WO2008064520A1 - Enterococcus faecalis cms-h001 et son utilisation - Google Patents

Enterococcus faecalis cms-h001 et son utilisation Download PDF

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WO2008064520A1
WO2008064520A1 PCT/CN2006/003223 CN2006003223W WO2008064520A1 WO 2008064520 A1 WO2008064520 A1 WO 2008064520A1 CN 2006003223 W CN2006003223 W CN 2006003223W WO 2008064520 A1 WO2008064520 A1 WO 2008064520A1
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cms
group
enterococcus faecalis
gastric
disease
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PCT/CN2006/003223
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Chinese (zh)
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Fanggen Lu
Gang Lin
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Pharmapep Research & Development (Shenzhen) Co., Ltd
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Priority to PCT/CN2006/003223 priority Critical patent/WO2008064520A1/fr
Publication of WO2008064520A1 publication Critical patent/WO2008064520A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to the field of microbial technology, and in particular to a novel strain of Enterococcus faecalis and the use of the strain.
  • HHP infection is one of the most common infections in humans, with infection rates reaching more than 50% of the global population.
  • Warren and Marshall successfully cultured Helicobacter pylori from human gastric mucosa specimens and identified HP as a pathogen of gastritis and peptic ulcer. It changed chronic gastritis, peptic ulcer, and gastric mucosa-associated lymphoma (MALT).
  • MALT gastric mucosa-associated lymphoma
  • the treatment and the prevention pattern of gastric cancer have identified these four diseases as HP-related diseases. Numerous studies have shown that HP infection is the main cause of chronic active gastritis and peptic ulcer, and is closely related to gastric cancer and gastric mucosa-associated lymphoma (MALT).
  • the combination of antibiotics can cause a series of problems such as antibiotic-associated diarrhea, intestinal flora imbalance, and even pseudomembranous colitis, which can reduce the efficacy, reduce patient compliance, and easily relapse and reinfection after stopping the drug, and The rapid increase in resistant strains has led to a decline in eradication rates. Therefore, looking for non-antibiotic treatment HP is imperative.
  • the object of the present invention is to solve the above problems and to provide a novel strain of Enterococcus faecalis which can effectively treat Helicobacter pylori (HP) infection and its application in preparing medicines and the like.
  • a further object of the present invention is to provide a Helicobacter pylori inhibitor, a pharmaceutical composition, a food, a health care product and a food additive containing the above strain.
  • the present invention adopts the following technical solutions:
  • the present invention discloses an Enterococcus faecium ( ⁇ teracocc3 ⁇ 4s/ «ecafe) CMS-H001 having a deposit number of CCTCC No. M 206108.
  • the present invention also discloses a Helicobacter pylori inhibitor comprising the Enterococcus faecalis CMS-H001 with the accession number CCTCC No. M 206108.
  • the present invention also discloses the use of the Enterococcus faecalis CMS-H001 for the preparation of a medicament for treating a disease caused by Helicobacter pylori infection.
  • the disease is a stomach disease.
  • the disease is chronic gastritis.
  • the disease is a peptic ulcer. - or specifically, the disease is a gastric mucosa-associated lymphoma.
  • the disease is gastric cancer.
  • the invention further discloses a pharmaceutical composition comprising a pharmaceutically effective amount of the above-described Enterococcus faecalis CMS-H001.
  • the invention also discloses a foodstuff comprising the above-mentioned Enterococcus faecalis CMS-H001.
  • the food product is a beverage.
  • the present invention further discloses a health care product comprising the above-mentioned Enterococcus faecalis CMS- ⁇ 1.
  • the invention further discloses a food additive comprising the above-described Enterococcus faecium CMS-H001.
  • the Enterococcus faecalis CMS-H001 of the present invention is an isolated novel strain which can effectively antagonize Helicobacter pylori (HP) in vivo and in vitro; and has good tolerance to acid and bile. It can alleviate the chronic inflammatory injury caused by HP infection and reduce the infection of lymphocytes and plasma cells. The eradication rate of UP reaches 75%-87.5%, which is not significantly different from the conventional PPI triple therapy at present.
  • the novel strain of the present invention can be used for the treatment of HP infection with HP-related diseases such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoma and gastric cancer.
  • CTCC China Center for Type Culture Collection
  • Fig. 1 is a view showing the morphology of Enterococcus faecalis CMS-H001 of the present invention under a light microscope.
  • Figure 2 is a 5000X SEM image of Enterococcus faecalis CMS-H001 of the present invention.
  • Figure 3 is a 2000x X-ray scanning electron microscope image of Enterococcus faecalis CMS-H001 of the present invention.
  • Figure 4 is a 6000X-time transmission electron micrograph of Enterococcus faecalis CMS-H001 of the present invention.
  • Figure 5 is a 12000 X-fold transmission electron micrograph of Enterococcus faecalis CMS-H001 of the present invention.
  • Fig. 6 is a histogram of the body and antral injury in Example 2, A represents a histogram of the corpus callosum, and B represents a histogram of the antrum of the antrum.
  • Figure ⁇ is a histogram of the scores of Giemsa-stained bacteria in Example 2, and A represents the corpus
  • Hp integral histogram B represents the gastric antrum Hp integral histogram
  • Figure 8 is a modified Giemsa staining of gastric mucosa in each treatment group in Example 2, blank control group (A) no obvious bacilli in the gastric pit, model group (B) showed a large number of bacilli adhesion; and Enterococcus faecalis CMS-H001 treatment Group (C) and PPI treatment groups (D) showed only a small amount of bacillus adhesion (Giemsa staining, X 1000).
  • Figure 9 is a graph showing the results of homogenization of gastric tissue in the model group in Example 2.
  • a and B are the morphology of the gastric tissue homogenate smear of the model group and the HE staining of the bacterial culture of the pulp tissue, respectively, and Gram's staining can be seen.
  • Negative bacteria, Campylobacter or curved Gram's stain, oil mirror).
  • Microecology studies the relationship between normal microbial populations and their hosts.
  • Microecological therapy can be used to treat bacterial infections by antagonizing live bacterial preparations for pathogen activity. The effects are specific, direct, long-lasting, and have no obvious side effects.
  • Microecological regulators – Probiotics have a beneficial effect on the host by increasing the number and activity of certain health-promoting bacteria and rebuilding the flora balance.
  • Common probiotics mainly include Lactobacillus, Streptococcus mutans, Bifidobacteria, etc. These bacteria are normal bacteria in the gastrointestinal tract of healthy people. The metabolites contain acetic acid, lactic acid, hydrogen peroxide, etc., which have certain effects on pathogenic bacteria. Antagonism.
  • these bacteria are capable of producing a variety of bacteriocins with a high degree of bacteriostatic or bactericidal action. Therefore, probiotics antagonize pathogens in a variety of ways, reducing the occurrence of drug resistance, and providing a new way to treat bacterial infectious diseases.
  • the new strain of the present invention Enterococcus faecalis, CMS-H001, is a probiotic which can effectively antagonize Helicobacter pylori (HP) and can be used for the treatment of HP-related diseases such as chronic gastritis and digestion. Ulcer, gastric mucosa-associated lymphoma and gastric cancer.
  • the new strain of the present invention Enterococcus faecalis CMS-H001, deposited on October 23, 2006 at the China Center for Type Culture Collection (CCTCC) in Wuhan, China, under the accession number CCTCC No. M 206108.
  • CTCC China Center for Type Culture Collection
  • the Enterococcus faecalis strain CMS-H001 of the present invention is a purple-stained flat colony on the EC medium, having a diameter of 1 to 2 mm, a uniform edge, a smooth surface, and a Gram-positive cocci under light microscopy; scanning electron microscopy and transmission electron microscopy It is spherical or beaded, has no spores, has a complete cell wall structure, no buds, and uniform cytoplasm.
  • VFA is mainly acetic acid
  • NVFA is mainly lactic acid
  • succinic acid is mainly succinic acid.
  • the above studies showed that the Enterococcus faecalis CMS-H001 strain selected from the anaerobic strains isolated from human gastric antrum and duodenum has good resistance to simulated gastric acid and duodenal bile, and can significantly inhibit Hp clinically in vitro. Strain, and this role depends on the direct involvement of live bacteria. These studies indicate that the Enterococcus faecalis CMS-H001 strain of the present invention has been indispensable for becoming a medicinal bacterium, and can be used as a new drug, a health care product, and a food additive for the application of microecological therapy to prevent Hp infection.
  • the present invention further uses the Enterococcus faecalis strain CMS-H001 to conduct an in vivo treatment study on HP-induced gastritis model rats, and compares it with the current conventional treatment method "PPI triple therapy".
  • the study found that the probiotic bacterium Enterococcus faecium CMS-H001 of the present invention can alleviate the chronic inflammatory injury caused by HP infection, reduce the infiltration of lymphocytes and plasma cells, and has significant difference compared with the model group, and there is no significant difference compared with the PPI treatment group. , and has the effect of inhibiting HP. After treatment, HP bacteria detection is significantly reduced, and the eradication rate to HP is 75%-87.5%. There is no significant difference compared with PPI triple therapy.
  • the results of the present invention indicate that there are symbiotic bacteria antagonistic to Hp in the stomach of a normal human, and there is strain specificity.
  • the Enterococcus faecalis CMS-H001 selected from the anaerobic strains isolated from healthy human gastric antrum has a significant inhibitory effect on Hp clinical strains in vivo, and the eradication rate of HP is not significantly different from the triple therapy of PPI plus antibiotics. Sexual differences. This result provides a new approach to the treatment of Hp infection, providing a theoretical basis for microecology in the treatment of HP-related diseases.
  • a pharmaceutical composition can be prepared using the Enterococcus faecalis CMS-H001 of the present invention.
  • the pharmaceutical composition contains a pharmaceutically effective amount of a live bacterial form of Enterococcus faecalis CMS-H001.
  • the pharmaceutical composition may also contain a suitable pharmaceutical carrier.
  • the pharmaceutical composition of the present invention may be in the form of a capsule, a solution or a drinkable suspension, a bagged powder or the like, and each single dose generally contains a strain of Enterococcus faecalis CMS-H001 of about 10 6 to 10 1 cells.
  • the pharmaceutical composition of the present invention can be used for the prevention and treatment of HP-related diseases such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoma and gastric cancer.
  • the Enterococcus faecalis CMS-H001 of the present invention can also be prepared into a form of food, health care product or food additive, which can be used for preventing and treating HP-related diseases such as chronic gastritis, peptic ulcer, gastric mucosa Related lymphoma and gastric cancer, etc., improve the health of users.
  • the food of the present invention may be in the form of a beverage containing the live bacteria of Enterococcus faecalis CMS-H001 of the present invention, or may be in the form of a dairy product containing such a live bacteria, fermented milk, sour milk or the like.
  • Example 1 Isolation, Identification and Biological Characteristics of Enterococcus faecalis CMS-H001
  • MRS liquid medium 5 g of peptone, 5 g of beef flour, 2.5 g of yeast dipping powder, 10 g of glucose, 2.5 g of K2HPO4, lg of ammonium citrate, 2.5 g of NaCl, 0.25 g of MgS0 4 , 0.1 g of MnSO 4 , Tween-80 0.5 g After adding 500 ml of distilled water, it was dissolved in a high-pressure steam at 121 ° C, and placed in a refrigerator at 4 ° C for use.
  • MRS solid medium peptone 5g, beef powder 5g, yeast dipping powder 2.5g, glucose 10g, K 2 HP0 4 2.5g, ammonium citrate lg, NaCl 2.5g, MgS0 4 0.25g, MnS0 4 O.lg, Tween- 80 0.5 g of distilled water was dissolved in 500 ml, 10 g of agar powder was added, and autoclaved at 121 ° C. The sterile petri dish was poured and placed in a refrigerator at 4 ° C for use.
  • Gastroscope sampling and specimen transfer Gastroscope aspirate healthy adult duodenal descending liquid 0.5ml into 2ml MRS liquid medium, take three specimens according to the above method, and transfer the anaerobic box to the laboratory within 30 minutes.
  • CMS-H001 Enterococcus faecalis bacteria isolated after removing seed from a healthy adult 4h descending part of duodenum fluid specimen temperature at 37 ° C in an anaerobic box culture, according to the dilution series of 10-1 to 10-7, and the liquid Each dilution of each dilution was added to the MRS plate, and the L-shaped glass rod was evenly coated. After anaerobic incubation for 48 hours at 37 °C, the colony morphology was taken out, and the monoclonal gram staining microscopy was performed to obtain different morphology and staining properties.
  • the colony of the colony was picked into MRS liquid medium, and cultured at 37 °C for 24 hours in an anaerobic environment.
  • the staining microscopy was taken out, the morphology under the microscope was observed, and the MRS solid medium was streaked, and anaerobic again at 37 °C: I*
  • the morphology of the colonies was observed for 48 hours, and the monoclonal staining microscopic examination was carried out, and the passage was repeated four times, until the colony morphology of the solid plate was consistent, and the Gram stain microscopic examination was consistently determined to be pure.
  • the pure bacteria of each strain can be added to the antifreeze and stored in a -70 V refrigerator at low temperature for use.
  • Enterococcus faecalis CMS-H001 is a flat purple colony on the EC medium, with a diameter of 1 ⁇ 2 mm, a uniform edge and a smooth surface. Under the light microscope, Gram-positive cocci, spherical or beaded, no spores. see picture 1.
  • Fig. 4 and 5 The results are shown in Fig. 4 and 5.
  • the image shows that the morphology of the bacteria is regular under the transmission electron microscope.
  • the cell wall structure is intact, there is no swelling and bud phenomenon, the cytoplasm is uniform, no abnormal particles are found, and no virus, mycoplasma, etc. are found in all the slices.
  • Factor, Figure 4, Figure 5 represent 6000X and 12000x times, respectively.
  • NVFA Methanol sulfuric acid method Take 2ml of standard solution or culture and add 0.2 ml of 50% sulfuric acid, adjust the pH below 2.0, take 1ml of acidic culture of sulfuric acid, add 1ml of methanol solution and 0.1ml of 50% sulfuric acid, boil the sample for 5 minutes or overnight at room temperature, add 0.5 ml of chloroform, mix and let stand for a while, 1000 rpm, 4 centrifugation for 2 min, take chloroform layer ⁇ for injection analysis.
  • the extracted samples and the standard ⁇ injection analysis were respectively taken, and the retention time of each substance was measured by a Shimadzu GC2010 gas chromatography detector (GC) and the standard was used as a quantitative standard for the substance in the sample.
  • GC Shimadzu GC2010 gas chromatography detector
  • VFA of Enterococcus faecalis CMS-H001 was mainly acetic acid
  • the peak time of acetic acid was 3 ⁇ 4 7.970 min
  • NVFA was mainly lactic acid
  • succinic acid there was a small amount of succinic acid.
  • the peak time of lactic acid was 7.300 min
  • the peak time of succinic acid was 9.775min.
  • API-20 STREP (French Enzyme) is a standardization method that combines 20 biochemical tests in all aspects. The system is capable of stabilizing the species or population of most streptococcus in medical bacteria.
  • the API-20 STREP test strip consists of 20 small tubes containing a dry substrate capable of enzymatic determination and sugar fermentation, and can be used to determine enzyme activity or sugar fermentation.
  • the enzyme activity assay is inoculated with a concentrated, pure bacterial suspension onto a dry enzyme substrate. During the culture, the resulting metabolite produced is visualized by natural discoloration or discoloration by the addition of a reagent.
  • the fermentation test is to inoculate a medium consisting of a sugar substrate. The fermented carbohydrate is shown as a PH indicator.
  • the MRS liquid medium was taken out after anaerobic incubation at 37 ° C for 24 hours, centrifuged at 5000 rpm / min for 4 minutes at 4 ° C, and the supernatant was taken, and the precipitate was discarded.
  • Hp was collected from the blood plate of heart and brain infusion by aseptic inoculating loop, inoculated into 0.2 ml of heart and brain infusion containing 50% fetal bovine serum, and the concentration of bacterial solution was determined by turbidimetry to be loUCFU/mL.
  • the punching plate was taken out to measure the diameter of the inhibition zone.
  • the supernatant of the autoclaved supernatant, the supernatant of the filter sterilization, and the inner wall of the corresponding pores of the MRS liquid medium and the pericellular area were free of bacteria, and the diameter of the inhibition zone was 0;
  • the culture supernatant of the enterococci CMS-H001 strain had corresponding bacteria growth on the inner wall of each well.
  • the diameter of the inhibition zone of the three plates was 18 mm, 18 mm, 11 mm, and the average was 15.7 mm.
  • Acid resistance test The frozen E. faecalis CMS-H001 strain was taken out from a 70 °C refrigerator, inoculated in MRS liquid medium, and incubated at 37 °C for 24 h, centrifuged (4300xg, lOmin, 4°). C), the supernatant was discarded, and the bacterial cell pellet was collected and washed three times with sterile physiological saline. Then, it was inoculated into a MRS liquid medium having a pH of 2.0 (titrated with hydrochloric acid) at a volume ratio of 1%. At the same time, MRS liquid medium having a pH of 6.8 was inoculated as a control.
  • the ⁇ broth was diluted in the 1-10 sequence after Omin and 120 min after inoculation.
  • the dilution gradient was taken from ⁇ to the MRS plate of ⁇ 6.8, and the viable count was performed after 48 hours of anaerobic culture. repeat three times.
  • Bile resistance test Inoculation of Enterococcus faecalis CMS-H001 strain from the -70 °C refrigerator After incubation for 24 hours at 37 ° C in MRS liquid medium, centrifugation (4300 x g, 10 min, 4 ° C), the supernatant was discarded, and the cell pellet was collected and washed 3 times with sterile physiological saline. Then, it was inoculated into a MRS liquid medium containing 0.3% of bile salt in a volume ratio of 1%. At the same time, MRS liquid medium containing no bile salt was inoculated as a control.
  • Crystal violet dye solution crystal violet lg
  • the crystal violet is dissolved in ethanol and then mixed with an ammonium oxalate solution.
  • Lugol's iodine solution iodine lg
  • Dilute charcoal red dyeing solution Weigh alkaline reddish 10g, grind finely, add 95% ethanol 100ml, place overnight, filter paper filter. 10 ml of this solution was taken and mixed with 90 ml of a 5% aqueous solution of carbolic acid to form a red carbonate solution of carbolic acid. Then take 10 ml of this solution and add 90 ml of water to form a red carbon solution of dilute carbonic acid.
  • Antifreeze DMSO and glycerin are mixed in a volume ratio of 1:2.
  • HP bacterial culture Prepare the medium for use in the laboratory with heart and brain infusion.
  • mice 24 normal BALB/c mice that had been successfully modeled as chronic gastritis, plus 8 blank control groups, 32 in total, male and female, weighing ⁇ 2 g.
  • the Enterococcus faecalis CMS-H001 strain on the MRS culture plate was collected by a sterile inoculating loop, and the suspension was prepared with sterile physiological saline, and the final concentration of the bacterial suspension was adjusted by turbidimetry to be lxl0 9 CFU/ml.
  • Proton pump inhibitor ⁇ (40mg/ ⁇ ) (Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., Batch number Q40805);
  • Antibiotics clarithromycin tablets (250mg/tablet) (Jiangxi Huiren Pharmaceutical Co., Ltd., batch number 0510019), ampicillin injection (0.5g/piece) (Huabei Pharmaceutical Co., Ltd., batch number 0404307), gentamicin ( Tianjin Pharmaceutical Co., Ltd., batch number 05052802), all purchased from the Department of Pharmacy, Second Xiangya Hospital, Central South University. ⁇
  • the doses of the various drugs were calculated by the above formula as follows - ⁇ (40 mg) was diluted with 100 ml of sterile physiological saline into a solution having a concentration of 0.4 mg/nil, and each mouse was intragastrically administered with 0.25 ml per day.
  • Ampicillin (0.5 g) was diluted with 25 ml of sterile physiological saline to a solution having a concentration of 20 mg/ml, and each mouse was administered 0.25 ml per day.
  • Clarithromycin (0.25 g) was diluted with 5 mT of sterile physiological saline to a concentration of 50 mg/ml, and each mouse was orally administered with 0.1 ml per day.
  • Balb/c mice were randomly divided into 3 groups, 8 in each group.
  • the first group was the PPI plus antibiotic treatment group
  • the second group was the Enterococcus faecalis CMS-H001 treatment group
  • the third group was the model control group
  • the other 8 normal Bdb/c mice were added as the blank control group (Group 4). .
  • Group 1 (PH+ antibiotic triple therapy group): From day 3-10, fasting 121 ⁇ per day, 0.25 ml of PPI solution, 0.25 ml of ampicillin and 0.1 ml of clarithromycin, and then banned after filling the stomach. Eat 3-4 hours, once a day, for 7 days. Mice were sacrificed at 4 weeks after the last gavage.
  • Group 2 E. faecalis CMS-H001 treatment group: From the first 1-10 days, fasting for 12 hours every day, and giving CMS-H001 fresh bacterial suspension 0.5ml/supply, the number of bacteria is 10 9 CFU, After the stomach is finished, fasting for 3-4 hours, once a day, for 10 days. Mice were sacrificed at the end of the last gavage for 4 weeks.
  • Group 3 (model control group): From day 1 to day 10, 0.5 ml per saline was administered per day, once a day. All mice were sacrificed at 4 weeks after the last gavage for 10 days.
  • Group 4 (blank control group): From day 1 to day 10, 0.5 ml per saline was administered per day, once a day. All mice were sacrificed at 4 weeks after the last gavage for 10 days.
  • mice Immediately after the mice were sacrificed by cervical dislocation, the gastric cavity was removed, the anterior stomach was removed, and the large curved side was cut open to obtain intact gastric tissue (including duodenum, gastric antrum, stomach, etc.). Half of the gastric mucosa was fixed with 10% formalin solution, HE staining and Giemsa staining, and the other half of the tissue was subjected to rapid urease test (observation time 5 min), smear and bacterial culture.
  • Hp culture gastroduodenal homogenized with a tissue sample ground into brain heart infusion of 500ul containing 10% FBS, with a calculated mass of the specimen before and after the amount of the difference method, a series of appropriately diluted 10-1, each of the gradient taken lOOul heart-infused brain blood plate, 37 ° C micro-aerobic culture for 3 to 7 days, observe the colony morphology, take suspected colonies Gram stain microscopy and rapid urease test.
  • Paraffin sections were placed in the staining rack in the same direction, and baked at 60 ° C for 10 minutes, or the wax on the slices was melted by using a hair dryer.
  • mice Immediately after dissection of the mice, half of the gastric mucosa was fixed with 10% formalin solution and HE stained: mouse stomach tissue was fixed with 10% formalin, embedded in paraffin, sectioned, HE stained (hematoxylin staining) 7 minutes - wash the excess dye solution with tap water - 1% hydrochloric acid alcohol for 5 seconds, make the nucleus purple blue - wash the tap water component - 1% eosin stain, about 3 minutes). A double-blind reading of the pathologist. .
  • Hp colonization 1 minute small amount of Hp colonization and not observed in each gastric pit (1-2 bacteria/gastric pits) 2 points Hp colonization is low but can be observed in most gastric pits (3-10 bacteria) / stomach small concave) 3 points all the small pits have a moderate amount of Hp (ll-20 Hp / stomach pit)
  • Hp eradication criteria One month after the treatment was stopped, the bacterial culture was negative or the rapid urease test and the histological staining examination were negative for Hp eradication.
  • Hp infection improvement criteria reduced Hp count per unit mass organization or reduced histological staining bacteria.
  • Pathological improvement criteria Generalized congestion and edema were lessened than before, erosive ulcer lesions were smaller than before treatment (the vernier card measured lesion diameter), or pathology was not completely normal, but the score was reduced before treatment.
  • Cure criteria (1) plus (3), that is, Hp eradication plus pathology results are normal.
  • Improvement criteria (1) plus (4), or (2) plus (3), or (2) plus (4).
  • a and B of Figure 6 represent the injury scores of the gastric sinus and the gastric body, respectively.
  • the score of inflammatory injury of gastric antrum mucosa was slightly higher than that of gastric mucosa.
  • There were significant differences in the scores of inflammatory injury between gastric mucosa in the 4 groups including group 1 (PH+ antibiotic treatment group) and group 2 (feces enterococcus CMS-
  • group 1 PH+ antibiotic treatment group
  • group 2 feces enterococcus CMS-
  • the scores of antral mucosal inflammatory lesions in group H001 and group 4 (blank control group) were lower than those in group 3 (model control group), the difference was significant ( ⁇ 0.05), group 1 and group 2 There was no significant difference in the comparison between the two groups (>0.05).
  • Hp colonization score Giemsa staining clearly shows that Hp is S-shaped, more colonized in the upper part of the gastric pit and in the gastric gland lumen.
  • the colonization scores of each group of Hp are shown in Table 8 and the histogram of Fig. 7, and A and B of Fig. 7 represent the Hp colonization scores of the gastric antrum and the gastric body, respectively.
  • Figure 8 shows the modified Giemsa staining of gastric mucosa in each group. No obvious bacilli were observed in the gastric pits of the blank control group. A large number of bacilli were observed in the model group, while only a small amount of bacilli were observed in the PPI-treated group and the Enterococcus faecalis CMS-H001 treatment group. Sticking.
  • Group 1 12.5% (1/8) 12.5% (1/8) 12.5%% (1/8) 0.0% (0/8)
  • Group 2 25.0% (2/8) 12.5% (1/8) 12.5 % (1/8) 12.5% (1/8)
  • Group 3 87.5% (7/8) 75% (6/8) 62.5% (5/8) 50% (4/8) Group 4 0 (0 /8) 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8)
  • the morphology of the gastric tissue homogenate smear and homogenate tissue culture in the model group was observed by HE staining under light microscopy. Gram's staining negative bacteria were observed, showing Campylobacter or curved, as shown in Figures 9A and B.
  • the above results indicate that the Enterococcus faecalis CMS-H001 of the present invention can alleviate the chronic inflammatory injury caused by HP infection, reduce the infection of lymphocytes and paddle cells, and is significantly different from the model group, and the PPI treatment group (conventional treatment) Methods) There was no significant difference; and there was inhibition of HP. After treatment, HP bacteria detection was significantly reduced, and HP eradication rate reached 75%-87.5%. There was no significant difference compared with PPI triple therapy.

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  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne une souche de Enterococcus faecalis CMS-H001, présentant le numéro d'entrée CTCC No. M 206108. La souche de Enterococcus faecalis CMS-H001 de l'invention peut être utilisée pour traiter efficacement une infection à Helicobacter pylori(HP).
PCT/CN2006/003223 2006-11-30 2006-11-30 Enterococcus faecalis cms-h001 et son utilisation WO2008064520A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2006/003223 WO2008064520A1 (fr) 2006-11-30 2006-11-30 Enterococcus faecalis cms-h001 et son utilisation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2006/003223 WO2008064520A1 (fr) 2006-11-30 2006-11-30 Enterococcus faecalis cms-h001 et son utilisation

Publications (1)

Publication Number Publication Date
WO2008064520A1 true WO2008064520A1 (fr) 2008-06-05

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2006/003223 WO2008064520A1 (fr) 2006-11-30 2006-11-30 Enterococcus faecalis cms-h001 et son utilisation

Country Status (1)

Country Link
WO (1) WO2008064520A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1304313A (zh) * 1998-06-05 2001-07-18 若素制药株式会社 含乳酸菌的组合物、药物和食物
JP2001258549A (ja) * 2000-02-19 2001-09-25 Korea Yakult Co Ltd ヘリコバクターピロリに対して抗菌活性を有するラクトバシルスアシドフィルスｈｙ２１７７、ラクトバシルスカセイｈｙ２７４３及びそれらを用いた乳酸菌製剤と発酵乳
KR20040013654A (ko) * 2002-08-07 2004-02-14 신차경 항 헬리코박터 유산균을 이용한 수산발효 식품 제조방법과그 식품
WO2004041854A2 (fr) * 2002-11-05 2004-05-21 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides bacteriens essentiels

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1304313A (zh) * 1998-06-05 2001-07-18 若素制药株式会社 含乳酸菌的组合物、药物和食物
JP2001258549A (ja) * 2000-02-19 2001-09-25 Korea Yakult Co Ltd ヘリコバクターピロリに対して抗菌活性を有するラクトバシルスアシドフィルスｈｙ２１７７、ラクトバシルスカセイｈｙ２７４３及びそれらを用いた乳酸菌製剤と発酵乳
KR20040013654A (ko) * 2002-08-07 2004-02-14 신차경 항 헬리코박터 유산균을 이용한 수산발효 식품 제조방법과그 식품
WO2004041854A2 (fr) * 2002-11-05 2004-05-21 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides bacteriens essentiels

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