CN100506974C - Fecal enterococcus CMS-II001 and its application - Google Patents
Fecal enterococcus CMS-II001 and its application Download PDFInfo
- Publication number
- CN100506974C CN100506974C CNB2006101571886A CN200610157188A CN100506974C CN 100506974 C CN100506974 C CN 100506974C CN B2006101571886 A CNB2006101571886 A CN B2006101571886A CN 200610157188 A CN200610157188 A CN 200610157188A CN 100506974 C CN100506974 C CN 100506974C
- Authority
- CN
- China
- Prior art keywords
- cms
- enterococcus
- group
- stomach
- fecal
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses one kind of Enterococcus faecalis CMS-H001 in the preservation number of CCTCC No. M 206108. The Enterococcus faecalis CMS-H001 of the present invention may be used in treating Helicobacter pylori (HP) infection effectively.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to the new bacterial strain of enterococcus faecalis and the application of this bacterial strain.
Background technology
Helicobacter pylori (Helicobacter pylori, HP) infecting is one of human modal infection, infection rate reaches more than 50% in global population.Nineteen eighty-two Warren and Marshall successfully turn out helicobacter pylori from people's gastric mucosa sample, and since definite HP is the pathogenic bacterium of gastritis and peptide ulceration, changed chronic gastritis, peptide ulceration, the treatment of gastric mucosa dependency lymphoma (MALT) and the prevention general layout of cancer of the stomach, it is diseases related above-mentioned four kinds of diseases to be decided to be HP.Studies show that in a large number, HP infect be chronic active gastritis and peptide ulceration main diseases because of, very close with the relation of cancer of the stomach and gastric mucosa dependency lymphoma (MALT), and confirmed as the one-level procarcinogen by the World Health Organization in 1996.Eradicate the incidence that the HP treatment can reduce gastropathy.At present both at home and abroad the standard scheme that infects of treatment Hp eradicate HP infect be with proton pump inhibitor (PPI) and (or) the bismuth agent adds two kinds antibiotic " three " or " tetrad " therapy (MalfertHeiner P, MegraudF, O ' Morain C et al.Current concepts in the management of Helicobacterpylori infection-tHe Maastricht 2-2000 Consensus Report.AlimentaryPharmacology and Therapeutics 2002; 16:167-180).Pressing down extremely with microbiotic, HP has obtained satisfied curative effect and has shortened treatment time.
Although it is very effective press down to kill HP with microbiotic at present, expense is expensive, and people must to bear the harm that microbiotic brings also quite big.Unite widely-usedly along with antibiotic, the HP Resistant strain increases, and kind, time and amount that microbiotic uses will increase thereupon, and this will cause following harm: 1, a large amount of long-time and unite multiple microbiotic, will increase the toxic side effect of human body; 2, bacterial strain variation and Resistant strain increase, and the bacterial strain of variation will be not limited only to HP; 3, little ecological damage of other chamber portions and skin causes each cavity self-purification function to reduce, and diseases related even incidence tumour of other mucosal atrophies is increased.Microbiotic is united extensive application, can produce a series of problems such as antibiotic-associated diarrhea, alteration of intestinal flora even generation pseudomembranous enteritis, curative effect is descended, patient's compliance reduces, easily recurrence and defective such as infections again after the drug withdrawal, and increasing sharply of Resistant strain more makes eradication rate decline.Therefore it is imperative to seek non-antibiotic treatment HP.
(Nomura A such as Adamasson, Stemmemann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ.Helicobacter pylori infection and gastric carcinomaamong Japanese Americans in Hawaii.N Engl J Med 1991; 325:1132-1136.) reported the flora imbalance that several examples use antibiotic therapy Hp to cause: resistance suis and staphylococcus appear in the oral cavity; Bacterial count increases in the stomach mucous membrane, and the resistance fungal component occurs; The change of the interior micro-ecological environment of enteron aisle is the most obvious, and resistance enterococcus bacteria, enterobacteria, genera bacillus all increase after treatment to some extent.So it is few to seek a kind of toxic side effect, the acceptable methods of treatment of patient becomes the focus in the present medical research.
Summary of the invention
Purpose of the present invention is exactly in order to overcome the above problems, and provides a kind of and can effectively treat helicobacter pylori (Helicobacter pylori, HP) the new enterococcus faecalis bacterial strain of Gan Raning and in the application for preparing aspects such as medicine.
A further object of the present invention provides pyloric spirobacterium inhibitor, pharmaceutical composition, food, healthcare products and the foodstuff additive that contain above-mentioned bacterial strains.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses a kind of enterococcus faecalis (Enterococcus faecalis) CMS-H001, its preserving number is CCTCC No.M 206108.
The invention also discloses pyloric spirobacterium inhibitor, described inhibitor contains the fecal enterococcus CMS-II 001 that preserving number is CCTCCNo.M 206108.
The invention also discloses described fecal enterococcus CMS-II 001 and be used for the treatment of application in the medicine of the disease that helicobacter pylori infection causes in preparation.
Concrete, described disease is a stomach trouble.
Perhaps concrete, described disease is a chronic gastritis.
Perhaps concrete, described disease is a peptide ulceration.
Perhaps concrete, described disease is a gastric mucosa dependency lymphoma.
Perhaps concrete, described disease is a cancer of the stomach.
The present invention further discloses a kind of pharmaceutical composition, described pharmaceutical composition contains the above-mentioned fecal enterococcus CMS-II 001 of pharmacy effective dose.
The invention also discloses a kind of food, described food contains above-mentioned fecal enterococcus CMS-II 001.
Preferably, described food is beverage.
The present invention further discloses a kind of healthcare products, described healthcare products comprise above-mentioned fecal enterococcus CMS-II 001.
The present invention further discloses a kind of foodstuff additive, described foodstuff additive comprise above-mentioned fecal enterococcus CMS-II 001.
Fecal enterococcus CMS-II 001 of the present invention is a kind of isolating brand-new bacterial strain, this bacterial strain in vivo, external all effectively the antagonism helicobacter pylori (Helicobacter pylori, HP); And acid and bile all there is good tolerability; Can alleviate HP and infect the mucous membrane chronic inflammatory diseases damage cause, reduce lymphocyte, plasmacytic dip-dye, the eradication rate of HP is reached 75%-87.5%, the PPI triple therapy comparing difference of routine does not have significance with present stage.It is diseases related with HP that new bacterial strain of the present invention can be used for treating the HP infection, as chronic gastritis, peptide ulceration, gastric mucosa dependency lymphoma and cancer of the stomach.
Preservation information:
Strain name: enterococcus faecalis (Enterococcus faecalis) CMS-H001
Preservation date: 2006 10 years 23 days
Depositary institution: Chinese typical culture collection center (CCTCC)
Deposit number: CCTCC No.M 206108
Description of drawings
Fig. 1 is the aspect graph of fecal enterococcus CMS-II 001 of the present invention under light microscopic.
Fig. 2 is 5000 * times scanning electron microscope image of fecal enterococcus CMS-II 001 of the present invention.
Fig. 3 is 20000 * times scanning electron microscope image of fecal enterococcus CMS-II 001 of the present invention
Fig. 4 is 6000 * times transmission electron microscope picture of fecal enterococcus CMS-II 001 of the present invention.
Fig. 5 is 12000 * times transmission electron microscope picture of fecal enterococcus CMS-II 001 of the present invention.
Fig. 6 is body of stomach, the stomach hole damage integration histogram among the embodiment 2, and A represents body of stomach damage integration histogram, and B represents stomach hole damage integration histogram.
Fig. 7 is the histogram of Giemsa dyeing bacterium numeration integration among the embodiment 2, and A represents body of stomach Hp integration histogram, and B represents stomach hole Hp integration histogram.
Fig. 8 is each treatment group stomach mucous membrane improvement Giemsa colored graph among the embodiment 2, does not see obvious bacillus in blank group (A) gastric pit, and model group (B) is sticked by visible a large amount of bacillus; The then only visible a small amount of bacillus of fecal enterococcus CMS-II 001 treatment group (C) and PPI treatment group (D) is sticked (Giemsa dyeing, * 1000).
Fig. 9 is model group stomach-tissue homogenate coloration result figure among the embodiment 2, A, B are respectively form under the HE dyeing light microscopic that model group stomach-tissue homogenate smear and homogenate tissue bacterial cultivate, all visible Gram ' s dyeing negative bacteria is Campylobacter or arc (Gram ' s dyeing, oily mirror).
Embodiment
Microecology research normal microflora and its host's mutual relationship, but little ecological therapy use the active active bacteria formulation of antagonism pathogenic bacteria and treat infectation of bacteria, act on special, direct, permanent, no obvious toxic-side effects.Probiotics---probiotic bacterium helps healthy number of bacteria and activity by improving some, rebuilds colony balance and the host is played beneficial effect.Common probiotic bacterium mainly comprises Bacterium lacticum, streptococcus acidi lactici, bifidus bacillus etc., and these bacteriums are normal microfloras of healthy human gastrointestinal tract, contains acetate, lactic acid, hydrogen peroxide etc. in its meta-bolites, and pathogenic bacteria is had certain antagonistic action.In addition, these bacteriums can produce the various bacteria element, and the antibacterial or germicidal action of high special is arranged.Therefore probiotic bacterium has reduced chemical sproof generation by number of ways antagonism pathogenic bacteria, for the treatment bacterial infection disease provides new approach.
New bacterial strain enterococcus faecalis of the present invention (Enterococcus faecalis) CMS-H001, it is a kind of probiotic bacterium, effective antagonism helicobacter pylori (Helicobacter pylori, HP), it is diseases related with HP to can be used for treating the HP infection, as chronic gastritis, peptide ulceration, gastric mucosa dependency lymphoma and cancer of the stomach.
New bacterial strain of the present invention: enterococcus faecalis (Enterococcus faecalis) CMS-H001, carried out preservation in 2006 10 years 23 days at the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Wuhan City, preserving number is CCTCC No.M 206108.
Fecal enterococcus CMS-II 001 bacterial strain of the present invention is dark reddish purple look flat colony on the EC substratum, 1～2 millimeter of diameter, and the edge is neat, and smooth surface shows as the Gram-positive coccus under the light microscopic; Be spherical or beading under scanning electron microscope and the transmission electron microscope, no gemma coccus, cell wall structures is complete, no blastogenesis phenomenon, kytoplasm is even.In the bacterial strain metabolic end product, VFA is mainly acetate, and NVFA is mainly lactic acid, and a small amount of amber platinic acid.
In the external antagonism Hp test of fecal enterococcus CMS-II 001 bacterial strain, find, the fecal enterococcus CMS-II 001 supernatant liquor unrestraint Hp effect of autoclaved and filtration sterilization, the fecal enterococcus CMS-II 001 culture supernatant then has tangible antagonism Hp effect, illustrates that the performance of the anti-Hp effect effectiveness of fecal enterococcus CMS-II 001 depends on the effective constituent of viable bacteria state or metabolic end product.
Further in-vitro simulated gastroduodenal environmental resistance is discovered, it is the no significant difference in live bacterial count and the nearly neutral environment that is inoculated into pH6.8 in 2.0 the sour environment that fecal enterococcus CMS-II 001 is inoculated into pH, shows the MRS liquid nutrient medium well-tolerated of fecal enterococcus CMS-II 001 bacterial strain to pH2.0; Simultaneously, the fecal enterococcus CMS-II 001 bacterial strain is inoculated in the MRS there was no significant difference that does not contain cholate at the MRS liquid nutrient medium live bacterial count that contains 0.3% biliary salts.Show that fecal enterococcus CMS-II 001 bacterial strain of the present invention all has good tolerability to acid and bile.
More than studies show that, detest the fecal enterococcus CMS-II 001 bacterial strain that filters out the bacteria strain from people's stomach hole and duodenum are isolating, good to simulation hydrochloric acid in gastric juice and duodenum bile environmental resistance, externally can obviously suppress the Hp clinical strain, and this effect relies on participating in directly of viable bacteria.These studies show that fecal enterococcus CMS-II 001 bacterial strain of the present invention has possessed the essential condition that becomes medicinal fungus, can be used for being developed to new drug, healthcare products and the foodstuff additive etc. of using little ecological therapy control Hp infection.
The present invention further carries out interior therapeutic research with the fecal enterococcus CMS-II 001 bacterial strain to HP inductive gastritis model mouse, and compares with the methods of treatment " PPI triple therapy " of present stage routine.Discover that probiotic bacterium fecal enterococcus CMS-II 001 of the present invention can alleviate HP and infect the mucous membrane chronic inflammatory diseases damage that causes, reduce lymphocyte, plasmacytic dip-dye, with model group significant difference is arranged relatively, compare no significant difference with PPI treatment group, and the effect that suppresses HP arranged, treatment back HP Bacteria Detection obviously reduces, and the eradication rate of HP is reached 75%-87.5%, does not have significance with PPI triple therapy comparing difference.
Existing in the result of study explanation normal people stomach of the present invention has the fungal component of antagonistic action to Hp, and has the bacterial strain specificity.The present invention detests the fecal enterococcus CMS-II 001 that filters out the bacteria strain from healthy people's stomach hole is isolating, and the effect of obvious inhibition Hp clinical strain is arranged in vivo, the triple therapy comparison there was no significant difference that it adds antibiotic to eradication rate and the PPI of HP.This result provides new method to the treatment that Hp infects, for little ecology provides theoretical basis at treatment HP aspect diseases related.
Utilize fecal enterococcus CMS-II 001 of the present invention can be prepared into pharmaceutical composition.This pharmaceutical composition contains the viable bacteria form of the fecal enterococcus CMS-II 001 of pharmacy effective dose.In addition, described pharmaceutical composition can also contain suitable pharmaceutical carrier.Pharmaceutical composition of the present invention can be capsule, solution or forms such as drinkable suspension, pockaged powder, and each single dose generally contains the fecal enterococcus CMS-II 001 bacterial strain and is about 10
6～10
10Cell.It is diseases related that pharmaceutical composition of the present invention can be used for preventing and treating HP, as chronic gastritis, peptide ulceration, gastric mucosa dependency lymphoma and cancer of the stomach etc.
Utilize fecal enterococcus CMS-II 001 of the present invention can also be prepared into the form of food, healthcare products or foodstuff additive, it is diseases related that these food, healthcare products or foodstuff additive can be used for preventing and treating HP, as chronic gastritis, peptide ulceration, gastric mucosa dependency lymphoma and cancer of the stomach etc., improve user's health level.Food of the present invention can be the form that contains the beverage of fecal enterococcus CMS-II 001 viable bacteria of the present invention, also can be forms such as the milk-product that contain this viable bacteria, fermented-milk, soybean milk yoghurt.
The present invention is described in further detail below by specific embodiment.
The isolation identification of fecal enterococcus CMS-II 001 and biological characteristics thereof
1, substratum prescription and configuration
The MRS liquid nutrient medium: get peptone 5g, beef powder 5g, yeast soak powder 2.5g, glucose 10g, K
2HPO
42.5g, ammonium citrate 1g, NaCl 2.5g, MgSO
40.25g, MnSO
40.1g Tween-80 0.5g adding distil water 500ml dissolves back 121 ℃ of autoclavings, it is standby to put 4 ℃ of refrigerators.
The MRS solid medium: peptone 5g, beef powder 5g, yeast soak powder 2.5g, glucose 10g, K
2HPO
42.5g, ammonium citrate 1g, NaCl 2.5g, MgSO
40.25g, MnSO
40.1g Tween-800.5g adding distil water 500ml dissolving adds agar powder 10g, 121 ℃ of autoclavings, and the cast sterile petri dish, it is standby to put 4 ℃ of refrigerators.
2, gastroscope is drawn materials and sample passes on
The gastroscope suction duodenum section of the falling liquid 0.5ml of health adult is down gone into the 2mlMRS liquid nutrient medium, gets three samples altogether by last method, and the anaerobism box transferred to the laboratory in 30 minutes.
3, the separation and Culture of fecal enterococcus CMS-II 001 kind daughter bacteria
Fecal enterococcus CMS-II 001 kind daughter bacteria separates from the health adult's duodenum section of falling liquid, and sample takes out behind 37 ℃ of constant temperature culture 4h in the anaerobism box, by 10
-1Serial dilution to 10
-7Stoste and diluent at different levels are respectively got 100ul and are added drop-wise to the MRS flat board, the coating of L type glass rod evenly, 37 ℃ of anaerobism are taken out the observation colonial morphology after cultivating 48h, choose the capable gram stain microscopy of mono-clonal, different shape and chromatic bacterium colony mono-clonal are chosen in the MRS liquid nutrient medium, after cultivating 24h, 37 ℃ of anaerobic environments take out dyeing microscopic examination, form under the sight glass, and streak inoculation is in the MRS solid medium, 37 ℃ of anaerobism are cultivated 48h and are observed colonial morphologies once more, choose the mono-clonal dyeing microscopic examination, the quadruplication purifying that goes down to posterity, chief's colonial morphology unanimity on solid plate, the gram stain microscopy form is consistent determine to divide pure.It is standby in-70 ℃ of refrigerator cryopreservation to divide each pure strain bacterium can add behind the frostproofer.
3, colony characteristics:
Fecal enterococcus CMS-II 001 is dark reddish purple look flat colony on the EC substratum, 1～2 millimeter of diameter, and the edge is neat, smooth surface.Light microscopic is Gram-positive coccus, sphere or beading, no gemma down.See Fig. 1.
4, scanning electron microscopic observation
Get 1g bacterium mud physiological saline and wash 3 times, get the centrifugal 5min of 600ul bacteria suspension 2500rpm/min, add the 600ul2.5% glutaraldehyde, break up precipitation, fixedly 24h; 0.1M PBS cleans, 3 times * 5min; 1% OSO
4Fixing 1h; 50%, 70%, 90%, 100% acetone gradient dehydration, every grade of 10min * 2 time; 50%, 70%, 90%, 100% isoamyl acetate, every grade of displacement 5min * 2 time; Critical point drying instrument drying; Ion sputtering instrument, spray platinum plated film; JSM5600-LV type scanning electron microscopic observation, photograph.The result is shown in Fig. 2,3, and fecal enterococcus CMS-II 001 strain bacterium surface is smooth, complete, and the form homogeneous is spherical or beading, and no gemma, Fig. 2, Fig. 3 represent 5000 respectively * and 20000 * doubly.
5, transmission electron microscope observing:
Getting 1g bacterium mud physiological saline washes 3 times, get the centrifugal 5min of 600ul bacteria suspension 2500rpm/min, add fixedly 24h of 2.5% glutaraldehyde along tube wall, 2% osmic acid is 2h fixedly, 50%, 70%, 90%, 100% acetone gradient dehydration, Resins, epoxy mixed solution: pure acetone=1:1,37 ℃, soak 24h, Epon812 (Epoxiaquivalentgewicht145-160), DDSA (DodecenylsuccinicAnhydride), MNA (Methyl Nadic Anhydride), DMP30 (Tris-(dimethylaminomethgl)-phenc), 60 ℃, embedding becomes piece; Repair piece, location; Semithin section; The section of Sweden LKB-III type ultramicrotome, thick about 500A; Acetic acid uranium, lead nitrate double staining, the H-600 of Hitachi type transmission electron microscope observing, photograph.The result is shown in 4,5, and pictorial display ne ar under transmission electron microscope is regular, and cell wall structures is complete, no swelling and blastogenesis phenomenon, kytoplasm is even, no abnormal particle, not seeing exogenous factors such as virus, mycoplasma in all sections, Fig. 4, Fig. 5 represent 6000 respectively * and 12000 * doubly.
6, the fecal enterococcus CMS-II 001 metabolic end product is measured
(1) instrument: day island proper Tianjin GC2010 type gas chromatographic detection instrument (GC), hydrogen flame ionization sensor (FID), DB-5MS chromatographic column; Day island proper Tianjin UV-2501PC;
(2) preparation of reference liquid:
The preparation of pure and mild voltaile fatty acid (VFA) reference liquid: formic acid 37 μ l, acetate 57 μ l, propionic acid 74 μ l, isopropylformic acid 93 μ l, butyric acid 92 μ l, isovaleric acid 109 μ l, valeric acid 108 μ l, caproic acid 125 μ l, ethanol 100 μ l, propyl alcohol 5 μ l, isopropylcarbinol 5 μ l, butanols 10 μ l, primary isoamyl alcohol 0.5 μ l, amylalcohol 0.5 μ l adding distil water are settled to 100ml, are the reference liquid of 10mmol/L.
The preparation of non-volatile fatty acid (NVFA) reference liquid: lactic acid 84 μ l, toluylic acid 60mg, succsinic acid 60mg adding distil water to 100ml, are the reference liquid of 10mmol/L.
(3) VFA preparation: select the metaphosphoric acid method for use
Get reference liquid or culture 1ml adds metaphosphoric acid 0.5ml, adjust the pH value below 2.0,37 ℃ of homogenize 2h, 10000 rev/mins, 4 ℃ of centrifugal 2min get supernatant 1 μ l and are used for the sample introduction analysis.
(4) NVFA preparation: select the methyl alcohol sulfuric acid process for use
Get reference liquid or culture 2ml and add 50% sulfuric acid 0.2ml, adjust pH value below 2.0, get sulfuric acid culture 1ml, add methanol solution 1ml and 50% sulfuric acid 0.1ml, sample was boiled 5 minutes or room temperature under spend the night, add chloroform 0.5ml, leave standstill a moment behind the mixing, 1000 rev/mins, 4 ℃ of centrifugal 2min get chloroform layer 1 μ l and are used for the sample introduction analysis.
(5) analysis of each material in sample and the standard substance:
Get sample and standard substance 1 μ l sample introduction analysis after the extraction respectively, detect the retention time of each material and with the quantitative criterion of standard substance as this material in the sample by day island proper Tianjin GC2010 type gas chromatographic detection instrument (GC).
The result shows: fecal enterococcus CMS-II 001 bacterial strain VFA is mainly acetate, and the acetate appearance time is 7.970min, and NVFA is mainly lactic acid, and a small amount of amber platinic acid is arranged, and the lactic acid appearance time is 7.300min, and amber platinic acid appearance time is 9.775min.
7, the biochemical identification of fecal enterococcus CMS-II 001
API-20 STREP (dust in the French enzyme) is a standardized method in conjunction with 20 biochemical tests of each side.This system can firm medical science bacterium in most streptococcic kinds or group.
API-20 STREP test bar is made up of 20 tubules that contain the dry substrate that can carry out enzymatic determination and sugar-fermenting, can measure enzyme and live or sugar-fermenting.Enzyme activity determination is in the exsiccant enzyme substrates with dense, pure bacterial suspension inoculation.In the training period, the final meta-bolites that is produced is by the nature variable color or add reagent and variable color shows.Fermentation test is the substratum of being made up of sugared substrate to be inoculated in.The carbohydrate of fermentation is to show with the PH indicator.
Get the capable API-20 STREP of microscopy Gram ' s stained positive coccus bacterial strain biochemical identification, the specification sheets that authentication method is equipped with referring to manufacturer in the API-20 STREP identification kit.The results are shown in Table 1.
Table 1 fecal enterococcus CMS-II 001 strains A PI-20 STREP biochemical reaction result
Fecal enterococcus CMS-II 001 is to the body outer suppressioning test of helicobacter pylori (Hp)
(1) fecal enterococcus CMS-II 001 bacterial strain supernatant liquor preparation: bacterial strain is inoculated in the MRS liquid nutrient medium by 1% volume ratio, takes out after 37 ℃ of anaerobism are cultivated 24h, and 4 ℃ of centrifugal 10min of 5000rpm/min leave and take supernatant, discard precipitation.
Fecal enterococcus CMS-II 001 bacterial strain sterilization supernatant liquor preparation: obtain 121 ℃ of autoclaving 20min after the supernatant liquor by last method.
Fecal enterococcus CMS-II 001 bacterial strain supernatant liquor filtered solution preparation: supernatant liquor is not contained the supernatant liquor of bacterium with the filter filtration sterilization of 0.22 micron pore size.
(2) Hp bacterium liquid preparation: the aseptic inoculation ring is collected Hp on the brain-heart-infusion blood agar, is inoculated in the brain-heart-infusion that 0.2ml contains 50% foetal calf serum, and turbidimetry judges that bacterial concentration is 10
11CFU/ml.
(3) bacteriostatic test: inhale 100 μ l helicobacter pylori bacteria suspensions and be added drop-wise to the brain-heart-infusion blood agar that diameter is 9cm, aseptic L shaped glass rod coating evenly.Treat that bacterium liquid permeates blood agar fully, on flat board, punch aperture 7mm with aseptic 200 μ lTip heads, hole depth 5mm, 4 holes of every plate with behind the 0.8% agar solution back cover, add fecal enterococcus CMS-II 001 bacterial strain supernatant liquor, sterilization supernatant liquor, filterable supernatant liquor respectively at the bottom of the hole in the hole, add MRS liquid nutrient medium hole in contrast, liquid level must not overflow every hole liquid feeding 120 μ l as far as possible near the blood agar surface, in triplicate, place to detest and support 37 ℃ of constant temperature culture 72h of the little aerobic environment of case.
Take out punching plane table surveying antibacterial circle diameter behind the 72h, the corresponding aperture inwall of the supernatant liquor of autoclaving supernatant liquor, filtration sterilization and MRS liquid nutrient medium and Kong Zhouwu bacterial growth, antibacterial circle diameter are 0; Each hole inwall of fecal enterococcus CMS-II 001 strain culturing supernatant liquor all has the respective fine bacteria growing, and the antibacterial circle diameter of three flat boards is respectively 18mm, 18mm, 11mm, average out to 15.7mm.
External acidproof, the anti-bile test of fecal enterococcus CMS-II 001
(1) acid resistance test: from-70 ℃ of refrigerators, take out frozen fecal enterococcus CMS-II 001 bacterial strain, be inoculated in the MRS liquid nutrient medium, 37 ℃ detest support cultivate 24h after, centrifugal (4300 * g, 10min, 4 ℃), supernatant discarded is collected bacterial sediment, with stroke-physiological saline solution washing 3 times.Be inoculated in the MRS liquid nutrient medium that the pH value is 2.0 (using the salt acidometric titration) with 1% volume ratio then.Be inoculated into the pH value simultaneously and be 6.8 MRS liquid nutrient medium in contrast.
0min and 120min get 100 μ l bacterium liquid by 10 after inoculation respectively
-1Serial dilution, each dilutes gradient and gets the MRS flat board that 100 μ l are inoculated into pH6.8, row live bacterial count behind the anaerobism cultivation 48h.Triplicate.
The result is as shown in table 2, the fecal enterococcus CMS-II 001 inoculation is in 2.0 the sour environment during 120min to pH, live bacterial count be inoculated into no significant difference in the nearly neutral environment of pH6.8 (P〉0.05), all the trend of increasing is arranged slightly than the bacterial count before the inoculation, but there was no significant difference (P〉0.05).Show the MRS liquid nutrient medium well-tolerated of fecal enterococcus CMS-II 001 bacterial strain to pH2.0.
Before the table 2. fecal enterococcus CMS-II 001 inoculation and be inoculated in the live bacterial count logarithmic value that pH is 120min behind 6.8 and 2.0 the MRS liquid nutrient medium
* P＜0.05vs. control group
(2) anti-bile test: from-70 ℃ of refrigerators, take out the fecal enterococcus CMS-II 001 inoculation in the MRS liquid nutrient medium, 37 ℃ detest support cultivate 24h after, centrifugal (4300 * g, 10min, 4 ℃), supernatant discarded, collect bacterial sediment, with stroke-physiological saline solution washing 3 times.Be inoculated in the MRS liquid nutrient medium that contains 0.3% biliary salts with 1% volume ratio then.Be inoculated into simultaneously do not contain biliary salts the MRS liquid nutrient medium in contrast.
0min and 120min get 100 μ l bacterium liquid by 10 after inoculation respectively
-1Serial dilution, each dilutes gradient and gets 100 μ l and be inoculated into the MRS flat board that pH6.8 does not contain biliary salts, detests to support and cultivates row live bacterial count behind the 48h.Triplicate.
The result is as shown in table 3, the fecal enterococcus CMS-II 001 bacterial strain when containing the MRS liquid nutrient medium 120min of 0.3% biliary salts, live bacterial count be inoculated in the MRS liquid nutrient medium that does not contain biliary salts and compare there was no significant difference (P〉0.05).
Before the table 3. fecal enterococcus CMS-II 001 inoculation and be inoculated in 120min live bacterial count logarithmic value behind the MRS that contains biliary salts and do not contain cholate
● P＜0.05vs. control group.
Study on the efficiency in the body of fecal enterococcus CMS-II 001 treatment HP infectious gastritis
Destroy micro-ecological environment in the cleaning level BalB/C mouse stomach with microbiotic, make under the situation that bacteria content obviously reduces in the stomach, infect HP again and successfully formed HP infectious gastritis model.The acidproof anti-bile experimental study of environment in the simulation stomach proves that fecal enterococcus CMS-II 001 has certain resistance to acid and bile.Existing its of this bacterial strain stomach hair transplant decided at the higher level but not officially announced can be stablized field planting in stomach.The proof fecal enterococcus CMS-II 001 has the condition of interior therapeutic HP.Below fecal enterococcus CMS-II 001 interior therapeutic HP inductive gastritis model mouse is carried out study on the efficiency.
One, material and method
1, the HP clinical strain separates and the required material of purifying
1.1 the preparation of common agents and solution
Anaerobism propellant: composition quality (g)
Sodium bicarbonate 0.8
POTASSIUM BOROHYDRIDE 1.1
Citric acid 1.08
Gram stain
Viola crystallina dye liquor: Viola crystallina 1g
95% ethanol 20ml
1% ammonium oxalate aqueous solution 80ml
Viola crystallina is dissolved in the ethanol, mixes with ammonium oxalate solution then.
Lu Ge Shi iodine dye liquor: iodine 1g
Potassiumiodide 2g
Distilled water 300ml
95% alcohol: 95ml ethanol adds 5ml distilled water
Rare carbolfuchsin dye liquor: take by weighing basic fuchsin 10g, porphyrize adds 95% ethanol 100ml, and placement is spent the night, filter paper filtering.Get this liquid 10ml, add 5% aqua carbolisata solution 90ml and mix, be carbolfuchsin liquid.Get this liquid 10ml again, add water 90ml, be rare carbolfuchsin liquid.
Deicing fluid: DMSO mixes by the volume ratio of 1:2 with glycerine.
2, HP expert evidence
2.1, RUT test kit (Fujian Sanqiang Biochemical Co., Ltd.);
2.2, the histopathologic examination of gastric mucosa: carry out paraffin section, HE dyeing, double blinding by experienced doctor and read sheet.
2.3, Giemsa dyeing and double blinding read the sheet scoring.
2.4, the HP microbial culture: it is standby to prepare substratum by this laboratory with brain-heart-infusion.
Capable Gram ' the s of the tissue juice of 3, milling stain smear
4, experimentation on animals material
Set up cleaning level animal rearing room, laboratory animal and cage tool, feed are responsible for raising and the management of animal by the special messenger available from Agricultural University Of Hunan experimentation on animals center;
Laboratory animal: modeling successfully is 24 of the regular grade BALB/c mouse of chronic gastritis, adds 8 of blank groups, totally 32, and male and female half and half, body weight ± 2g.
5, the preparation of bacteria suspension, soup:
The aseptic inoculation ring is collected the fecal enterococcus CMS-II 001 bacterial strain on the MRS culture plate, is made into suspension with stroke-physiological saline solution, and it is 1 * 10 that turbidimetry is adjusted the bacteria suspension final concentration
9CFU/ml.
Medicine:
Proton pump inhibitor: dissolve upright Soviet Union injection (40mg/ props up) (Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou, lot number 040805);
Microbiotic: clarithromycin tablet (250mg/ sheet) (Jiangxi Huiren Pharmaceutical Co., Ltd, lot number 0510019), penbritin injection (0.5g/ props up) (Huabei Pharmaceutic Co., Ltd, lot number 0404307), gentamicin (Tianjin Pharmaceutical Jiaozuo Co., Ltd., lot number 05052802), all available from Central South University Hunan Pharmacy of the refined Second Academy.
The dosage of the medicine of mouse converts by the dosage between people and the various animal: known A kind animal per kg body weight dosage, when desire is calculated B kind animal per kg body weight dosage, can table look-up (referring to Sun Jingfang work " animal experiment method ", Beijing, People's Health Publisher calendar year 2001,116 to 125 pages), find out conversion factor (W) and be calculated as follows again: the dosage (mg/kg) of dosage (mg/kg)=W of B kind animal * A kind animal.The dosage that is calculated various medicines by above-mentioned formula is as follows:
Dissolve upright Soviet Union's injection (40mg) and be diluted to the solution that concentration is 0.4mg/ml with the 100ml stroke-physiological saline solution, every mouse is irritated stomach to 0.25ml every day at every turn.
Penbritin (0.5g) is diluted to the solution that concentration is 20mg/ml with the 25ml stroke-physiological saline solution, and every mouse is irritated stomach to 0.25ml every day at every turn.
Clarithromycin (0.25g) is diluted to the solution that concentration is 50mg/ml with the 5ml stroke-physiological saline solution, and every mouse is irritated stomach to 0.1ml every day at every turn.
6, laboratory animal grouping and methods of treatment:
Through the microbiotic pre-treatment, give PPI again and add HP and irritate stomach, 24 Balb/c mouse of modeling success are divided into 3 groups at random, 8 every group.The 1st group is PPI added with antibiotic treatment group, and the 2nd group is the fecal enterococcus CMS-II 001 treatment group; The 3rd group is model control group; Increase by 8 normal Balb/c mouse in addition as blank group (the 4th group).
The 1st group (PPI+ microbiotic triple therapy treatment group): from 3-10 days, the fasting 12h of every day elder generation gave PPI solution 0.25ml and penbritin 0.25ml and adds clarithromycin 0.1ml and irritate stomach, irritated behind the stomach fasting 3-4 individual hour again, once a day, and logotype 7 days.The 4th week was all put to death mouse behind last filling stomach.
The 2nd group (fecal enterococcus CMS-II 001 treatment group): from 1-10 days, the fasting 12h of elder generation gave the fresh bacteria suspension 0.5ml/ of CMS-H001 and only irritated stomach every day, and bacterial count is 10
9CFU irritated behind the stomach fasting 3-4 hour again, once a day, and logotype 10 days.4 weeks of the end are all put to death mouse behind last filling stomach.
The 3rd group (model control group):, give physiological saline 0.5ml/ every day and only irritate stomach, once a day from the 1st～10 day.Logotype 10 days, the 4th week was put to death whole mouse behind last filling stomach.
The 4th group (blank group):, give physiological saline 0.5ml/ every day and only irritate stomach, once a day from the 1st～10 day.Logotype 10 days, the 4th week was put to death whole mouse behind last filling stomach.
The processing of mouse: with dissecting immediately behind the disconnected neck method execution mouse, take out gastral cavity, remove glandular stomach, cut off, get complete stomach-tissue (comprising duodenum, stomach hole, body of stomach etc.) along greatly curved side; Half stomach mucous membrane dyes the capable rapid urease test of second half tissue (5min observing time), smear and microbial culture with row HE dyeing of the fixing back of 10% formalin solution and Giemsa.
7. Bacteria Detection:
7.1 HP detects
1) rapid urease test: get stomach mucous membrane after modeling group mouse is put to death and place liquid area enzyme reagent, observe 5min under the room temperature, indicator reddens positive.
2) Hp cultivates: stomach and duodenum tissue sample grind to form homogenate with the brain-heart-infusion 500ul that contains 10% foetal calf serum, with the front and back difference assay calculate specimen quality, 10
-1Series is dilution suitably, and each gradient is got 100ul and is coated with the brain-heart-infusion blood agar, and colonial morphology is observed in 37 ℃ of little aerobic cultivations 3～7 days, gets capable gram stain microscopy of suspicious bacterium colony and rapid urease test.
3) Giemsa dyeing: step is as follows:
1. paraffin section is by the same direction staining rack of packing into, 60 ℃ of roasting sheets 10 minutes, more than or with hair dryer the paraffin in the section is dissolved.
2. go into to dewax 2～5 minutes in the dimethylbenzene, repeat 1 time, the dewaxing time in summer is short, and winter is then opposite.
3. 100%, 100%, 95%, 90%, 80%, 70% ethanol each 1 minute is successively gone into tap water, and flowing water is placed about 3 minutes, and is clear to cutting into slices.
4. directly into 30 minutes (dye liquor is reusable) of dyeing in the 2%Giemsa dye liquor.
5. tap water flush away dye liquor
6. directly into dewatering in 100% ethanol.
7. conventional dehydration, transparent, mounting.
4) Gram ' s staining procedure: 1. violet staining: 1min, flowing water flushing; 2. Lu Ge Shi iodine mordant dyeing: dyeing 1min, flowing water flushing; 3. alcohol decolouring: the decolouring of 95% alcohol, 15～30 seconds, till not having purple and taking off, the flowing water flushing; 4. rare carbolfuchsin is redyed, 1min, flowing water flushing.
In the centralized detecting method, positive bacterial culture promptly is diagnosed as the HP positive, and rapid urease test, improvement Giemsa dyeing and two positive diagnosis of Gram ' s dyeing are the Hp positive.
8. pathology
After the execution mouse is dissected immediately, getting half stomach mucous membrane dyes with the fixing back of 10% formalin solution row HE: mouse stomach-tissue 10% formalin fixed, paraffin embedding, section, HE dyeing (haematoxylin dyeing 7 minutes---dye liquor that the tap water flushing is unnecessary---1% hydrochloride alcohol differentiation 5 seconds, dyeing in---washing of tap water composition---1% Yihong to make nucleus be hyacinthine, about 3 minutes).Sheet is read in Pathology Deparment's doctor's double blinding.
9. judging criterion
9.1 the judging criterion of Hp field planting (International Agency for Research on CancerWorking Group on the Evaluation of Carcinogenic Risks to Humans.Schistosomes, Liver Flukes, and Helicobacter pylori.Lyon:InternationalAgency for Research on Cancer, 1994:177-240.) (MalfertHeiner P, Megraud F, O ' Morain C et al.Current concepts in the management of Helicobacter pyloriinfection-tHe Maastricht 2-2000 Consensus Report.Alimentary Pharmacologyand Therapeutics 2002; 16:167-180.)
High power lens is observed 10 visuals field of Giemsa stained down, judges its field planting situation with how much scoring of Hp quantity.Standard such as table 4:
The standards of grading of table 4.Hp field planting amount
9.2 the standards of grading of stomach mucous membrane chronic inflammatory diseases (MalfertHeiner P, Megraud F, O ' Morain C et al.Current concepts in the management of Helicobacter pyloriinfection-tHe Maastricht 2-2000 Consensus Report.Alimentary Pharmacologyand Therapeutics 2002; 16:167-180.)
Observe proper mucous membrane chronic inflammatory cells (lymphocyte, plasmocyte and eosinophilic granulocyte) and neutrophil leucocyte, respectively lamina propria chronic inflammatory diseases and reactivity inflammation degree are scored standard such as following table 5.The same proper mucous membrane of submucosal inflammation standards of grading.The inflammation scoring of whole mucous membrane is the mean value of mucous membrane lamina propria and Submucosa.
Table 5 pathology of gastric mucosa Histological injury standards of grading
9.3 curative effect is judged
(1) Hp eradicates standard: treatment stops to do in back one month bacterium and cultivates feminine gender or rapid urease test and histological stain and check that 2 all negatively are the Hp elimination.
(2) Hp infects the improvement standard: unit mass organizes the Hp counting to reduce or the histological stain amount of bacteria reduces.
(3) pathology criterion of cure: see substantially transfer to normal and the histopathology result normal.
(4) pathology improvement standard: see congestion and edema substantially and before alleviate, the due to intestine erosion and ulcer kitchen range dwindles (the vernier card is measured the focus diameter) before the treatment, or pathology are abnormal fully, but scoring reduces before the treatment.
Validity judging criterion in the body:
Criterion of cure: (1) adds (3), and promptly Hp eradicates and adds that the pathology result is normal.
The improvement standard: (1) adds (4), or (2) add (3), or (2) add (4).
Efficient=curative ratio+improvement rate
10, statistical method
Utilization SPSS11.0 statistical software carries out data processing, the result with mean ± standard deviation (x ± s) expression, mean relatively adopts t check and variance analysis between two groups, P＜0.05 is considered as significant difference.
Two, result
1, generalized case
In experimentation, observe and respectively to organize diet situation, activity and the hair of mouse, the situation of body weight, weigh weekly, find respectively to organize that diet and activity do not have considerable change, the variation there was no significant difference of body weight (P＜0.05) (seeing Table 6) between the mouse.
Table 6: mouse body weight change table (x ± s, n=8)
2. gross examination of skeletal muscle
2.1 observe from gastric mucosa face along the greater gastric curvature longitudinal incision, can see that model group Hp infects congestion and edema and the erosion of part stomach hole mucous membrane that causes, most of gastric mucosa is organized in treatment does not have obvious congestion and edema, and treatment is seen visible gastric mucosa inflammation and the apparent in view improvement of model group after finishing substantially.
2.2 histopathology damage integration
Each group damage integration is referring to the histogram of table 7 and Fig. 6, and the A of Fig. 6 and B represent the damage integration of stomach hole, body of stomach respectively.
The a little higher than body of stomach mucous membrane of stomach hole mucous membrane inflammation damnification integration generally, stomach hole mucous membrane inflammation damnification integration relatively has significant difference between 4 groups, the integration of the stomach hole mucous membrane inflammation damnification of wherein the 1st group (PPI+ antibiotic treatment group), the 2nd group (fecal enterococcus CMS-II 001 treatment group), the 4th group (blank group) all is lower than the 3rd group (model control group), difference has significance (P＜0.05), the 1st group, the 2nd group, the 4th group in twos comparing difference do not have significance (P〉0.05).
Body of stomach inflammation damnification integration relatively has significant difference for 4 groups, the integration of wherein the 1st group, the 2nd group, the 4th group body of stomach mucous membrane inflammation damnification all is lower than the 3rd group, difference has significance (P＜0.05), the 1st group, the 2nd group, the 4th group in twos comparing difference do not have significance (P〉0.05).
Table 7:Balb/c mouse handle each treatment group gastric mucosa chronic inflammatory diseases damage integration (x ± s, n=8)
Annotate: with the 3rd group of comparison,
*P＜0.05
3, calculate the eradication rate of HP
3.1 the field planting integration of Hp
Giemsa dyeing can clearly illustrate that Hp is S-shaped, is colonizated in gastric pit top and the gastric gland chamber more.Each organizes the histogram of the field planting integration of Hp referring to table 8 and Fig. 7, and the A of Fig. 7 and B represent the Hp field planting integration of stomach hole, body of stomach respectively.
Stomach hole mucous membrane Hp field planting integration relatively has significant difference between 4 groups, and wherein the 1st group, the 2nd group has significance (P＜0.05) with the 3rd group of comparing difference, the 1st group, the 2nd group in twos comparing difference do not have significance (P〉0.05).
Body of stomach mucous membrane Hp field planting integration relatively has significant difference between 4 groups, and wherein the 1st group, the 2nd group has significance (P＜0.05) with the 3rd group of comparing difference, the 1st group, the 2nd group in twos comparing difference do not have significance (P〉0.05).
Table 8: each organize Balb/c mouse Hp field planting integral result (x ± s, n=8)
Annotate: each organizes Hp field planting integration and the 3rd group of comparison,
*P＜0.05
Fig. 8 does not see obvious bacillus for each group stomach mucous membrane improvement Giemsa colored graph in the visible blank group gastric pit, and the visible a large amount of bacillus of model group are sticked, and then only visible a small amount of bacillus is sticked for PPI treatment group and fecal enterococcus CMS-II 001 treatment group.
3.2 the rapid urease test of HP and cultivation results:
The results are shown in Table 9.1st, 2 groups of stomach holes and body of stomach rapid urease test and microbial culture and the 3rd group more all have significant difference (P＜0.05).
Table 9: rapid urease test and bacteria cultivation results
3.3 the eradication rate of each treatment group different sites HP is relatively:
The results are shown in Table 10.
Stomach hole portion: the 1st group and the 2nd group have significance (P＜0.05) with the 3rd group, the 4th group comparing difference, and comparing difference does not have significance (P〉0.05) between the 1st, 2 group.
Body of stomach: the 1st group, the 2nd group with the 3rd group, the 4th group comparing difference significance (P＜0.05) is arranged.
The eradication rate of table 10 different treatment group, different sites HP relatively
3.4 the HP that cultivates makees Gram ' s stain smear and observes ne ar
Form under the HE dyeing light microscopic that model group stomach-tissue homogenate smear and homogenate tissue bacterial are cultivated, all visible Gram ' s dyeing negative bacteria is Campylobacter or arc, shown in Fig. 9 A, B.
Above result shows, fecal enterococcus CMS-II 001 of the present invention can alleviate HP and infect the mucous membrane chronic inflammatory diseases damage that causes, reduce lymphocyte, plasmacytic dip-dye, significant difference is relatively arranged, compare no significant difference with PPI treatment group (conventional treatments) with model group; And the effect that suppresses HP is arranged, treatment back HP Bacteria Detection obviously reduces, and the eradication rate of HP is reached 75%-87.5%, does not have significance with PPI triple therapy comparing difference.
Claims (8)
1, a kind of enterococcus faecalis (Enterococcu faecalis) CMS-H001, its preserving number is CCTCC No.M206108.
2, helicobacter pylori (Helicobacter pylori, HP) inhibitor is characterized in that: described inhibitor contains the enterococcus faecalis that preserving number is CCTCC No.M 206108 (Enterococcufaecalis) CMS-H001.
3, the described fecal enterococcus CMS-II 001 of claim 1 is used for the treatment of helicobacter pylori in preparation (Helicobacter pylori HP) infects application in the medicine of the disease cause, and described disease is helicobacter pylori infection gastritis or peptide ulceration.
4, a kind of pharmaceutical composition is characterized in that: described pharmaceutical composition contains the fecal enterococcus CMS-II 001 that preserving number is the pharmacy effective dose of CCTCC No.M206108.
5, a kind of food is characterized in that: described food contains the fecal enterococcus CMS-II 001 that preserving number is CCTCC No.M206108.
6, food according to claim 5 is characterized in that: described food is beverage.
7, a kind of healthcare products is characterized in that: described healthcare products contain the fecal enterococcus CMS-II 001 that preserving number is CCTCC No.M206108.
8, a kind of foodstuff additive is characterized in that: described foodstuff additive contain the fecal enterococcus CMS-II 001 that preserving number is CCTCC No.M206108.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006101571886A CN100506974C (en) | 2006-11-29 | 2006-11-29 | Fecal enterococcus CMS-II001 and its application |
| HK08104478.5A HK1110098A1 (en) | 2006-11-29 | 2008-04-23 | Enterococcus faecalis, cms-h001 and its applying |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006101571886A CN100506974C (en) | 2006-11-29 | 2006-11-29 | Fecal enterococcus CMS-II001 and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101067120A CN101067120A (en) | 2007-11-07 |
| CN100506974C true CN100506974C (en) | 2009-07-01 |
Family
ID=38879819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006101571886A Expired - Fee Related CN100506974C (en) | 2006-11-29 | 2006-11-29 | Fecal enterococcus CMS-II001 and its application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN100506974C (en) |
| HK (1) | HK1110098A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031235B (en) * | 2010-11-09 | 2012-07-25 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
| CN102389568B (en) * | 2011-04-25 | 2014-02-19 | 通威股份有限公司 | Vaccine for preventing golden pompano ulcer |
| CN112618576B (en) * | 2021-01-19 | 2021-12-21 | 慕恩（广州）生物科技有限公司 | Enterococcus lactate, medicine for preventing or treating tumors and application of medicine |
| CN117511773B (en) * | 2023-10-13 | 2024-07-09 | 健合香港有限公司 | Enterococcus faecalis, composite microbial inoculum and application thereof |
-
2006
- 2006-11-29 CN CNB2006101571886A patent/CN100506974C/en not_active Expired - Fee Related
-
2008
- 2008-04-23 HK HK08104478.5A patent/HK1110098A1/en not_active IP Right Cessation
Non-Patent Citations (2)
| Title |
|---|
| Antagonistic activity against Helicobacter pylori infection invitro by a strain of Enterococcus faecium TM39. Cheng-Chih Tsai et al.International Journal of Food Microbiology,Vol.96 No.1. 2004 |
| Antagonistic activity against Helicobacter pylori infection invitro by a strain of Enterococcus faecium TM39. Cheng-Chih Tsai et al.International Journal of Food Microbiology,Vol.96 No.1. 2004 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167785A (en) * | 2016-08-29 | 2016-11-30 | 内蒙古工业大学 | A kind of E. Faecium strains and application thereof |
| CN106167785B (en) * | 2016-08-29 | 2019-05-28 | 内蒙古工业大学 | A kind of E. Faecium strains and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101067120A (en) | 2007-11-07 |
| HK1110098A1 (en) | 2008-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ogbolu et al. | In vitro antimicrobial properties of coconut oil on Candida species in Ibadan, Nigeria | |
| CN102210717B (en) | Compositions comprising Lactobacillus plantarum strains in combination with tannin and new Lactobacillus plantarum strains | |
| KR101796423B1 (en) | Composition and ladies genital area cleanser composition comprising composition for inducing Antiviral and antibacterial cause vaginitis and method for manufacturing the same | |
| CN100506974C (en) | Fecal enterococcus CMS-II001 and its application | |
| Temerk et al. | Antibacterial effect of phytochemial extracts from Ziziphus-spina christi against some pathogenic bacteria | |
| Ahumuza et al. | Qualitative (phytochemical) analysis and antifungal activity of Pentas decora (De wild), a plant used traditionally to treat skin fungal infections in Western Uganda | |
| CN108486002B (en) | Momordica grosvenori endophyte strain capable of producing exopolysaccharides, method for producing exopolysaccharides and application of exopolysaccharides | |
| CN101134051A (en) | Application of clostridium butyricum, condensate bacillus and bifidobacteria in the preparation of medicament for treating irritable bowel syndrome, cold diarrhoea and drink diarrhoea | |
| CN108853358A (en) | A kind of antibacterial Chinese medicine composition and its preparation method and application | |
| CN101322746B (en) | Secondary fermentation method natural medicinal preparation, preparation and use | |
| KR100782500B1 (en) | Antifungal agent comprising natural plant extracts | |
| CN111419829A (en) | Application of honokiol in inhibiting streptococcus suis or biofilm thereof | |
| CN103272134B (en) | Medicine for killing acid-fast staining positive (red) bacillus | |
| Idu et al. | Microbial load of some polyherbal products from Lagos State, Nigeria | |
| Alwan et al. | Comparing the effect of some plant extracts and commercial washes against some isolated microorganisms from vagina of pregnant women | |
| Nchabeleng | Determination of biological activity of Celtis africana extracts and its endophytic microflora and mycoflora | |
| CN104248637A (en) | Application of dictamine in preparation of anti candida albicans drugs | |
| CN106719849A (en) | Insecticide associated with a kind of matrine and Verticillium lecanii | |
| Narkwa | Antifungal Susceptibility of Candida Species and Cryptococcus neoformans Isolated from Patients at the Komfo Anokye Teaching Hospital in Kumasi | |
| Al-qertani et al. | Antifungal Activity of Acetonic Extracts of Syzygium aromaticum’Flowers and Mentha longifolia’s Leaves Against Clinical Isolates of Candida albicans | |
| Le et al. | Isolation and characterization of Xanthomonas axonopodis causing canker disease of lime tree and evaluation of ability of extraction from stem of Euphorbia tirucalli against X. axonopodis | |
| Nasution et al. | Phytochemical and Antibacterial Analysis of Mangkokan Leaf Extract Against Salmonella typhimurium Bacteria | |
| Yulianti et al. | Antibacterial activity of jamblang leaf ethanol extract (Syzygium cumini) against the growth of Propionibacterium acnes | |
| Asaiya et al. | Antibacterial Effect of Pongamia pinnata Leaf Extract Against Some Human Pathogenic Bacteria | |
| Pohan et al. | Effect of Putri Malu (Mimosa pudica Linn) Leaf Extract as an Inhibitor of Vibrio cholerae Growth |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1110098 Country of ref document: HK |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1110098 Country of ref document: HK |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090701 Termination date: 20131129 |