CN104293718B - Lactobacillus jensenii and application thereof - Google Patents
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Abstract
The invention discloses a lactobacillus jensenii and application thereof. The lactobacillus jensenii disclosed by the invention is named as IMJ-19, and is collected in the China General Microbiological Culture Collection Center on July 14th, 2014, with the collection number of CGMCC No.9439. The lactobacillus jensenii IMJ-19 has strong capability of generating H2O2, can effectively inhibit the growth of foreign pathogenic bacteria and other conditioned pathogens, also has strong adhesion action to vagina epithelial cells, and ensures the colonization of strains on vaginal epithelial, and the lactobacillus jensenii IMJ-19 has certain application prospects on the aspect of prevention and treatment of urogenital tract infection.
Description
Technical field
The present invention relates to microorganism field is and in particular to one plant of Lactobacillus Jensenii and its application.
Background technology
Urogenital infections are that women is most commonly seen but be not affected by the frequently-occurring disease paid attention to, and the incidence of disease is only second to respiratory tract
Infection, may be up to 40%;Female urogenital tract infection is urinary tract infections and the general name of RTI, and it not only directly affects
The physical and mental health of women, and numerous family's wellbeings can be affected and stablize, and then affect quality of the people and the warp of the whole society
Ji development.The common cause of disease of women urinary tract infections is gram-Negative bacillus, wherein common with EHEC, staphylococcus
Take second place;These majorities are the normal flora on human body intestinal canal and skin and mucosa, and because immunity of organisms declines, these bacteriums can be by
The enteron aisle and position of living away from home such as mucocutaneous migrates to urinary system and amount reproduction causes urinary tract infections.The common genital tract of women
Pathogenic infection includes trichomonad, mould and Chlamydia etc., and RTI is the overwhelming majority first per vaginam infect and rise;Because of protracted inflammation
Diffusion can cause pelvic infecton, and leads to infertility.Urogenital infections are " little disease, burdens ", also can increase AIDS
Danger with venereal diseases such as gonorrhoeas;Patients ' life quality is decreased obviously, and mental burden is very heavy.Gravid woman may occur in which a series of gestation
Complication, the such as Averse pregnancy outcomes such as premature labor and chorioamnionitis;Also neonate can be directly made to be subject to by birth canal during childbirth similar
Pathogenic infection and cause a disease.
At present, the treatment of urogenital infections relies primarily on various antibiotic, but therapeutic effect is not good, and cure rate exists
Less than 40%, and the ratio recurring is very high, up to more than 40%.Reason is mainly drug resistance pathogen in Patients with Female Genital
Exist and cause antibiotic to lose efficacy;Even if in addition, antibiotic therapy can kill pathogen, but also kill beneficial bacterium (benefit simultaneously
Raw bacterium), accordingly, it is difficult to the urogenital tract dominant microflora under state of getting well.
There is substantial amounts of bacterium in healthy women of reproductive age urogenital tract, be generically and collectively referred to as urogenital tract flora, wherein wrap
Include multiple different types of bacteriums, " beneficial bacterium " and " pathogenic bacteria " two big class can be roughly classified into, wherein Bacillus acidi lactici is to maintain to secrete
The urodaeum most common and most important beneficial bacterium (" just bacterium ") of health, EHEC, staphylococcus, trichomonad, mould and
Chlamydia is then pathogenic bacteria (" evil bacterium ").Some factors lead to a certain pathogenic bacteria abnormality proliferation, cause urogenital tract sense
Dye.The change of vagina bacterial species is closely related with the health of women and fetus, lactobacillus curvatus therein and Zhan Shi breast bar
Bacterium is the main advantage " just bacterium " maintaining vaginal health micro-ecological environment, can account for healthy urogenital tract flora bacteria total amount
More than 90%.They mainly pass through to suppress the growth of external pathogen and other conditions pathogenic bacteria (" evil bacterium ") and invade
Maintain urogenital tract flora health environment, " just bacterium (beneficial bacterium) " defeat the mechanism of action of " evil bacterium " mainly include with
Lower several respects: the glycogen that (1) passes through to decompose storage in vaginal mucosa epithelium produces the acidic materials such as lactic acid, acetic acid, maintains vagina
Sour environment, with suppress pathogen growth;(2) produce multiple antibacterial substance such as hydrogen peroxide and lactein, in order to
Kill the pathogen of invasion;(3) Bacillus acidi lactici class beneficial bacterium is by, after the field planting of vaginal mucosa epithelium, forming and first impressions are strongest
Occupy-place protective effect, stops the invasion of pathogenic microorganism.
The women of child-bearing age increased with the age, and vagina lactobacillus curvatus or Lactobacillus Jensenii quantity can gradually decrease.Post menopausal,
Ovarian estrogen secretion tapers off, and vaginal epithelial cell glycogen deposit lacks, and vagina ph rises, and lactobacillus curvatus or Zhan Shi are newborn
Bacillus can reduce further, pathogenic bacteria thus increase.So lactobacillus curvatus or Lactobacillus Jensenii give birth to the uropoiesis of elderly woman
Grow health and also there is important function.
Microecology theory thinks, the composition adjusting therapy righting by ecological agent and protecting intravaginal dominant microflora
The invasion of external " evilness " pathogenic bacteria can be driven away with ratio, improve the self-protection of women's " strengthening vital QI to eliminate pathogenic factors " inherently
Effect.Lactobacillus is the dominant microflora of healthy women vaginal flora, accounts for vaginal bacteria total amount more than 90%, urogenital infections
Patient's Vaginal lactobacilli significantly reduces, and have even can't detect completely, therefore loses and resists external pathogenic bacterial infection
Ability.And pass through the lactobacillus that supplement lacks, then be conducive to rebuilding the dominant microflora of female genitourinary tract, recover it intrinsic
The protective effect of " strengthening vital QI to eliminate pathogenic factors ".
Urogenital infections are the common disease of women, frequently-occurring disease, are easy to recur, though having preferable short-term to treat with antibiotic
Effect, but have 40% recurrence rate.Research has shown that: has the dominant strain lactobacillus curvatus of protective effect or Lactobacillus Jensenii
Preparation will be easier to settle down in vagina and breed, and produce antibacterial material, play lasting effect.Therefore exploitation adapts to China
The specific Lactobacillus vaginalis preparation wide market of women's feature, changes in the past pure on treatment urogenital infections
Pure sterilized medication guide thought, it is to avoid by widely applying a series of bad reactions that antibiotic brought as treatment and disadvantage
End, is conducive to recovering the dominant microflora of female genitourinary tract and rebuilding its microecological balance;Thus effectively reducing urogenital
The recurrence rate of road infection, really cures Female urogenital tract infection.
At present, domestic Female urogenital tract infection only has one kind with probio microbial inoculum, and its main component is De Shi breast bar
Bacterium viable bacteria.Although it is a kind of lactobacillus separated from healthy women vagina, in Bu Shi China woman vagina flora
Main beneficial bacterium.
Content of the invention
It is an object of the invention to provide one plant of Lactobacillus Jensenii (lactobacillus.jensenii) and its application.
Lactobacillus Jensenii provided by the present invention, is named as imj-19, has been preserved in China on July 14th, 2014 micro-
Biological inoculum preservation administration committee common micro-organisms center (abbreviation cgmcc, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is cgmcc no.9439.
It is a further object to provide a kind of microbial inoculum, its active component is Lactobacillus Jensenii described above
(lactobacillus.jensenii)imj-19cgmcc no.9439.
The preparation method of microbial inoculum described above is also protection scope of the present invention.
The preparation method of microbial inoculum described above comprises the steps: above-mentioned Lactobacillus Jensenii (lactobacillus.jensenii)
Imj-19cgmcc no.9439, as active component, obtains described microbial inoculum.
The preparation method of above-mentioned Lactobacillus Jensenii or microbial inoculum or microbial inoculum maintains the sour environment of urogenital tract in preparation
Application in product falls within protection scope of the present invention.
The preparation method of above-mentioned Lactobacillus Jensenii or microbial inoculum or microbial inoculum preparation suppression urogenital tract in pathogen and/or
Application in the product of the growth of conditioned pathogen and/or breeding falls within protection scope of the present invention.
The preparation method of above-mentioned Lactobacillus Jensenii or microbial inoculum or microbial inoculum has to the epithelial cell of urogenital tract in preparation
Application in the product of Colonization ability falls within protection scope of the present invention.
The urogenital tract that the preparation method of above-mentioned Lactobacillus Jensenii or microbial inoculum or microbial inoculum produces hydrogen peroxide in preparation is beneficial
Application in bacterium falls within protection scope of the present invention.
The preparation method of above-mentioned Lactobacillus Jensenii or microbial inoculum or microbial inoculum is in preparation prevention and/or treatment urogenital infections
Product or the application in the product of disease that preparation prevention and/or treatment are caused by urogenital infections fall within this
Bright protection domain.
Lactobacillus Jensenii (lactobacillus.jensenii) imj-19 that the present invention provides, has stronger generation
h2o2Ability, can effectively suppress the growth of external pathogen and other conditions pathogenic bacteria, have simultaneously again stronger to vagina
The adhesive attraction of epithelial cell it is ensured that bacterial strain vagina epithelium field planting.Lactobacillus Jensenii imj-19 is in urogenital tract sense
Dye is prevented and treated aspect and is had certain application prospect.
Brief description
Fig. 1 is Lactobacillus Jensenii imj-19 bacterium colony.
Fig. 2 is the adhesive attraction of Lactobacillus Jensenii imj-19 and vaginal epithelial cell.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Healthy women vaginal fluid sample is provided by vast thinking healthy health management organization.
The preparation of mrs fluid nutrient medium: by 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, magnesium sulfate (mgso4·7h2O) 0.58g, manganese sulfate (mnso4·h2O) 0.25g is dissolved in distilled water, and uses distilled water
It is settled to 1000ml, be adjusted to ph to 6.2-6.6.
Embodiment 1, the separation of Lactobacillus Jensenii bacterial strain and identification
First, the separation of Lactobacillus Jensenii bacterial strain
The healthy women vaginal fluid sample of collect more than 200 example clinical detection absence of vagina inflammation symptom and signs is compiled successively
Number, and it is inoculated in mrs fluid nutrient medium, 37 DEG C of Anaerobic culturel 24h.Each sample selects 5 single bacterium colonies at random in 2ml mrs liquid
In body culture medium, culture increases bacterium 24h, and to bacterium colony number consecutively.By hundreds of plants be separated to bacteriums carry out respectively strain idenfication,
Produce hydrogen peroxide and the detection with vaginal epithelial cell adhesive attraction, wherein one plant is had stronger adhesive attraction and produces peroxidating
The Lactobacillus Jensenii of hydrogen is named as imj-19.
2nd, the identification of Lactobacillus Jensenii bacterial strain
(1) Morphological Identification
Above-mentioned steps one are separated the bacterial strain imj-19 obtaining and is inoculated in culture on mrs Bacillus acidi lactici culture medium flat plate.Knot
Fruit shows: it is neat, relatively that bacterium colony shows as diameter about 1-2 millimeter, opaque, projection milky bacterium colony, and colony edge
Moistening, size and form stable (as shown in Figure 1).The described Lactobacillus Jensenii being in exponential phase is identified as: Grain stain
The positive, asporous bacillus, atrichia.
(2) analysis of physio biochemical characteristics
Bacterial strain imj-19 amphimicrobian, grows optimum temperature 37-42 DEG C, optimum ph 5.8-6.2.According to api Bacteria Identification
Standard, using api 50chl culture medium and api 50ch reagent strip (purchased from biome ' rieux, inc., marcy l ' etoile,
France) physiological and biochemical property that sugared fermentation reaction interpretation detects bacterial strain imj-19 is carried out to detection bacterial strain imj-19.api
The simple culture medium that 50chl is made up of the api 50ch test bar of 49 kinds of fermentable carbohydrates.To detect bacterial strain imj-
19 each tubule making suspension inoculation test bar.When culture, because fermentable carbohydrates produce acid, ph declines, and makes finger
Show agent variable color.
Result shows: bacterial strain imj-19 can multiple sugar such as glucose fermentation, fructose, sucrose, trehalose, maltose.
(3) 16s rdna sequence homology analysis
The total dna extracting bacterial strain imj-19, as template, carries out pcr amplification using universal primer 27f and 1492r, obtains
Fragment containing bacterial strain imj-1916s rrna gene conserved region.Primer sequence is as follows:
27f (upstream primer): 5 '-agagtttgatcctggctcag-3 ';
1492r (downstream primer): 5 '-tacggttaccttgttacgactt-3 '.
Pcr system (25 μ l): pcr amplification buffer, final concentration of 200 μm of ol/l dntps, final concentration is 0.2 μ
The upstream of mol/l and downstream primer, 1u taq dna polymerase, 1 μ l template dna, balance of ddh2o.
Pcr condition: 94 DEG C of denaturations 5min;94 DEG C of denaturation 30s, 55 DEG C of annealing 40s, 72 DEG C of extension 30s, totally 25 are followed
Ring;72 DEG C of extension 10min.
The sequence of the 16s rdna of bacterial strain imj-19 that pcr amplification obtains is as shown in seq id no.1.
Comprehensive above identification and analysis, the Classification And Nomenclature of bacterial strain imj-19 is Lactobacillus Jensenii (lactobacillus
Jensenii), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on July 14th, 2014
Center (abbreviation cgmcc, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
100101), preserving number is cgmcc no.9439.
Embodiment 2, the functional analysis of Lactobacillus Jensenii imj-19
First, Lactobacillus Jensenii imj-19 produces h2o2Detection
(1) h2o2The making of calibration curve: the piperazine-n being 100mmol/l with concentration first, n'- bis--ethyl sulfonic acid
Volumn concentration is 30% by the aqueous solution of (piperazine-n, n-bis 2-ethanesulfonic acid, pipes)
H2o2The aqueous solution (being equivalent to 9.128mol/l) storing liquid is diluted to 1mol/l, then will with the aqueous solution of 100mmol/l pipes
The h of above-mentioned 1mol/l2o2It is diluted to 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 respectively, will be above-mentioned
The aqueous solution of each dilution and 100mmol/l pipes is denoted as h2o2Working solution;Then take the above-mentioned h of 100 μ l respectively2o2Work
Liquid and 100 μ l concentration are the aqueous solution of tmb (tetramethyl benzidine) of 20mmol/l, 2 μ l concentration are the horseradish peroxide of 1mg/ml
Compound enzyme (purchased from takara company) mixes, 16 DEG C of incubation 10min, measures od630Value.With h2o2Concentration (unit: μm ol/l)
For abscissa, od630For ordinate, make calibration curve, obtain calibration curve formula y=0.0033x-0.0496, r2=
0.992.
(2) Lactobacillus Jensenii imj-19 Liquid Culture h2o2Concentration Testing: picking Lactobacillus Jensenii imj-19 bacterium colony, by it
It is inoculated in mrs fluid nutrient medium, carry out under anaerobic cultivating 48h, take 1ml to be inoculated in 10ml mrs fluid nutrient medium,
The concentration making bacterium in nutrient solution is 105Cfu/ml, under anaerobic environment, after 37 DEG C of quiescent culture 21h, is converted to 37 DEG C of vibration trainings
Foster 3h, rotating speed is 220r/min, and radius of turn is 2.54cm.After culture terminates, collect nutrient solution, 12000r/min is centrifuged
2min, obtains supernatant.Take the aqueous solution of tmb (tetramethyl benzidine) that 100 μ l supernatants are 20mmol/l with 100 μ l concentration, 2 μ l dense
Spend horseradish peroxidase (purchased from the takara company) mixing for 1mg/ml, 16 DEG C of incubation 10min, measure od630Value, by it
Bring the calibration curve formula that step () obtains into, calculate the h in supernatant2o2Concentration.Test in triplicate, results averaged.
Result is as shown in table 1.
Table 1 Lactobacillus Jensenii imj-19 produces h2o2Ability detection
Repeat 1 | Repeat 2 | Repeat 3 | Mean value | |
h2o2Yield | 310.8 | 357.8 | 298.3 | 322.3 |
Result shows: Lactobacillus Jensenii imj-19 can produce substantial amounts of h in fermentation culture2o2, its concentration reaches
322.3μm.
2nd, the adhesive attraction to vaginal epithelial cell for the Lactobacillus Jensenii imj-19
(1) vaginal epithelial cell is taken from more than 20 example health (clinical detection display non-reproductive system infection) child-bearing period woman
Female, will be separately added into mem culture medium (ph is 4.0), 120g is centrifuged 10min, repeats centrifugation 2 times after each cell sample label,
Wash surface bacteria off, be 10 with mem culture medium adjustment vaginal epithelial cell concentration5Individual/ml.
(2) the Lactobacillus Jensenii imj-19 of activation incubated overnight makes its concentration is 108cfu/ml.
(3) concentration obtaining step () is 105The concentration that the vaginal epithelial cell of individual/ml and step (two) obtain
For 108Be placed in after each 500 μ l equal-volumes mixing of the Lactobacillus Jensenii imj-19 of cfu/ml 37 DEG C of shaking tables (rotating speed is 100r/min,
Radius of turn is 2.54cm) incubation 1h, then with 0.8 μm of membrane filtration, removes free Lactobacillus Jensenii imj-19, and will filter
Film is gently pressed on cover glass, and after fixing cell carries out Gram's staining, oil mirror is observed, and counts three vaginal epithelial cells respectively and glues
Attached Lactobacillus Jensenii imj-19 bacterial population, calculates the Lactobacillus Jensenii imj-19 bacterium of each vaginal epithelial cell average adhesion
Number.Vaginal epithelial cell is carried out with oil mirror after Gram's staining simultaneously observe, as comparison.
Result is as shown in Fig. 2 the right side is for adhering to the vaginal epithelial cell of Lactobacillus Jensenii imj-19 in Fig. 2;Left is to be located away from
The vaginal epithelial cell (comparison) of healthy Women of Childbearing Age.Result shows: Lactobacillus Jensenii imj-19 is good for being isolatable from more than 20 examples
The vaginal epithelial cell of health Women of Childbearing Age is respectively provided with stronger adhesive attraction.
From more than 20 examples, randomly select 4, carry out statistical analysis, result is as shown in table 2.Result shows: on each vagina
The quantity of chrotoplast average adhesion Lactobacillus Jensenii imj-19 reaches 65.
Table 2 vaginal epithelial cell adheres to Lactobacillus Jensenii imj-19 quantity statistics
Based on the above results, Lactobacillus Jensenii imj-19 had both had stronger generation h2o2Ability (can effectively suppress cause of disease
Bacterium and the growth of other conditions pathogenic bacteria), there is the stronger adhesive attraction to vaginal epithelial cell again and (ensure that bacterial strain in the moon
The field planting of road epithelium), this allows for Lactobacillus Jensenii imj-19 becomes effective candidate probio preventing and treating urogenital infections
Bacterial strain.
Claims (8)
1. one plant of Lactobacillus Jensenii (lactobacillus.jensenii), this bacterial strain is named as imj-19, in 2014 7
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 14th, and its deposit number is cgmcc
no.9439.
2. a kind of microbial inoculum, its active component is the Lactobacillus Jensenii (lactobacillus.jensenii) described in claim 1
imj-19cgmcc no.9439.
3. the preparation method of microbial inoculum described in claim 2, comprises the steps: the Lactobacillus Jensenii described in claim 1
(lactobacillus.jensenii) imj-19cgmcc no.9439, as active component, obtains described microbial inoculum.
4. the system of the Lactobacillus Jensenii described in claim 1 or the microbial inoculum described in claim 2 or the microbial inoculum described in claim 3
Preparation Method maintains the application in the product of sour environment of urogenital tract in preparation.
5. the system of the Lactobacillus Jensenii described in claim 1 or the microbial inoculum described in claim 2 or the microbial inoculum described in claim 3
In the Preparation Method growth of pathogen and/or conditioned pathogen and/or product of breeding in preparation suppression urogenital tract should
With.
6. the system of the Lactobacillus Jensenii described in claim 1 or the microbial inoculum described in claim 2 or the microbial inoculum described in claim 3
Preparation Method has the application in the product of Colonization ability in preparation to the epithelial cell of urogenital tract.
7. the system of the Lactobacillus Jensenii described in claim 1 or the microbial inoculum described in claim 2 or the microbial inoculum described in claim 3
Preparation Method produces the application in the urogenital tract beneficial bacterium of hydrogen peroxide in preparation.
8. the system of the Lactobacillus Jensenii described in claim 1 or the microbial inoculum described in claim 2 or the microbial inoculum described in claim 3
Preparation Method prevents in the product of preparation prevention and/or treatment urogenital infections or in preparation and/or treatment is by urogenital
Application in the product of disease that road infection causes.
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