CN105238727A - Lactobacillus plantarum capable of antagonizing campylobacter jejuni and inhibiting expression of flaA gene of campylobacter jejuni - Google Patents
Lactobacillus plantarum capable of antagonizing campylobacter jejuni and inhibiting expression of flaA gene of campylobacter jejuni Download PDFInfo
- Publication number
- CN105238727A CN105238727A CN201510777864.9A CN201510777864A CN105238727A CN 105238727 A CN105238727 A CN 105238727A CN 201510777864 A CN201510777864 A CN 201510777864A CN 105238727 A CN105238727 A CN 105238727A
- Authority
- CN
- China
- Prior art keywords
- campylobacter jejuni
- plant lactobacillus
- cgmccno
- jejuni
- flaa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses lactobacillus plantarum. The Lactobacillus plantarum can antagonize campylobacter jejuni and inhibit expression of a flaA gene of the campylobacter jejuni, has the good acid resistance and bile salt resistance, can inhibit growth of the campylobacter jejuni in vitro and has the good adhesive capacity on the intestinal epithelial cells. A fermentation supernatant of the lactobacillus plantarum enables the expression amount of the flaA gene of the campylobacter jejuni to be reduced by 8.2 times compared with a control group in vitro, and synthesis of campylobacter jejuni flagella is effectively inhibited; the lactobacillus plantarum enables the expression amount of the flaA gene of the campylobacter jejuni to be reduced by 1.7 times compared with a control group in a cell model. The lactobacillus plantarum can be used for preparing medicine for preventing and treating diseases caused by the campylobacter jejuni and participate in preparing fermentation milk products and the like and has the wide application prospect.
Description
Technical field
The present invention relates to a strain antagonism campylobacter jejuni and suppress the plant lactobacillus of its flaA genetic expression, belong to microbial technology field.
Background technology
Campylobacter jejuni (Campylobacterjejuni) is Gram-negative bacteria, be distributed widely in nature, by propagation such as animal, food, water, milk, can be colonizated in the enteron aisle of various wildlife and poultry, domestic animal, contact poultry class, eats and not to boil or contaminated chicken, beef, thoroughly the milk of sterilization and the water of pollution can cause human infection.In the last few years, C. jejuni infec-tion rate was generally in rising trend all over the world.In some developed countries, the dysentery number of cases that C. jejuni infec-tion causes even has exceeded Salmonellas and Shigellae, becomes modal diarrhea pathogens; In developing country, campylobacter jejuni is that infant infection is suffered from diarrhoea modal pathogenic bacteria.After people infects campylobacter jejuni, modal meeting causes gastroenteritis, diarrhoea, fever and abdominal colic; Immunocompromised person can cause a series of complication further, such as cholecystitis, peritonitis, meningitis, septicemia and osteomyelitis etc.; The most serious complication that campylobacter jejuni causes is Guillain Barre syndrome, i.e. GBS, and it can cause the damage of aixs cylinder and irreversible nerve injury, can cause paralysis of respiratory muscle and dead.
C.jejuni by differentiating environmental factors, regulating and expressing or suppress some specific gene of self to adapt to the intestinal environment of pin main body inner height competition in host.Only in this way could survive, grow, final implement that it is pathogenic, and amount reproduction.Namely the first step of C.jejuni invasion and attack sticks and field planting, and this is inseparable with the expression of its virulence factor flaA, and the expression of flaA also invades target cell with C.jejuni close contacting, if the expression of C.jejuni virulence factor flaA is suppressed, it is pathogenic will weaken greatly.The research of 2008 such as QuocV.Tu finds that C.jejuni utilizes MUC2 can suppress the expression of its flaA gene as environmental triggers; The research in 2013 such as A.Mundi finds that the cell-free extract after having the fermentation suppressing the LactobacillusacidophilusStrainLa-5 of C.jejuni activity can suppress the expression of flaA gene in vitro.But the research at present not about regulation and control campylobacter jejuni flaA genetic expression in vitro level suppresses its flaA genetic expression multiple very low mostly, the regulating and controlling effect of milk-acid bacteria in cell model to campylobacter jejuni flaA genetic expression be it be unclear that, also do not verify the function of its antagonism campylobacter jejuni in vivo.
The common drug that current clinical treatment C.jejuni infects is microbiotic, but antibiotic use can bring body and C.jejuni to antibiotic resistance, and excessive use also can cause the residual of drug disposition.
Milk-acid bacteria is the important member of body innate immune system, is that body maintains the indispensable part of immune homeostasis.Can maintain intestinal health, thus reach the object of growth promoting effects, disease preventing and treating, its use value comes into one's own day by day.Utilize milk-acid bacteria to intervene at present and treat the key areas that digestive tract bacterial infection has become domestic and international Food science and biological medicine research, research shows that milk-acid bacteria can infect by effective prevention C.jejuni in vivo and in vitro.The mechanism that milk-acid bacteria intervenes C.jejuni infection mainly comprises the growth producing antimicrobial substance and suppress C.jejuni; The milk-acid bacteria that adhesion is high can suppress C.jejuni sticking host cell by space steric effect or generation inhibitory substance; The integrity of milk-acid bacteria to Intestinal Morphology has significant provide protection, can alleviate the infection symptoms of C.jejuni to intestinal epithelial cell; Milk-acid bacteria some can also have the material of immunoregulatory activity to cell exocrine, as exocellular polysaccharide and other secretory protein, these compositions participate in immunomodulatory or directly cause immunne response, thus constitute the basic substance that milk-acid bacteria plays beneficial function, play a role in bacteria planting, immuno-stimulating, prevention canceration etc.
At present, some patent document antagonisms relating to campylobacter jejuni and uses thereof, such as CN104870472A discloses a kind of antibody and the fragment thereof that are specific to campylobacter jejuni; CN102497875A discloses a kind of antigen for reducing campylobacter jejuni field planting; CN103381220A discloses a kind of medicine can treating chicken campylobacter jejuni; But, relate to milk-acid bacteria antagonism campylobacter jejuni at present and suppress the Patent Application Publication of its virulence gene expression also few, CN102533618A discloses a kind of plant lactobacillus with vitro inhibition campylobacter jejuni ability, but this patent does not relate to the expression of the flaA gene of campylobacter jejuni and the effect in cell model and body in antagonism campylobacter jejuni.
Therefore, filter out the milk-acid bacteria of a strain antagonism campylobacter jejuni, prove that it in vitro and effectively can suppress the expression of campylobacter jejuni flaA gene in cell model, and there is the effect of good alleviation C. jejuni infec-tion in animal model, and study this milk-acid bacteria practical use and just seem very necessary.
The present inventor, on the basis of summing up prior art, by lot of experiments, completes the present invention finally.
Summary of the invention
The invention provides a kind of plant lactobacillus (Lactobacillusplantarum), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on September 24th, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11445.
Described plant lactobacillus CGMCCNo.11445 has following biological characteristics:
(1) thalline feature: Gram-positive, elongated rod-shaped, micro-chain that curves occurs, atrichia, without gemma;
(2) colony characteristics: bacterium colony oyster white, edge is irregular, sheet, center projections, and surface wettability is smooth;
(3) growth characteristics: the minimum growth temperature of this bacterial strain is 15 DEG C, and maximum growth temperature is 40 DEG C, at temperature 30-37 DEG C, growth is best, and the most suitable growth PH is 6.5, enters stationary phase after cultivating 12h;
(4) have acid resistance, well-grown under pH3.0-9.0 envrionment conditions, under pH2.0 environment, survival is good;
(5) have Bile salt resistance, in the scope that gallbladder salinity is 0.1% one 0.4%, strain growth is good;
(6) vitro inhibition campylobacter jejuni growth;
(7) to intestinal epithelial cell, there is good adhesive capacity;
(8) plant lactobacillus CGMCCNo.11445 fermentation supernatant significantly can suppress the expression of campylobacter jejuni flaA virulence factor in vitro;
(9) plant lactobacillus CGMCCNo.11445 fermentation supernatant can affect the synthesis of campylobacter jejuni flagellum in vitro;
(10) plant lactobacillus CGMCCNo.11445 fermentation supernatant significantly can suppress the expression of campylobacter jejuni flaA virulence factor in cell model.
Plant lactobacillus CGMCCNo.11445 of the present invention can be used for pharmaceutical composition or the health functional food of preparing suppression campylobacter jejuni.By acquisition thalline centrifugal after plant lactobacillus CGMCCNO.11445 activation culture, compositely with pharmaceutically acceptable carrier after centrifuge washing obtain pharmaceutical composition or obtained nourishing function product composite with available support in food.Described pharmaceutical composition is made up of plant lactobacillus CGMCCNo.11445 microbial inoculum and pharmaceutically acceptable carrier.Described pharmaceutically acceptable carrier is that one or more are selected from pharmaceutically normally used tackiness agent, weighting agent, wetting agent, disintegrating agent, lubricant or correctives.Described pharmaceutical composition is capsule, granule, tablet, pill or oral liquid formulation.
Described plant lactobacillus CGMCCNo.11445 can be used for preparing leavened food.Described leavened food be use starter containing plant lactobacillus CGMCCNo.11445 bacterial classification to produce milk-product, fruit and vegetable product and, bean product.Described milk-product are yogurt milk, smetana or cheese; Described fruit and vegetable product is cucumber, beet, celery, Radix Dauci Sativae or Caulis et Folium Brassicae capitatae goods; Described bean product are soy sauce, soybean milk yoghurt, fermented soya bean or beans sauce.
The present invention also provides a kind of method preparing plant lactobacillus CGMCCNo.11445 microbial inoculum, be to be prepared by normal freeze-drying technique containing described plant lactobacillus biological bacteria CGMCCNo.11445 bacterium liquid or other technique prepares obtained pulvis, it contains and is greater than 10
6the active plant Bacterium lacticum CGMCCNo.11445 of CFU/g.
A preferred embodiment of the invention, described starter adopts following method to prepare:
The preparation of A, substratum:, 0.3% yeast extract old with this substratum total weight 10% enzymic hydrolysis skimming milk, 0.5% glucose, 1.5 Trypsins and excess water, the pH of the prepared substratum of adjustment is to 6.8.
B, protective material: the protective material of 100g/L skim-milk, 30mL/L glycerine, 100g/L maltodextrin, 150g/L trehalose, 10g/LL-Sodium Glutamate.
C. plant lactobacillus CGMCCNo.11445 bacterial classification is according to being inoculated in the substratum at temperature 110-120 DEG C after sterilizing 8-12min in the inoculum size of the quality 2% of described substratum; then at temperature 37 DEG C, 18h is cultivated; with pH7.2 phosphate buffered saline buffer cleaning 2-4 time, make that bacterium is dense reaches 10 with described protective material is resuspended
10cFU/ml; Then, allow this suspension preculture 60min at temperature 37 DEG C, then carry out lyophilize and obtain described fermenting agent.
Plant lactobacillus CGMCCNo.11445 of the present invention has acidproof, bile tolerance characteristic, to intestinal epithelial cell, there is good adhesion properties, effectively can suppress the growth of campylobacter jejuni, effectively can suppress the expression of campylobacter jejuni flaA virulence factor in vitro and in cell model.Described plant lactobacillus CGMCCNo.11445 is used for the treatment of and prevents C. jejuni infec-tion, has widely application prospect.
Biomaterial preservation
A kind of plant lactobacillus (Lactobacillusplantarum), China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is preserved on September 24th, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11445.
Accompanying drawing explanation
Fig. 1 is the adhesion situation of plant lactobacillus CGMCCNO.11445 on HT-29 cell.
Fig. 2 is that plant lactobacillus CGMCCNO.11445 fermentation supernatant is in vitro on the impact that campylobacter jejuni flaA virulence factor is expressed.
Fig. 3 is that plant lactobacillus CGMCCNO.11445 fermentation supernatant is in vitro on the impact of campylobacter jejuni flagellar production.
Fig. 4 is the impact on the expression of campylobacter jejuni flaA virulence factor in cell model of plant lactobacillus CGMCCNO.11445 thalline.
Embodiment
Embodiment 1 screening lactobacillus from Pickles, Sichuan Style
Sample is taken from Pickles, Sichuan Style, is stored in 30% glycerine.Drawing 1ml pickles suspension after homogeneous joins in the test tube containing 9ml sterile saline, shaken well, spread plate method is adopted after gradient dilution, these dilution bacterium liquid are coated on and with the addition of acid base indicator and smell on MRS substratum (the Qingdao Hai Bo Bioisystech Co., Ltd product) flat board of cresol purple, Nostoc commune Vanch and Anaerobic culturel 48h is carried out respectively at temperature 37 DEG C, observe colony growth situation, picking can make the single bacterium colony of typical case smelling cresol purple flavescence in substratum, and gramstaining is carried out to single bacterium colony, picking gram-positive microorganism is as preliminary object bacterial strain, then the separation and purification of MRS plate streaking is forwarded to, until obtain pure lactobacillus strain, preserve as experimental strain and at temperature-20 DEG C.A kind of plant lactobacillus (Lactobacillusplantarum), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on September 24th, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11445.
Embodiment 2 adopts Odontothrips loti to screen the milk-acid bacteria that campylobacter jejuni can be suppressed to grow
The preparation of lactobacillus-fermented supernatant liquor: milk-acid bacteria separation and purification obtained is according to being inoculated in MRS liquid nutrient medium with MRS liquid nutrient medium volumeter 2%, 18h is cultivated at 37 DEG C, cultured continuously three generations in the same way, then by milk-acid bacteria bacteria suspension centrifugal 8min under 8000r/min, 4 DEG C of conditions, Aspirate supernatant, use 0.22 μm of water system filter membrane to carry out filtration sterilization, obtain lactobacillus-fermented supernatant liquor.
Campylobacter jejuni (Campylobacterjejuni) NCTClll68 bacterial strain (purchased from American Type Tissue Collection ATCC) on two substratum (i.e. brucella agar (Qingdao Hai Bo Bioisystech Co., Ltd product) with brain heart infusion broth substratum (Oxoid company substratum)) at temperature 37 DEG C and 5%O by volume
2, 10%CO
2and 85%N
2environment under cultivate 48h, in the same way after Secondary Culture three generations, centrifugal 6min under 2800r/min condition, use phosphate buffered saline buffer (PBS, pH are 7.2) washing after, adjust this bacteria concentration to 10
8cFU/mL.
Draw C.jejuni (numbering NCTC11168) bacteria suspension (250 μ L, 10
8cFU/mL) be spread evenly across in Columbia Blood Agar flat board (Oxoid company substratum), after the seasoning of bacterium liquid, place Oxford cup, in the cup of Oxford, add 100 μ L lactobacillus-fermented supernatants.Three gas incubator (5%O are put into after 4 DEG C of diffusion 2h
2, 10%CO
2and 85%N
2), measure the diameter of inhibition zone after cultivating 48h through 37 DEG C.Using pH be the MRS of 3.8 as negative control, 0.28mg/ml norfloxicin Broad spectrum antibiotics is positive control, detect antibacterial circle diameter (mm)
Table 1: the measuring result of antibacterial circle diameter
Table 1 shows that milk-acid bacteria CGMCCNO.11445 fermented supernatant fluid significantly suppress the growth of campylobacter jejuni.
Embodiment 3 plant lactobacillus CGMCCNo.11445 is to the tolerance test of simulated intestinal fluid and simulated gastric fluid
Simulated gastric fluid: take that 3g stomach en-(1:10000, Sigma, USA) is dissolved in 1L physiological saline (0.5%, w/v), adjustment pH is 2.0, this solution is crossed 0.45/0.22 μm of sterilized double layer millipore filtration, Preservation in sterile condition is for subsequent use.
Simulated intestinal fluid: take 1g trypsin P-1500, Sigma, USA) and 0.3g bovine bile, be dissolved in 1L physiological saline (0.5%, w/v) in, adjustment pH be 8.0, this solution is crossed 0.45/0.22 μm of sterilized double layer millipore filtration, Preservation in sterile condition is for subsequent use.
PBS solution (phosphate buffer soln): potassium primary phosphate 0.27g, Repone K 0.2g, disodium hydrogen phosphate dodecahydrate 2.85g, NaCl8.5g, distilled water 1L, pH regulator to 7.2, crosses 0.45/0.22 μm of sterilized double layer millipore filtration, 121 DEG C, 20min sterilizing, is stored in 4 DEG C of refrigerators.
(1) artificial simulation gastric juices tolerance test
Choosing lactic acid bacteria number magnitude is 10
9the PBS solution of CFU/mL carry out this experiment, and centrifugal (5000r/min, 10min, 4 DEG C), add isopyknic simulation simulated gastric fluid, arrange blank group, control group directly uses 10
9the PBS phage solution of CFU/mL, cultivate two hours in water-bath (37 DEG C), after completing, employing gradient dilution method carries out plate count, and intermediate demand is it is to be noted that on average rocked 1 time every 30 minutes.
(2) artificial simulation intestinal juice tolerance test
Choosing lactic acid bacteria number magnitude is 10
9the PBS solution of CFU/mL carry out this experiment, and centrifugal (5000r/min, 10min, 4 DEG C), add isopyknic simulation simulated intestinal fluid, arrange blank group, control group directly uses 10
9the PBS phage solution of CFU/mL, cultivates two hours in water-bath (37 DEG C), notes all shaking 1 time every 30 minutes, terminate rear use gradient dilution method and carry out plate count.
Table 2: plant lactobacillus CGMCCNO.11445 is to the tolerance test of simulated gastric fluid and simulated intestinal fluid
Right table 2 is known, and plant lactobacillus CGMCCNO.11445 has good tolerance to simulated gastric fluid and simulated intestinal fluid.
Embodiment 4 plant lactobacillus CGMCCNo.11445 is to the adhesive capacity of intestinal epithelial cell HT-29
RPMI1640 nutrient solution (Gibco company) (adding 10% foetal calf serum and 1% mycillin) is used to cultivate Intestinal epithelium cell HT-29 cell (purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank).HT-29 cell in incubator in 5%C0
2cultivate under the condition of atmosphere and temperature 37 DEG C, often cultivate 48h and just change nutrient solution 1 time, cultured continuously.
The HT-29 cell of growth fusion to 70%-80% is digested, adjustment concentration to 2 × 10
5individual/mL, be placed on by sterile cover slips in 6 porocyte culture plates, every hole adds 2mL suspension culture, and in 5%CO2 incubator, 37 DEG C of cultivations, when cell grows to individual layer, clean three times with PBS, and every hole adds 1mL milk-acid bacteria bacteria suspension (1 × 10
8cFU/mL), add RPMI-1640 cell culture fluid (not containing serum and microbiotic) and, to 2mL, hatch 2h.After hatching end, clean three times with PBS, to remove the milk-acid bacteria do not adhered to, then fix 20min with methyl alcohol, carry out gramstaining after PBS cleans three times, microscopy under 100 times of oily mirrors.Random selecting 20 visuals field calculate the bacterial count that every 100 cells adhere to, as adhesion index.Adhesion experiment the results are shown in table 3, and the adhesion situation of plant lactobacillus CGMCCNO.11445 on HT-29 cell is shown in accompanying drawing 1.
As shown in Table 3: plant lactobacillus CGMCCNo.11445 has stronger Adhering capacity to intestinal epithelial cell HT-29, and can to stick on intestinal epithelial cell be the prerequisite that it plays a role further, there is the plant lactobacillus CGMCCNO.11445 of stronger adhesive capacity after intestinal colonisation, effectively can prevent pathogenic micro-organism from contacting and adhere to intestinal mucosa cells, thus pathogen enterobacteria can be prevented to cause intestinal tract disease.
Embodiment 5 plant lactobacillus CGMCCNO.11445 is in vitro on the impact that campylobacter jejuni flaA virulence factor is expressed
C.jejuni is placed on two substratum (brucella agar+9ml brain heart infusion broth liquid nutrient medium) and cultivates 36h, 1ml plant lactobacillus fermentation supernatant is added under aseptic condition, control group adds 1mlMRS liquid nutrient medium, shakes up and is placed on (5%O in three gas incubators
2, 10%CO
2and 85%N
2) cultivate 4h, at 4 DEG C, the centrifugal 6min of 2800r/min collects thalline, extracts thalline total serum IgE, becomes cDNA with test kit (Takara company) reverse transcription, after carry out quantitative fluorescent PCR, and select rpoD as reference gene.Carry out quantitative fluorescent PCR, quantitative fluorescent PCR the primer sequence is as shown in table 4.
Table 4: the few core former times acid sequence of quantitative fluorescent PCR
Quantitative fluorescent PCR reaction conditions: (1) 95 DEG C, for30s; (2) 95 DEG C, 5s, 60 DEG C, 30s, 40 circulations; With 2
-Δ Δ Ctmethod calculates plant lactobacillus CGMCCNO.11445 fermentation supernatant in vitro on the impact that campylobacter jejuni flaA virulence factor is expressed.Experimental result is shown in accompanying drawing 2
From accompanying drawing 2: plant lactobacillus CGMCCNO.11445 fermentation supernatant can effectively suppress campylobacter jejuni flaA virulence factor to be expressed in vitro, and namely the first step of C.jejuni invasion and attack sticks and field planting, the expression of this and flaA gene is closely related, flaA genetic expression is suppressed, the survival of C.jejuni, growth, breeding and to be pathogenicly all affected.
Embodiment 6 plant lactobacillus CGMCCNO.11445 is in vitro on the impact of campylobacter jejuni flagellar production
C.jejuni is placed on two substratum (brucella agar+9ml brain heart infusion broth liquid nutrient medium) and cultivates 36h (logarithmic phase), 1ml plant lactobacillus fermentation supernatant is added under aseptic condition, control group adds 1mlMRS liquid nutrient medium, shakes up and is placed on (5%O in three gas incubators
2, 10%CO
2and 85%N
2) cultivate 4h, at 4 DEG C, the centrifugal 6min of 2800r/min collects thalline.
Abandon supernatant, add PBS solution washing and remove substratum, at 4 DEG C, the centrifugal 6min of 2800r/min collects thalline; In 2.5% glutaraldehyde, fix 4h, wash three times by PBS solution; At 4 DEG C, the centrifugal 6min of 2800r/min collects thalline, and use the Gradient elution using ethanol of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 85%, 90%, 100% respectively, centrifugal condition is 4 DEG C, the centrifugal 6min of 2800r/min; Ethanol is replaced twice with Isoamyl Acetate FCC; By sample at-80 DEG C of pre-freeze 12h postlyophilizations; Scanning electron microscope (FDAC, S-4700 cold field emission) observes campylobacter jejuni flagellum.
From accompanying drawing 3, after plant lactobacillus CGMCCNO.11445 fermentation supernatant process C.jejuni, the flagellum quantity of C.jejuni and length are much smaller than untreated fish group, and this illustrates that the synthesis of plant lactobacillus CGMCCNO.11445 fermentation supernatant to the flagellum of C.jejuni truly has restraining effect.
Embodiment 7 plant lactobacillus CGMCCNO.11445 in cell model on campylobacter jejuni flaA virulence factor express impact
The HT-29 cell of growth fusion to 70%-80% is digested, adjustment concentration to 2 × 10
5individual/mL, be placed on by sterile cover slips in 6 porocyte culture plates, every hole adds 2mL suspension culture, and 37 DEG C of cultivations in 5%CO2 incubator, when cell grows to individual layer, clean three times with PBS, every hole adds 0.5mL logarithmic phase C.jejuni (2 × 10
8cFU/mL), 0.5mL PBS cleans 2 resuspended milk-acid bacteria suspensions (2 × 10
8cFU/mL) control group adds 0.5mlMRS liquid nutrient medium, adds RPMI-1640 cell culture fluid (not containing serum and microbiotic) and, to 2mL, hatches 4h.After hatching end, outwell supernatant, extract RNA.Remaining steps is identical with experiment in vitro.Experimental result is shown in accompanying drawing 4
From accompanying drawing 4: plant lactobacillus CGMCCNO.11445 fermentation supernatant can effectively suppress campylobacter jejuni flaA virulence factor to be expressed in cell model.
Embodiment 8 plant lactobacillus CGMCCNO.11445 is on the impact infecting campylobacter jejuni Mouse Weight
Get female BLAB/c mouse in 2 week age (SPE level) 30, be divided into three groups at random, often organize 10: blank group, campylobacter jejuni negative control group, plant lactobacillus and campylobacter jejuni intervention group.Concrete grouping situation is as shown in table 5, by the situation shown in table 5 to mouse stomach, once a day, and each gavage 200ul, continuous gavage 4 weeks.
Table 5: mice group situation
Weigh and calculate the change of Mouse Weight, test-results is listed in table 6.These results show, feeding 1.0 × 10
9the plant lactobacillus CGMCCNO.11445 of CFU/mL significantly can alleviate the reduction of the Mouse Weight that the diarrhoea that causes owing to infecting campylobacter jejuni causes.
Table 6: lactobacillus reuteri CGMCCNO.11447 is on the impact infecting campylobacter jejuni Mouse Weight
Note: * represents blank group and plant lactobacillus intervention group and the remarkable P<0.05 of campylobacter jejuni group gap
Embodiment 9 utilizes plant lactobacillus CGMCCNO.11445 of the present invention to manufacture the fermentation cow's milk containing this bacterium
Raw dairy skimmed milk 50g is dissolved in 300ml water, 95 DEG C of thermal sterilization 20min; 45g white sugar is dissolved in 300ml water, 100 DEG C of 20min; Then be cooled to room temperature, by syrup and the milk mixing of boiling, add plant lactobacillus CGMCCNO.11445 bacterium powder, make its concentration reach 10
6more than CFU/ml, places 4h at 37 DEG C, as 4 DEG C of refrigerations, so obtain the fermentation cow's milk containing plant lactobacillus CGMCCNo.11445 viable bacteria of the present invention.
Embodiment 10 utilizes plant lactobacillus CGMCCNO.11445 of the present invention to manufacture the ferment carrot containing this bacterium
Select fresh Radix Dauci Sativae to clean and dry rear slitting, load in altar, according to the weight ratio 100:0.05 of Radix Dauci Sativae and plant lactobacillus CGMCCNO.11445 starter of the present invention, plant lactobacillus CGMCCNO.11445 starter is added in Radix Dauci Sativae, the salt solution that concentration is 5% (weight percent) is added again by Radix Dauci Sativae and salt solution weight ratio 1:30, and add appropriate food flavouring, push down Radix Dauci Sativae by bamboo cane to avoid floating, sealing, in temperature 30 DEG C of bottom fermentations 10 days, obtain pickling carrot product containing plant lactobacillus CGMCCNO.11445.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (9)
1. a plant lactobacillus (Lactobacillusplantarum), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on September 24th, 2015, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and deposit number is CGMCCNo.11445.
2. plant lactobacillus CGMCCNO.11445 according to claim 1, is characterized in that it has following characteristic:
(1) there is acid resistance, Bile salt resistance;
(2) vitro inhibition campylobacter jejuni growth;
(3) to intestinal epithelial cell, there is good adhesive capacity;
(4) plant lactobacillus CGMCCNo.11445 fermentation supernatant significantly can suppress the expression of campylobacter jejuni flaA virulence factor in vitro;
(5) plant lactobacillus CGMCCNo.11445 fermentation supernatant can affect the synthesis of campylobacter jejuni flagellum in vitro;
(6) plant lactobacillus CGMCCNo.11445 fermentation supernatant significantly can suppress the expression of campylobacter jejuni flaA virulence factor in cell model.
3. prepare the method for the biotechnological formulation suppressing campylobacter jejuni for one kind, it is characterized in that, by acquisition thalline centrifugal after plant lactobacillus CGMCCNO.11445 activation culture, compositely with pharmaceutically acceptable carrier after centrifuge washing obtain pharmaceutical composition or obtained nourishing function product composite with available support in food.
4. method according to claim 3, is characterized in that, described pharmaceutical composition is made up of plant lactobacillus CGMCCNO.11445 microbial inoculum and pharmaceutically acceptable carrier.
5. the method according to claim 3 or 4, is characterized in that, described pharmaceutically acceptable carrier is one or more pharmaceutically normally used weighting agent, tackiness agent, wetting agent, disintegrating agent, lubricant or correctivess.
6. method according to claim 5, is characterized in that, described pharmaceutical composition is granule, capsule, tablet, pill or oral liquid formulation.
7. plant lactobacillus described in claim 1 is suppressing the application in campylobacter jejuni flagellar production.
8. plant lactobacillus CGMCCNO.11445 described in claim 1 is preparing the application in leavened food.
9. the application of plant lactobacillus CGMCCNO.11445 in fermentative production milk-product described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510777864.9A CN105238727B (en) | 2015-11-13 | 2015-11-13 | A kind of antagonism campylobacter jejuni and the lactobacillus plantarum for inhibiting its flaA gene expression |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510777864.9A CN105238727B (en) | 2015-11-13 | 2015-11-13 | A kind of antagonism campylobacter jejuni and the lactobacillus plantarum for inhibiting its flaA gene expression |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105238727A true CN105238727A (en) | 2016-01-13 |
| CN105238727B CN105238727B (en) | 2019-01-11 |
Family
ID=55036547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510777864.9A Active CN105238727B (en) | 2015-11-13 | 2015-11-13 | A kind of antagonism campylobacter jejuni and the lactobacillus plantarum for inhibiting its flaA gene expression |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105238727B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060174A (en) * | 2018-01-10 | 2018-05-22 | 信阳师范学院 | A kind of method for improving Escherichia coli Growth performance |
| CN110669690A (en) * | 2019-10-16 | 2020-01-10 | 衡阳师范学院 | Lactobacillus plantarum strain for expressing quorum sensing signal molecule AI-2 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533618A (en) * | 2012-02-28 | 2012-07-04 | 江南大学 | Lactobacillus plantarum CCFM8724 and application thereof |
| CN102586155A (en) * | 2012-03-07 | 2012-07-18 | 江南大学 | Lactobacillus plantarum N13 and use thereof |
-
2015
- 2015-11-13 CN CN201510777864.9A patent/CN105238727B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533618A (en) * | 2012-02-28 | 2012-07-04 | 江南大学 | Lactobacillus plantarum CCFM8724 and application thereof |
| CN102586155A (en) * | 2012-03-07 | 2012-07-18 | 江南大学 | Lactobacillus plantarum N13 and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| WU DING 等: "Probiotics Down-Regulate flaA σ28 Promoter in Campylobacter jejuni", 《JOURNAL OF FOOD PROTECTION》 * |
| 赵煜: "具有拮抗空肠弯曲杆菌功效的乳酸菌的研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060174A (en) * | 2018-01-10 | 2018-05-22 | 信阳师范学院 | A kind of method for improving Escherichia coli Growth performance |
| CN110669690A (en) * | 2019-10-16 | 2020-01-10 | 衡阳师范学院 | Lactobacillus plantarum strain for expressing quorum sensing signal molecule AI-2 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105238727B (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102533618B (en) | Lactobacillus plantarum CCFM8724 and application thereof | |
| CN102586155B (en) | Lactobacillus plantarum N13 and use thereof | |
| CN100489086C (en) | Lactic acid bacteria capable of stimulating mucosal immunity | |
| CN102174450B (en) | Lactobacillus plantarum for resisting helicobacter pylori infection and application thereof | |
| CN105779350B (en) | A kind of lactobacillus plantarum and application thereof of anti-Bacterium enteritidis infection | |
| CN106834187A (en) | A kind of bifidobacterium bifidum and application thereof | |
| CN107893044A (en) | One plant of bifidobacterium longum and its application | |
| CN110122877A (en) | Lactobacillus rhamnosus and application thereof | |
| CN106318880B (en) | One bacillus amyloliquefaciens, bacteriostatic agent prepared therefrom and purposes | |
| CN107058158A (en) | A kind of dog immunity that improves reduces the compound probiotic agent and its preparation and application of diarrhea disease percentage | |
| JP2007518693A (en) | Stable liquid probiotic composition, its preparation and application | |
| CN109628358A (en) | A kind of compound probiotic and its application | |
| CN103911305B (en) | Lactobacillus crispatus bacterial strain and uses thereof | |
| CN109481476A (en) | Application of the lactobacillus fermenti CQPC04 in the food or drug that preparation improves ulcerative colitis | |
| CN105920049A (en) | Application of lactobacillus rhamnosus grx19 in regulation of intestinal floras | |
| CN114908020A (en) | Lactobacillus plantarum for resisting helicobacter pylori infection and application of lactobacillus plantarum in edible herbal enzyme product | |
| CN101904411B (en) | Composite microbial ecological agent and application thereof | |
| CN105274032B (en) | A kind of antagonism campylobacter jejuni and the lactobacillus reuteri for inhibiting its flaA gene expression | |
| CN108018248B (en) | Lactobacillus casei capable of regulating flora structural disorder caused by antibiotics | |
| CN109652334A (en) | A kind of complex microbial inoculum and its preparation method and application | |
| CN105238727A (en) | Lactobacillus plantarum capable of antagonizing campylobacter jejuni and inhibiting expression of flaA gene of campylobacter jejuni | |
| CN103911307B (en) | Lactobacillus crispatus bacterial strain and uses thereof | |
| WO2022110281A1 (en) | Bifidobacterium bifidum i771, separation and purification method therefor, and application thereof | |
| CN116445360A (en) | Lactobacillus rhamnosus with effect of relieving chronic alcoholic liver injury and application thereof | |
| CN103266076B (en) | Space-induced efficient bifidobacterium bifidum and application thereof as well as preparation method of capsule preparation of space-induced efficient bifidobacterium bifidum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |