WO2008064521A1

WO2008064521A1 - Lactobacillus fermentum cms-h002 and use thereof

Info

Publication number: WO2008064521A1
Application number: PCT/CN2006/003224
Authority: WO
Inventors: Fanggen Lu; Gang Lin
Original assignee: Pharmapep Research & Development (Shenzhen) Co., Ltd.
Priority date: 2006-11-30
Filing date: 2006-11-30
Publication date: 2008-06-05

Abstract

A strain of Lactobacillus fermentum CMS-H002, with the accession number CCTCC No. M 206110. The use of the strain, the pharmic composition, food product and additive comprising the strain. The strain of Lactobacillus fermentum CMS-H002 in the present invention could be used to treat the rectal or colonic inflammatory conditions, such as ulserative colitis effectively.

Description

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.

Background technique

Ulcerative colitis (UC) 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 is quite common in Western countries, with a prevalence of 35 to 100/105. The incidence rate in China is lower than that in Europe and the United States, and mild cases are more common. However, the incidence of this disease has increased in the past 20 years, and domestic reports on UC have also increased significantly.

For the treatment of this disease, in recent years, comprehensive medical treatment has been mainly used to control acute attacks, reduce recurrence, and prevent complications. 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. In addition, sulfasalazine (SASP) is also often used as the drug of choice, and it is suitable for patients with mild or severe adrenal glucocorticoid therapy. Although these drugs have desirable therapeutic effects, they have large side effects and are expected to develop safer alternative treatments.

Recent studies have shown that dysbacteriosis is one of the important causes of ulcerative colitis (UC). The role of probiotics in the treatment of UC has been paid more and more attention because of its superior safety and small side effects. However, the reported efficacy of probiotics in the treatment of UC has not yet reached the desired state. Choosing the best strain becomes the key to probiotics in the treatment of UC. .

Summary of the invention

SUMMARY OF THE INVENTION 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.

To achieve the above object, 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 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.

Preferably, 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. This strain is safe and effective in the treatment of rectal or colonic inflammatory diseases such as ulcerative colitis.

Deposit information:

Name of the strain: Lactobacillus fermentum Lactobacillusfermentum) CMS-H002

Date of deposit: 2006 10 years 23 days

Depository: China Center for Type Culture Collection (CCTCC)

Deposit number: CCTCC No. M 206110

DRAWINGS

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;

g is for treating three groups of colonic mucosa; h is for treating four groups of colonic mucosa;

i is the treatment of five groups of colonic mucosa; j is the high dose of 0501 enema group colonic mucosa;

k is the high dose of cl20 enema group colonic mucosa; 1 for the treatment of six groups of colonic mucosa.

detailed description

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.

The new strain of the present invention, 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). 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. Furthermore, 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.

The invention is further described in detail below by way of specific examples.

Example 1

Isolation, Identification and Biological Characteristics of Lactobacillus fermentum CMS-H002

1, material

Reagents: Beef powder (Beijing Aoboxing Biological Co., Ltd., batch number: 20040607), peptone (Beijing Aoboxing Biological Co., Ltd., batch number: ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Magnesium sulfate. (Hebei Chemical Industry No. 3 Plant, Jiaozuo City, Henan Province), Manganese Sulfate (Henan Jiaozuo Chemical Plant, Lot No.: 981014), Yeast Dipping Powder (Beijing Aoboxing Biotechnology Co., Ltd., Batch No.: 20030825), Ammonium Citrate (Hunan Xiangzhong Fine Chemicals Factory, batch number: 20030409), glucose (Hunan Normal University Chemical Reagent Factory, batch number: 20040208), Tween-80 (Xi'an Fuli Chemical Factory, batch number: 021125), dipotassium hydrogen phosphate (Jiaozuo City, Henan Province) Chemical Industry No.3, batch number: 20010404), glacial acetic acid (Xifu Chemical Plant, Shantou City, Guangdong Province, batch number: 050414) Agar powder (Beijing Aoboxing Biological Co., Ltd., batch number: 040415), X-gal (inalc), API 20A biochemical Identification strip (French enzyme Lie), API 50 CHL biochemical identification strip (French enzyme Lie)

Experimental equipment: Anaerobic incubator (Mérieux, France), Shimadzu GC2010 gas chromatograph (GC), hydrogen flame ion detector (FID), DB-5MS column, Shimadzu UV-2501PC, JSM5600- LV type scanning electron microscope, Hitachi H-600 transmission electron microscope,

Preparation of MRS-X-gal medium: '

MRS medium formula: 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. After the MRS medium was formulated according to the formula, 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.

2. Isolation and purification of Lactobacillus fermentum CMS-H002:

Use a sterile cotton swab to pick up a stool sample from healthy infants or children, or take duodenal juice in the gastroscope room, set 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. .

3. Colony characteristics:

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.

4, the shape under the microscope:

Lactobacillus fermentum CMS-H002 bacterial smear: Gram-positive bacilli, mostly short rod-shaped bacteria, single or in pairs, see Figure 1.

5, oxygen test

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.

6. Biochemical identification of Lactobacillus fermentum CMS-H002

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.

Operation according to API 20A and API 50 CHL anaerobic identification card instructions: The bacterial suspension of Lactobacillus fermentum CMS-H002 was inoculated into the reaction well of the identification card. Micro-aerobic culture at 35 ° C for 48 h, according to the color reaction (purple yellow) judgment results, coding, check the code search or computer software. The results of API20A biochemical identification showed that: Lactobacillus fermentum CMS-H002 can ferment glucose, maltose, lactose, sucrose, xylose, mannose, raffinose, code: 6504202, 81.5% in line with Lactobacillus fermentum. The results are shown in Table 1.

Table 1: Lactobacillus fermentum CMS- HG02 API2GA biochemical identification results

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.

7. Color reaction of MRS-X-gal medium

The 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.

The results showed that the colonies were light blue on the MRS-X-gal plate.

8. Electron microscopy of Lactobacillus fermentum CMS-H002 Scanning electron microscope:

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. ,

Transmission electron microscopy: '

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.

9, plasmid detection

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.

10, genetic characteristics

Collect the bacterial sludge of Lactobacillus fermentum CMS-H002 and extract the DNA according to the conventional method. PCR-amplify the lactic acid bacteria 16s rDNA with the specific primers of Lactobacillus, and detect the PCR product by 1.0% agarose gel electrophoresis. The band is the same size as the target segment.

11. Determination of metabolites

(1) Preparation of standard solution:

Preparation of alcohol and volatile fatty acid (VFA) standards: formic acid 37μ1, acetic acid 57μ1, propionic acid 74μ, isobutyric acid 93μ1, butyric acid 92μ1, isovaleric acid 109μ1, valeric acid 108μ1, hexanoic acid 125μ, ethanol 100μ1, propanol 5μ1, isobutanol 5μ1, butanol 10μ1, isoamyl alcohol 0.5μ1, pentanol 0.5μ1, and distilled water to 100ml, which is a standard solution of 10mmol/L.

Preparation of non-volatile fatty acid (NVFA) standard solution: Add lactic acid 84μ1, phenylacetic acid 60m _g , and succinic acid 60mg to distilled water to 100 ml, which is 10mmol/L standard solution.

(2) Preparation of VFA: Selective partial phosphoric acid method

Take 1 ml of standard solution or culture with 0.5 ml of metaphosphoric acid, adjust the pH to below 2.0, homogenize at 37 °C for 2 h, 10000 rpm, centrifuge at 4 °C for 2 min, and take supernatant Ιμΐ for injection analysis.

(3) Preparation of VFA: Methanol sulfuric acid method

Take 2ml of standard solution or culture and add 0.2 ml of 50% sulfuric acid, adjust the pH value 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, centrifuge at 4 ° C 2 min, chloroform layer l / l was taken for injection analysis.

(4) Analysis of each substance in specimens and standards:

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.

The results showed that the 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. Example 2

Therapeutic effect of Lactobacillus fermentum CMS-H002 enema on ulcerative colitis in mice

For the 5% DSS-induced Balb/c mouse colitis colitis model, the retention enema of normal saline, SASP, Lactobacillus fermentum CMS-H002, Bifidobacterium 0501, cl20, etc. were observed, and the body weight of each group of mice was observed. Death, stool traits, blood in the stool, length of the colon, DAI scores, and pathological changes in the intestinal mucosa. The results showed that Lactobacillus fermentum CMS-H002 can alleviate symptoms such as diarrhea, hemorrhage and weight loss in mice, and can also reduce intestinal mucosal damage and inflammatory cell infiltration. The effects are better than the model group, SASP group and double. Each group of the bacterium. It can effectively treat ulcerative colitis in mice.

First, experimental materials and methods:

1. Experimental materials:

1. 1 Experimental animals: 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.

1. 2 main reagents: sodium acetate (Taishan Chemical Plant, Guangdong Province, batch number 20030401), magnesium sulfate (Hebei Province Jiaozuo City Chemical Plant No. 3), manganese sulfate (Henan Jiaozuo Chemical Plant, batch number 981014), peptone (Beijing Aobo Star) Biotechnology Co., Ltd., Lot No. 20040615), Yeast Dipping Powder (Beijing Aoboxing Biotechnology Co., Ltd., Lot No. 20030825), Ammonium Citrate (Hunan Xiangzhong Fine Chemicals Factory, Lot No. 20030409), Glucose (Hunan Normal University Chemical Reagent) Factory, batch number 20040208), beef powder (Beijing Aoboxing Biotechnology Co., Ltd., 20040607), Tween-80 (Xi'an Fuli Chemical Factory, batch No. 021125), potassium dihydrogen phosphate (Hebei Province Jiaozuo City Chemical No. 3 Plant, batch number 20010404 ), agar (Beijing Aoboxing Biotechnology Co., Ltd.), sulfasalazine tablets (SASP, Shanghai Fuda Pharmaceutical Co., Ltd., batch number 041207), DSS (molecular weight 5000, Sigma). 2. Experimental method:

2.1 Experimental bacteria: Lactobacillus fermentum CMS-H002 and Bifidobacteria (0501 strain and cl20 strain), of which Bifidobacterium (0501 strain and cl20 strain) are isolated from healthy infantile feces and adult duodenal juice in our laboratory. , identification of the proceeds. The three strains were collected in the MRS medium for 24 hours after anaerobic culture, and the colonies were collected by spectrophotometer and diluted with sterile physiological saline to lxl0 ⁹ CFU/ml, l xlO ⁷ CFU/ml^ ΙχΙΟ ⁵ CFU/ml. After the backup.

2.2 Animal model: Balb/c 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.

2.3 Experimental grouping: 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

B, model control group: drinking DSS modeling

C, negative control group: only use sterile saline enema

D, positive control group: enema with SASP (20mg/ml)

E, treatment group: DSS modeling + l xl0 ⁵ CFU / ml 0501 bacterial liquid enema

F, treatment two groups: DSS modeling + lxl0 ⁷ CFU / ml 0501 bacterial liquid enema

G, treatment three groups: DSS modeling + lxl0 ⁹ CFU / ml 0501 bacterial liquid enema

H, treatment of four groups: DSS modeling + lx l0 ⁹ CFU / ml cl20 bacterial enema

I, treatment of five groups: DSS modeling + lx l0 ⁷ CFU / ml 0501 bacterial solution

J, high dose 0501 group: l xl0 ⁹ CFU/ml 0501 bacterial liquid enema

K, high dose cl20 group: l xl0 ⁹ CFU/ml cl20 bacterial enema

L, treatment of six groups: DSS modeling + lxl0 ⁹ CFU / ml CMS-H002 bacterial enema

2.4 Animal treatment: Observe daily general conditions such as eating and activities, weigh the body weight, observe fecal traits and fecal occult blood, and assess the severity of colitis. On the 9th day after the experiment, the mice were sacrificed, the free colon and the distal ileum were removed, and the entire colon and the rectum were removed from the anus to the end of the cecum. The gross changes in the colon of each group were observed, and the length of the entire colon was measured. The colon was rinsed with pre-cooled saline, and a lcm long colon was cut at the end of the colon (lcm from the anus), the middle part of the colon, and the upper part of the colon (1 cm from the cecum). Formalin was fixed, paraffin-embedded, and sliced. HE staining was performed to observe pathological changes. 2.5 Assessment of the degree of injury and inflammation

2.5.1 Assessment of disease activity: Daily observation of mouse body weight, stool traits and occult blood conditions were scored according to Table 3, and DAI scores for each mouse were obtained.

Table 3 DAI score sheet

Fecal occult blood / gross blood

Weight loss (%) stool traits ^: scoring

0 normal normal 0

1 -5 1

5- 10 Loose occult blood positive 2

10- 15 3

>15 loose stools, bloody stools 4

* Normal stool: forming stool; loose stool: paste-like, semi-formed stool that does not stick to the anus; loose stool: a thin watery stool that can adhere to the anus

2.5.2 Assessment of histological damage: Randomly select at least 27 high power fields per slice (400

I P ) Score according to the scoring standard of Table 4, and take the average value.

Table 4 Histological damage scoring criteria

Rating, organizational change

0 normal colonic mucosa

1 crypt defect 1/3

1. 5 crypt defect 1/2

2 crypt defect 2/3

3 The crypt is completely destroyed, and the lamina propria covers a single layer of epithelium with mild inflammatory cells.

4 mucosal erosion ulcer with significant inflammatory cell infiltration

Second, the results

1, weight change

There was no significant difference in body weight between the groups before the experiment. After the experiment, the model group, the negative control group, the positive control group, the treatment group 1, 2, 3, 4, and 5 groups all showed significant weight loss, and the body weight was poor (after the experiment and before the experiment). There was a significant difference between the difference between the body weight and the normal control group (P<0.05). There was no significant difference in body weight between the six groups (P>0.05). See Figure 4.

2. Mouse death A total of 15 mice died during the experiment, of which 10 died of lower gastrointestinal bleeding, 1 chronic intestinal perforation, and 4 died of unknown causes. A total of 11 patients died of gastrointestinal bleeding and perforation, including 1 in the model group, 1 in the negative control group, 1 in the positive control group, 3 in the treatment group, 2 in the treatment group, and 3 in the treatment group. There was no statistical difference in mortality between the groups of mice that died of gastrointestinal bleeding and perforation by ² tests, as shown in Figure 5.

3. Fecal traits and blood in the stool

In the model group and the negative control group, some 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. Positive, after 5 days of drinking DSS, there was a thin paste-like bloody stool; after treatment for 6 days of DSS, 2 mice showed positive fecal occult blood, but no gross bloody stools appeared; no change in the normal control group. See Table 5 for the blood in the mice of each group at 7 days after drinking DSS.

Bloodletting of mice in each group at the end of the experiment

Simple appearance

Group Other IJ Cases Number of deaths

Number of blood positive cases

Normal control group (A) 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

Treatment six groups (L) 10 2 0 0

4, mouse colon length changes

After the experiment, the DSS model group, the negative control group, the positive control group, the treatment one, two, 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. However, 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. There were significant differences between the four groups and the fifth group, compared with the model group, the negative control group, and the positive control group, as shown in Figure 6.

5, DAI score

The DAI scores of the six groups were significantly lower than those of the model group and the positive control group, and the difference was significant (P < 0.05), as shown in Figure 7.

6, histopathology score:

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. The above results indicate that 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. By using 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. However, when two strains of Bifidobacterium were enema in mice not induced by DSS, the general condition of the mice and the pathological examination of the intestinal mucosa did not differ from the normal control group, so some strains of Bifidobacterium or Bifidobacterium may be in UC. Colonic mucosal damage of UC was aggravated during the onset of the disease.

Claims

Rights request

1. A Lactobacillus fermentum CMS-H002 having a deposit number of CCTCC No. M 206110.

The use of the Lactobacillus fermentum CMS-H002 according to claim 1 for the preparation of a medicament for the treatment of a rectal or colonic inflammatory disease.

3. Use according to claim 2, characterized in that the rectal or colonic inflammatory disease is ulcerative colitis.

4. A pharmaceutical composition comprising a pharmaceutically effective amount of Lactobacillus fermentum 'CMS-H002, deposited under the accession number CCTCC No. M 206110, or a metabolite thereof, cell debris Or secretions.

A food product comprising: the food product comprising Lactobacillus fermentum CMS-H002 having a deposit number of CCTCC No. M 206110, or a metabolite thereof, a cell debris or a secretion.

6. The food according to claim 5, wherein: the food is a beverage.

A health care product, comprising: the health care product comprising Lactobacillus fermentum CMS-H002 having a deposit number of CCTCC No. M 206110, or a metabolite thereof, a cell debris or a secretion.

A food additive characterized in that the food additive comprises Lactobacillus fermentum LactobaciUus fermentum CMS-H002 having a deposit number of CCTCC No. M 206110, or a metabolite thereof, cell debris or secretion.