO4·H2O)0.25g；The 000mL of distilled water 1；It is adjusted to pH 6.2~6.6.
The healthy women vaginal fluid sample of more than 200 example clinical detection absence of vagina inflammation symptom and signs is have collected, by sample
Number consecutively, it is inoculated in MRS fluid nutrient mediums, 37 DEG C of Anaerobic culturel 24h.Each sample selects 5 single bacteriums and falls within 2mL at random
Culture increases bacterium 24h in MRS fluid nutrient mediums, and to bacterium colony number consecutively.Expand bacterium and be used for bacterium conservation and DNA preparations.Will
Hundreds of plants of bacteriums being separated to carry out strain idenfication, production hydrogen peroxide and the detection with vaginal epithelial cell adhesive attraction respectively,
There is stronger adhesive attraction wherein one plant and the lactobacillus curvatus of production hydrogen peroxide are named as IMC-79.
2nd, the identification of lactobacillus curvatus bacterial strain
1st, Morphological Identification
On MRS Bacillus acidi lactici culture medium flat plates, the isolated bacterial strain IMC-79 of above-mentioned steps one shows as size about
1-2 millimeters, opaque, projection milky bacterium colony, and colony edge is neat, relatively moistens, size and form is stable (Fig. 1).
2nd, analysis of physio biochemical characteristics
Bacterial strain IMC-79 amphimicrobians, grow 37-42 DEG C of optimum temperature, optimal pH 5.8-6.2.According to API Bacteria Identifications
Standard using API 50CHL culture mediums and API 50CH reagent strips (bioMe ' rieux, Inc., Marcy l ' Etoile,
France the physiological and biochemical property that sugared fermentation reaction interpretation detects bacterial strain IMC-79) is carried out to detection bacterial strain IMJ-1.API
The simple culture medium that 50CHL is made up of the API 50CH test bars of 49 kinds of fermentable carbohydrates.With determine bacterium be made it is outstanding
Each tubule of liquid inoculation test bar.When culture, because fermentable carbohydrates produce acid, pH declines, and makes indicator discoloration.
As a result show, bacterial strain IMC-79 can a variety of sugar such as glucose fermentation, fructose, sucrose, trehalose, maltose.
3rd, 16s rDNA sequence homology analysis
The isolated bacterial strain IMC-79 of conventional method culture above-mentioned steps one, the STb gene for extracting bacterial strain expand as gene
Increase template, using universal primer 27f：5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492r：5′-
TACGGTTACCTTGTTACGACTT-3 ' amplification bacterial 16 S rRNA genes conserved region.25 μ L amplification systems include：1 × PCR is anti-
Answer buffer solution, 200 μm of ol/L dNTPs, upstream and each 0.2 μm of ol/L of anti-sense primer, 1U Taq archaeal dna polymerases, 1 μ L templates
DNA.Reaction condition：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 30s, totally 25 circulate；72
DEG C extension 10min.1% detected through gel electrophoresis of PCR primer, positive findings 27f：5′-AGAGTTTGATCCTGGCTCAG-
3 ', 1492r：The two-way sequencing of 5 '-TACGGTTACCTTGTTACGACTT-3 ' carry out.Sequence assembly and similarity analysis use
DNAStar softwares are completed, and sequence alignment passes through American National Biotechnology Information center ncbi database (http://
Www.ncbi.nlm.nih.gov it is) online to complete.
Bacterial strain IMC-79 16s rDNA sequence refers to sequence 1 in sequence table.
The functional analysis of embodiment 2, lactobacillus curvatus (Lactobacillus jensenii) IMC-79
1st, lactobacillus curvatus (Lactobacillus jensenii) IMC-79 produces H2O2Detection
H2O2Standard curve making：Piperazine bis--the ethyl sulfonic acid of-N, N'- for being first 100mmol/L with concentration
(Piperazine-N, N_-Bis 2-ethanesulfonic acid, PIPES) is by 30% (v/v) H2O2(equivalent to
9.128mol/L) storing liquid is diluted to 1mol/L, then with 100mmol/L PIPES by 1mol/L H2O20 μm of ol/ is diluted to respectively
L, 20 μm of ol/L, 40 μm of ol/L, 60 μm of ol/L, 80 μm of ol/L, 100 μm of ol/L working solution；Then take 100 μ L above-mentioned respectively
H2O2Working solution and 100 μ L concentration are 20mmol/L TMB (tetramethyl benzidine), 2 μ L horseradish peroxidases (1mg/mL) are mixed
Close, 16 DEG C of incubation 10min, measure OD630Value.Finally give H2O2Concentration standard curve：Y=0.0033x-0.0496, R2=
0.992。
Lactobacillus curvatus (Lactobacillus jensenii) IMC-79 Liquid Cultures H2O2Concentration Testing：Picking is curved
Bent Bacillus acidi lactici (Lactobacillus jensenii) IMJ-1CGMCC No.6558 bacterium colonies, inoculation 1mL cultures 48h bacterium solution
In 10mL MRS fluid nutrient mediums, the concentration for making bacterial strain in nutrient solution is 105Cfu/ml, under anaerobic environment, 37 DEG C of standings
After cultivating 21h, 37 DEG C of shaken cultivation 3h, rotating speed 220r/min, radius of turn 2.54cm are converted to, after culture terminates, is received
Collect zymotic fluid, 12000r/min centrifugation 2min, take 100 μ L bacterium solutions supernatants to measure OD630Value, according to standard curve, calculate bacterium solution
H in supernatant2O2Concentration.Test in triplicate, results averaged.
As a result as shown in table 1, lactobacillus curvatus (Lactobacillus jensenii) IMC-79 is in fermentation culture
In can produce substantial amounts of H2O2, it is at concentrations up to 365.5 μM.
The lactobacillus curvatus of table 1 (Lactobacillus jensenii) IMC-79 produces H2O2Ability detection unit：μM
| Repeat 1 | Repeat 2 | Repeat 3 | Average value | |
| H2O2Yield | 365.9 | 345.6 | 385.1 | 365.5 |
Adhesive attractions of the lactobacillus curvatus IMC-79 of embodiment 3 to vaginal epithelial cell
Vagina epithelium cast-off cells are taken from more than 20 examples health (clinical detection shows non-reproductive system infection) child-bearing period woman
Female, MEM culture mediums (pH 4.0) are separately added into after each cell sample label, 120g is centrifuged 10 minutes, is repeated centrifugation 2 times, is washed
Fall surface bacteria, it is 79/mL to adjust vagina epithelium cast-off cells concentration with MEM culture mediums；Activation is incubated overnight bending lactic acid
Bacillus IMC-79 makes its concentration be (108CFU/mL).The vagina epithelium cast-off cells and the lactobacillus curvatus IMC-79
Each 500 μ L are placed in 37 DEG C of shaking tables (rotating speed 100r/min, radius of turn 2.54cm) and are incubated 1h, Ran Houyong after mixing in equal volume
0.8 μm of membrane filtration, remove free lactobacillus curvatus IMC-79, and filter membrane is gently pressed on cover glass, fixed cell is carried out
Oil mirror is observed after Gram's staining, is counted three cell adherence bacterial populations respectively, is calculated each cell average adhesion bacterial population.Together
When the other one plant same method separation identification using embodiment 1 obtained, but with vaginal epithelial cell Colonization power compared with
Weak lactobacillus curvatus are as negative control.
As a result show, lactobacillus curvatus IMC-79 is to being isolated from the vaginal epithelial cells of more than 20 example health Women of Childbearing Age
It is respectively provided with stronger adhesive attraction.Fig. 2 is oil after the adhesive attraction Gram's staining of lactobacillus curvatus and vaginal epithelial cell
Sem observation figure, figure left side are the vaginal epithelial cell for being located away from healthy Women of Childbearing Age；Figure right side is vaginal epithelial cell
With the adhesive attraction of lactobacillus curvatus, stronger adhesion (lactobacillus curvatus IMC-79) is represented.From more than 20 examples, random choosing
4 are taken, carries out statistical analysis, the quantity that each vaginal epithelial cell average adhesion lactobacillus curvatus IMC-79 is calculated reaches
113 (being shown in Table 2).According to The Journal of Infectious Diseases, Vol.171, No.5 (May, 1995),
Even if pp.1237-1243 adherent cells are strong more than 50 adhesive capacities.
The vaginal epithelial cell of table 2 adhesion lactobacillus curvatus IMC-79 quantity statistics
The result of integrated embodiment 1 and embodiment 2, lactobacillus curvatus IMC-79 of the invention both have stronger generation
H2O2Ability (growth that can effectively suppress pathogen and other conditions pathogenic bacteria), again have it is stronger to vaginal epithelial cell
Adhesive attraction (ensure that field planting of the bacterial strain in vagina epithelium), this is enough to illustrate, the lactobacillus curvatus that obtain of the present invention
IMC-79 is candidate's probiotics strain of effective preventing and treating bacterial vaginosis BV.
In addition, in bacteriostatic experiment, using staphylococcus aureus as indicator bacteria, the antibacterial ring size of IMC79 culture supernatants
1.2 millimeters of diameter average out to.
Claims (10)
1. one plant of lactobacillus curvatus (Lactobacillus.crispatus) bacterial strain IMC-79, its preserving number is CGMCC
No.9468。
2. the product for preventing and/or treating bacterial vaginosis BV, it is characterised in that：The active component of the product includes power
Profit requires the lactobacillus curvatus bacterial strain IMC-79 described in 1.
3. the product for preventing and/or treating bacterial vaginosis BV, it is characterised in that：The active component of the product is right
It is required that the lactobacillus curvatus bacterial strain IMC-79 described in 1.
4. the product according to Claims 2 or 3, it is characterised in that：Also containing the customary adjuvant composition for preparing microbial inoculum.
5. the product for suppressing pathogenic bacteria, it is characterised in that：The active component of the product is included described in claim 1
Lactobacillus curvatus bacterial strain IMC-79.
6. the product for suppressing pathogenic bacteria, it is characterised in that：The active component of the product is described in claim 1
Lactobacillus curvatus bacterial strain IMC-79.
7. the product according to claim 5 or 6, it is characterised in that：Also containing the customary adjuvant composition for preparing microbial inoculum.
8. the lactobacillus curvatus bacterial strain IMC-79 described in claim 1 is preparing prevention and/or is treating bacterial vaginosis BV
Purposes in product, it is characterised in that the active component of the product includes the lactobacillus curvatus bacterial strain described in claim 1
IMC-79。
9. the lactobacillus curvatus bacterial strain IMC-79 described in claim 1 is preparing prevention and/or is treating bacterial vaginosis BV
Purposes in product, it is characterised in that：The active component of the product is the lactobacillus curvatus bacterial strain described in claim 1
IMC-79。
A kind of 10. external method for suppressing pathogenic bacteria, it is characterised in that use with one plant of bending described in claim 1
Lactic acid bacillus mycopremna IMC-79 is that the product of active component suppresses to external existing pathogenic bacteria.
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| CN105400724A (en) * | 2015-12-17 | 2016-03-16 | 中国科学院微生物研究所 | Lactobacillus. crispatus strain, and microecological preparation thereof |
| CN105349474B (en) * | 2015-12-17 | 2019-07-02 | 中国科学院微生物研究所 | For preventing and treating the lactobacillus curvatus of women's urogenital infections |
| CN111088178B (en) * | 2018-10-23 | 2022-11-22 | 上海上药信谊药厂有限公司 | Produce lactic acid and H 2 O 2 Lactobacillus and application thereof |
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| CN101657534A (en) * | 2007-03-19 | 2010-02-24 | 科·汉森有限公司 | A new lactic acid bacteria strain and its use for the protection of food products |
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