WO2008064521A1 - Lactobacillus fermentum cms-h002 et son utilisation - Google Patents

Lactobacillus fermentum cms-h002 et son utilisation Download PDF

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Publication number
WO2008064521A1
WO2008064521A1 PCT/CN2006/003224 CN2006003224W WO2008064521A1 WO 2008064521 A1 WO2008064521 A1 WO 2008064521A1 CN 2006003224 W CN2006003224 W CN 2006003224W WO 2008064521 A1 WO2008064521 A1 WO 2008064521A1
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cms
lactobacillus fermentum
group
treatment
strain
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PCT/CN2006/003224
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English (en)
Chinese (zh)
Inventor
Fanggen Lu
Gang Lin
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Pharmapep Research & Development (Shenzhen) Co., Ltd.
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Priority to PCT/CN2006/003224 priority Critical patent/WO2008064521A1/fr
Publication of WO2008064521A1 publication Critical patent/WO2008064521A1/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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • C12R2001/225—Lactobacillus

Definitions

  • Lactobacillus fermentum CMS-H002 Lactobacillus fermentum CMS-H002 and its application
  • the present invention relates to the field of microbial technology, and in particular to a novel strain of Lactobacillus fermentum and the use of the strain.
  • Ulcerative colitis is an abbreviation for chronic non-specific ulcerative colitis, a chronic inflammatory disease of the rectum and colon that is unexplained.
  • the onset of UC is the result of complex, multi-environment, multi-factor interactions. There are many studies on it, but its etiology and pathogenesis are still not fully understood.
  • the main clinical manifestations were diarrhea, mucus pus and bloody stools, abdominal pain and urgency.
  • the severity of the illness varies from recurrent or prolonged to chronic exacerbations, often with complications and even cancer. The disease can occur at any age, and is more common in 20-50 years old. There was no significant difference in the incidence of men and women.
  • UC ulcerative colitis
  • the drugs currently treated include adrenal glucocorticoids, which are suitable for patients with fulminant or severe disease, can control inflammation, inhibit autoimmune processes, alleviate symptoms of poisoning, and have better therapeutic effects.
  • sulfasalazine (SASP) is also often used as the drug of choice, and it is suitable for patients with mild or severe adrenal glucocorticoid therapy.
  • An object of the present invention is to solve the above problems and to provide a novel Lactobacillus fermentum strain which can effectively treat a rectal or colonic inflammatory disease, particularly ulcerative colitis, and its use in the preparation of a medicament and the like.
  • It is still another object of the present invention to provide a pharmaceutical composition comprising the above novel Lactobacillus fermentum strain, Food, health products and food additives.
  • the present invention adopts the following technical solutions:
  • the invention discloses a Lactobacillus fermentum actobacillus fermentunO CMS-H002, which has the accession number CCTCC No. M 206110.
  • the present invention also discloses the use of the above-mentioned Lactobacillus fermentum for the preparation of a medicament for treating a rectal or colonic inflammatory disease.
  • the rectal or colonic inflammatory disease is preferably ulcerative colitis.
  • the present invention also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of the above-mentioned Lactobacillus fermentum CMS-H002, or a metabolite thereof, a cell fragment or a secretion thereof.
  • the invention also discloses a foodstuff comprising the above-mentioned Lactobacillus fermentum CMS-H002, or a metabolite thereof, a cell debris or a secretion thereof.
  • the food product is a beverage.
  • the invention also discloses a health care product comprising the above-mentioned Lactobacillus fermentum CMS-H002, or a metabolite thereof, a cell debris or a secretion.
  • the present invention also discloses a food additive comprising the above-mentioned actobadllus fermentum CMS-H002, or a metabolite thereof, cell debris or secretion.
  • the Lactobacillus fermentum CMS-H002 of the present invention is an isolated novel strain which can relieve diarrhea, hemorrhage, weight loss and the like, and can also reduce intestinal mucosal damage and inflammatory cell infiltration for ulcerative colitis. , the effect is better than sulfasalazine (SASP); and as a probiotic, the safety is superior and the side effects are small.
  • SASP sulfasalazine
  • This strain is safe and effective in the treatment of rectal or colonic inflammatory diseases such as ulcerative colitis.
  • CTCC China Center for Type Culture Collection
  • Figure 1 shows the morphology of the Lactobacillus fermentum CMS-H002 smear microscopy (Gram's staining, 2 is a scanning electron microscope result (x l0000) of Lactobacillus fermentum CMS-H002 of the present invention.
  • Fig. 3 is a transmission electron microscope result of the Lactobacillus fermentum CMS-H002 of the present invention (xl2000).
  • Fig. 4 is a graph showing the results of changes in body weight of mice in each experimental group before and after the experiment in Example 2.
  • Fig. 5 is a graph showing the results of mortality of mice in each group in Example 2.
  • Fig. 6 is a graph showing the results of colon length of each group of mice in Example 2.
  • Fig. 7 is a graph showing the results of DAI integral curve of each group of mice in Example 2.
  • Fig. 8 is a graph showing the results of histological damage scores of mice in each group in Example 2.
  • Figure 9a-1 is a colonic pathological section (HE x400) of each group of mice in Example 2, wherein: a is a colonic mucosa of a normal control group; b is a colonic mucosa of a model group;
  • c is the colonic mucosa of the negative control group
  • d is the colonic mucosa of the positive control group
  • e is for treating a group of colonic mucosa
  • f is for treating two groups of colonic mucosa
  • k is the high dose of cl20 enema group colonic mucosa; 1 for the treatment of six groups of colonic mucosa.
  • the new strain of the present invention Lactobacillus fermentum CMS-H002 has been deposited at the China Type Culture Collection (CCTCC) in Wuhan, China on October 23, 2006, under the accession number CCTCC No. M 206110.
  • CTCC China Type Culture Collection
  • the new strain of the present invention Lactobacillus fermentum CMS-H002
  • Lactobacillus fermentum CMS-H002 can be isolated from healthy infant feces or adult duodenal juice.
  • This strain can alleviate diarrhea, hemorrhage, weight loss and other symptoms of ulcerative colitis, and can also reduce intestinal mucosal damage and inflammatory cell infiltration, the effect is better than sulfasalazine (SASP).
  • SASP sulfasalazine
  • the strain is effective for treating rectal or colonic inflammatory diseases such as ulcerative colitis.
  • a pharmaceutical composition can be prepared using the Lactobacillus fermentum CMS-H002 of the present invention.
  • the pharmaceutical composition contains a pharmaceutically effective amount of Lactobacillus fermentum CMS-H002, or a metabolite thereof, cell debris or secretion.
  • 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 Lactobacillus fermentum CMS-H002 of about ⁇ ) 8 ⁇ ⁇ ) 11 cells.
  • the Lactobacillus fermentum CMS-H002 of the present invention can also be prepared in the form of a food, a health supplement or a food additive.
  • the food, health care product or food additive contains Lactobacillus fermentum CMS-H002, Or its metabolites, cell debris or secretions. These foods, health supplements or food additives can be used to prevent rectal or colonic inflammatory diseases such as ulcerative colitis and improve the health of users.
  • the food of the present invention may be in the form of a beverage containing the live bacteria of the Lactobacillus fermentum CMS-H002 of the present invention, or may be in the form of a dairy product containing the living bacteria, fermented milk, sour milk or the like.
  • Reagents Beef powder (Beijing Aoboxing Biological Co., Ltd., batch number: 20040607), peptone (Beijing Aoboxing Biological Co., Ltd., batch number: ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
  • MRS medium beef powder 10 g / liter, peptone 10 g / liter, sodium acetate 5 g / liter, magnesium sulfate 0.5 g / liter, manganese sulfate 0.2 g / liter, yeast dip powder 5 g / liter, ammonium citrate 2 g / liter, glucose 20 g / liter, Tween-80 (1 g / liter), dibasic potassium phosphate 5 g / liter, agar powder 15 g / liter.
  • the pH was adjusted to 4.5, 5.4, and 6.8 with acetic acid, respectively.
  • the X-gal solution (20 mg/ml) was prepared and sterilized by filtration through a 0.22 um-secondary filter. 50 ul of the solution was placed on a solid medium of MRS and uniformly coated.
  • MRS Anaerobic liquid medium cassette quickly transported to a laboratory to, 35 ° C incubation 2- 3h, the bacteria adapt to the environment of the medium, and then diluted 10-fold serial dilution of several 10-1, 10-2, 10-3, etc. 0.1 ml of each was placed on MRS solid medium, coated with L-shaped glass rods, and anaerobic cultured at 35 ° C for 48-72 hours. Typical colonies were picked up in an anaerobic environment of MRS liquid medium for 24 h, and Gram's staining was performed.
  • the morphology was observed under a microscope, and the microscopic morphology of the Gram's stain-positive bacilli was selected and streaked onto the MRS solid medium plate.
  • Anaerobic culture for 48 h according to the morphological characteristics of the colony on the plate and microscopic observation of the staining characteristics, size, bulb shape and distribution of the cells, to determine whether to purify. If the bacteria are not pure, continue to pick a single colony and inoculate the MRS solid medium plate to further separate and purify, and repeatedly separate and pass the passage to obtain a purified strain. .
  • Lactobacillus fermentum CMS-H002 showed round, white, convex, wetted colonies with a diameter of about 2-4 mm and neat edges after 48 hours of anaerobic incubation on MRS solid medium plates.
  • Lactobacillus fermentum CMS-H002 bacterial smear Gram-positive bacilli, mostly short rod-shaped bacteria, single or in pairs, see Figure 1.
  • Lactobacillus fermentum CMS-H002 showed a round, white, convex, wetted colony with a diameter of about 2-4 mm and a neat edge after 48 h of aerobic incubation on a MRS solid medium plate.
  • API 20A is an anaerobic identification system with 21 assays for rapid and easy biochemical identification of anaerobic bacteria.
  • the API 20A test strip consists of 20 small tubes containing dry substrates. The substrate suspension was reconstituted by dispensing the bacterial suspension into the tube. After 24 or 48 hours of incubation at 35 to 37 ° C, the metabolite produced is presented by an acidity (pH) indicator or by the addition of a reagent. The reaction results can be judged according to the reading table; they are identified by analytical spectrum indexing or identification software.
  • API 50 CHL is used for the identification of Lactobacillus actpbacUlus and related bacteria, which is a simple medium consisting of 49 50 CH test strips of fermentable carbohydrates. Each tube of the test strip was inoculated with a suspension of the assay bacteria. When cultured, the pH of the fermented pond water compound is lowered, causing the indicator to discolor. The results constitute the biochemical profile of the strain and are used for identification or typing.
  • API 50 CHL biochemical identification results show: Lactobacillus fermentum CMS-H002 can ferment ribose, galactose, glucose, fructose, mannose, maltose, lactose, melibiose, sucrose, raffinose, code: 44514002, in line with fermented milk Bacillus.
  • Lactobacillus fermentum CMS-H002 isolated and purified was inoculated on MRS-X-gal medium, anaerobic cultured for 48 hours, and the plate was taken out and placed in the air, and the colony color development was observed after a few minutes.
  • Lactobacillus fermentum CMS-H002 was fixed and subjected to gradient dehydration, gradient replacement, and platinum plating. After observation by electron microscopy, the surface of the bacteria was smooth, intact, uniform in shape, short stick-shaped, blunt at both ends, and no spores, as shown in Fig. 2. ,
  • Lactobacillus fermentum CMS-H002 was fixed and subjected to gradient dehydration, embedding, sectioning, double staining of uranium acetate and lead nitrate, and then observed by transmission electron microscopy.
  • the morphology of the bacteria was regular, the cell wall structure was intact, no swelling and budding, and the cytoplasm was uniform. No abnormal particles were found, and no exogenous factors such as virus and mycoplasma were observed, as shown in Figure 3.
  • the bacterial slime was collected and the plasmid was extracted by a conventional method, and the presence of a plasmid was detected by 1.0% agarose gel electrophoresis for Lactobacillus fermentum CMS-H002. No plasmid was found in the test results.
  • VFA volatile fatty acid
  • NVFA non-volatile fatty acid
  • the extracted samples and the standard 1/ ⁇ 1 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 Lactobacillus fermentum CMS-H002 was mainly lactic acid, and the peak time was 7.467 seconds.
  • NVFA is mainly acetic acid, and a small amount of succinic acid has a peak time of 7.947 seconds.
  • mice Balb/c mice, SPF grade, male, 6-8 weeks old, weighing 20 ⁇ 2 g, purchased from Hunan Agricultural University Dongchuang Experimental Animal Science and Technology Service Department, and kept in clean animal room.
  • mice 5% DSS solution was free to drink. The day caused an acute ulcerative colitis model.
  • the probiotics (Lactobacillus fermentum CMS-H002, Bifidobacterium 0501 and cl20 strains), saline, SASP and other enema were started from 2 days before drinking DSS, and each mouse was given once a day, 0.3 ml/time. 20g.
  • mice 120 Balb/c mice were randomly divided into 12 groups, and each group was divided into 10 groups as follows:
  • A, 'normal control group normal diet, no special treatment
  • model control group drinking DSS modeling
  • the crypt is completely destroyed, and the lamina intestinal covers a single layer of epithelium with mild inflammatory cells.
  • mice died during the experiment, of which 10 died of lower gastrointestinal bleeding, 1 chronic intestinal perforation, and 4 died of unknown causes.
  • mice showed positive fecal occult blood after 3 days of drinking DSS. Some mice showed thin paste-like gross bloody stool after 4 days of drinking DSS. The mice in the positive control group began to have gross bloody stool after drinking DSS for 4 days. One group, two groups, three groups, and four groups all had fecal occult blood positive after 2 days of drinking DSS, and thin paste-like gross bloody stools after drinking DSS for 3 days. Some groups of mice developed fecal occlusion after 4 days of drinking DSS.
  • Normal control group 100 0 0 model control group (B) 10 1 8 1 negative control group (C) 10 4 6 2 positive control group (D) 10 4 5 1 treatment group (E) 10 1 7 0 Treatment group 2 (F) 10 1 7 3 treatment three groups (G) 10 0 7 5 treatment four groups (H) 10 1 8 3 treatment five groups (I) 10 3 6 0 high dose 0501 enema group (J) 8 2 0 0 high dose cl20 enema group (K) 8 3 0 0 0 0
  • the colons of the third, fourth, fifth and sixth groups were shortened.
  • the length of the colon was significantly different from that of the normal control group.
  • the length of the colon in the six groups was longer than that in the model group, the negative control group, the positive control group, and the treatment one, two, three.
  • the model group, the negative control group, the positive control group, and the treatment group 1, 2, 3, 4, and 5 were extensively deficient in colonic mucosal epithelial cells, most of the glands were incomplete, and inflammatory cells were extensively infiltrated, showing typical inflammatory changes.
  • the treatment of the six groups of colonic mucosa gland was basically complete, local small amount of inflammatory cell infiltration or crypt destruction, histological score treatment of the six groups compared with the model group, the negative control group, the positive control group decreased, there was a significant difference (P ⁇ 0.05), see Figure 8. Colonic pathology of each group of mice, see Figure 9a ⁇ 1.
  • Lactobacillus fermentum CMS-H002 bacterial enema can alleviate diarrhea, hemorrhage, weight loss and other symptoms in mice with ulcerative colitis, and also reduce intestinal mucosal damage and inflammatory cell infiltration.
  • the group and the SASP enema group were significantly relieved.
  • Bifidobacterium liquid enema to treat DSS-induced UC mice, we found that two Bifidobacterium broth enemas can aggravate diarrhea, hemorrhage, and weight loss in mice with ulcerative colitis, and also aggravate their intestinal mucosa.
  • the injury and inflammatory cell infiltration were more severe than those in the model group and the negative control group.

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Abstract

L'invention concerne une souche de Lactobacillus fermentum CMS-H002, présentant le numéro d'entrée CTCC No. M 206110. L'invention concerne également l'utilisation de la souche, une composition pharmaceutique, un produit alimentaire et un additif contenant la souche. La souche de Lactobacillus fermentum CMS-H002 de la présente invention peut être utilisée pour traiter efficacement des états pathologiques inflammatoires du rectum ou du côlon, tels que la recto-colite hémorragique.
PCT/CN2006/003224 2006-11-30 2006-11-30 Lactobacillus fermentum cms-h002 et son utilisation WO2008064521A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011018080A3 (fr) * 2009-08-11 2011-04-21 Schrezenmeir Juergen Composition renfermant des souches de lactobacillus fermentum
RU2738803C2 (ru) * 2015-08-25 2020-12-17 Иммьюнбиотек Медикал Свиден Аб Композиция и способ лечения и профилактики кишечной инфекции и воспаления
CN112218646A (zh) * 2018-05-31 2021-01-12 深圳华大生命科学研究院 一种组合物及其应用
CN113234612A (zh) * 2021-02-05 2021-08-10 重庆第二师范学院 对结肠炎具有预防效果的Lactobacillus fermentum ZS40
CN113403212A (zh) * 2021-06-17 2021-09-17 大连医科大学 一种肠道真菌Candida metapsilosis M2006B及其应用

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CN86103736A (zh) * 1985-05-29 1987-05-20 海布雷德国际先锋公司 用于治疗动物胃肠疾病的细菌组合物的制备方法
WO2003013558A1 (fr) * 2001-07-30 2003-02-20 Claudio De Simone Traitement de diarrhees provoquees par des rayonnements, a l'aide d'une composition probiotique
WO2003022255A2 (fr) * 2001-09-05 2003-03-20 Vsl Pharmaceuticals, Inc. Utilisation de cytosine-guanine (cpg) non methylee
CN1701116A (zh) * 2002-09-06 2005-11-23 Vri生物医学有限公司 益生细菌:发酵乳杆菌
CN1853508A (zh) * 2005-04-19 2006-11-01 韩国养药多股份有限公司 有效改善肝功能、降低血液酒精浓度及加强抗氧化能力的组合物

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Publication number Priority date Publication date Assignee Title
CN86103736A (zh) * 1985-05-29 1987-05-20 海布雷德国际先锋公司 用于治疗动物胃肠疾病的细菌组合物的制备方法
WO2003013558A1 (fr) * 2001-07-30 2003-02-20 Claudio De Simone Traitement de diarrhees provoquees par des rayonnements, a l'aide d'une composition probiotique
WO2003022255A2 (fr) * 2001-09-05 2003-03-20 Vsl Pharmaceuticals, Inc. Utilisation de cytosine-guanine (cpg) non methylee
CN1701116A (zh) * 2002-09-06 2005-11-23 Vri生物医学有限公司 益生细菌:发酵乳杆菌
CN1853508A (zh) * 2005-04-19 2006-11-01 韩国养药多股份有限公司 有效改善肝功能、降低血液酒精浓度及加强抗氧化能力的组合物

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011018080A3 (fr) * 2009-08-11 2011-04-21 Schrezenmeir Juergen Composition renfermant des souches de lactobacillus fermentum
US11667977B2 (en) 2009-08-11 2023-06-06 Jurgen Schrezenmeir Method of using a medication or a food supplement with lactobacillus strains isolated from kimere for strengthining the immune system
RU2738803C2 (ru) * 2015-08-25 2020-12-17 Иммьюнбиотек Медикал Свиден Аб Композиция и способ лечения и профилактики кишечной инфекции и воспаления
CN112218646A (zh) * 2018-05-31 2021-01-12 深圳华大生命科学研究院 一种组合物及其应用
CN112218646B (zh) * 2018-05-31 2024-06-11 深圳华大生命科学研究院 一种组合物及其应用
CN113234612A (zh) * 2021-02-05 2021-08-10 重庆第二师范学院 对结肠炎具有预防效果的Lactobacillus fermentum ZS40
CN113403212A (zh) * 2021-06-17 2021-09-17 大连医科大学 一种肠道真菌Candida metapsilosis M2006B及其应用
CN113403212B (zh) * 2021-06-17 2022-05-10 大连医科大学 一种肠道真菌Candida metapsilosis M2006B及其应用

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