CN114107121B - Bacillus coagulans and application thereof in treatment of alcoholic liver disease - Google Patents

Abstract

The invention discloses bacillus coagulans and application thereof in treatment of alcoholic liver diseases, and belongs to the field of microorganisms. The invention provides application of bacillus coagulans FCYS01 in preparation of a medicine for preventing and/or treating alcoholic liver diseases, wherein the bacillus coagulans FCYS01 is preserved in China center for type culture collection, the preservation number is CCTCC NO: M20211273, the preservation address is university of Wuhan, china, the preservation date is 2021, 10 and 14 days.

Description

Bacillus coagulans and application thereof in treatment of alcoholic liver disease

Technical Field

The invention relates to the field of microorganisms, in particular to bacillus coagulans and application thereof in treatment of alcoholic liver diseases.

Background

Chronic alcohol abuse is a major cause of liver-related illnesses leading to death. The world health organization estimates that there are about 300 million deaths from alcoholic disease in 2016, accounting for 5.1% of global disease, in the world's reports of alcohol and health status. Continued drinking can lead to a variety of chronic Alcoholic Liver Diseases (ALD), including Alcoholic Steatohepatitis (ASH), alcoholic Fatty Liver (AFL), alcoholic Fibrosis (AF), alcoholic Hepatitis (AH), alcoholic Cirrhosis (AC), and hepatocellular carcinoma (HCC). ALD can gradually develop into ASH and eventually HCC, leading to death. The current treatment of ALD is mainly focused on phosphodiesterase inhibitors, glucocorticoids, polyenylphosphatidylcholine, etc. However, long-term use of these drugs often results in host drug resistance and increased risk of infection. To date, no drugs or therapeutic methods have been approved for the treatment of ALD patients. Therefore, there is an urgent need to develop more effective and safer methods of treating or preventing ALD.

A number of animal experiments and clinical trials have shown that certain probiotics have been reported to be useful in the treatment of alcoholic liver disease. Probiotics modulate gut microbiota, remediating alcohol-related gut barrier dysfunction by reducing gut mucosal permeability and preventing gut bacterial translocation. The method for treating alcoholic liver diseases by using probiotics is undoubtedly a new idea which is simple, effective and free of side effects.

Bacillus coagulans (Bacillus coagulans) is a gram-positive bacterium, is facultative anaerobic, non-pathogenic, can form spores and produce lactic acid, and has the industrial characteristics of high temperature resistance, acid resistance, bile resistance and the like. Has been widely applied to the industries of medicine, food, chemical industry and the like. Research reports that bacillus coagulans can be used for treating intestinal diseases such as acute diarrhea, irritable bowel syndrome, antibiotic-associated diarrhea, constipation, colitis and the like by regulating the composition of microbiota, host immunity and metabolism. However, no reports on the treatment of alcoholic liver disease by bacillus coagulans have been found so far.

Disclosure of Invention

The invention aims to provide bacillus coagulans and application thereof in treatment of alcoholic liver diseases, so as to solve the problems in the prior art, the bacillus coagulans can prevent and treat alcoholic liver diseases induced by alcohol, and has potential application prospects in the field of prevention and treatment of alcoholic liver diseases.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides Bacillus coagulans (Bacillus coagulans) FCYS01 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20211273, the preservation address of university of Wuhan, china and the preservation date of 2021, 10 and 14 days.

The invention also provides application of the bacillus coagulans FCYS01 in preparation of a medicine for preventing and/or treating alcoholic liver disease.

Further, the active ingredient of the medicament is the bacillus coagulans FCYS01.

Further, the medicine can prevent and/or treat the alcoholic liver disease through one or more of the following ways:

(1) Reducing the content of glutamic-pyruvic transaminase in serum of alcoholic liver patients;

(2) Reducing the content of glutamic-oxaloacetic transaminase in serum of alcoholic liver patients;

(3) Reducing the content of myeloperoxidase in the serum of patients with alcoholic liver;

(4) Reducing the content of lipopolysaccharide in serum of alcoholic liver patients;

(5) Reducing the weight of the liver of the alcoholic liver patient;

(6) Reducing hepatomegaly of patients with alcoholic liver;

(7) Reducing the content of inflammatory factor interleukin-1 beta in the liver of the alcoholic liver patient;

(8) Increasing the content of anti-inflammatory factor interleukin-10 in the liver of the alcoholic liver patient;

(9) Reducing the content of inflammatory factor tumor necrosis factor alpha in the liver of the alcoholic liver patient;

(10) Reducing the content of inflammatory factor interleukin-22 in the liver of the alcoholic liver patient;

(11) Reducing the content of triglyceride in the liver of the alcoholic liver patient;

(12) Reducing the vacuole content of liver tissues of patients with alcoholic liver;

(13) Reducing the cell infiltration content of liver tissues of alcoholic liver patients;

(14) Reducing the liver fat content of alcoholic liver patients;

(15) Recovering intestinal barrier of alcoholic liver patients;

(16) Increasing the content of connexin Occludin protein in colon epithelial cells of alcoholic liver patients;

(17) Increasing the abundance of beneficial intestinal bacteria of alcoholic liver patients;

(18) The abundance of harmful bacteria in the intestinal tract of the alcoholic liver patient is reduced;

(19) The abundance and diversity of intestinal microflora of alcoholic liver patients are improved.

Further, in the drug, the number of Bacillus coagulans FCYS01 is 1.0 × 10 ⁹ CFU。

Further, the medicine also comprises other medicines compatible with the bacillus coagulans FCYS01 and pharmaceutically acceptable carriers and/or auxiliary materials.

Furthermore, the other medicines comprise medicines which are compatible with the bacillus coagulans FCYS01, do not cause hydrolysis and damage failure physicochemical reaction of the bacillus coagulans FCYS01, and can improve the effect of the bacillus coagulans FCYS01 on treating the alcoholic liver disease after synergistic action with the bacillus coagulans FCYS01.

Further, the medicament is a pharmaceutically acceptable dosage form.

Further, the dosage form is powder, injection, capsule, tablet or oral liquid.

The invention discloses the following technical effects:

the bacillus coagulans is obtained by separating and screening from a healthy cow body, and is a new probiotic strain with potential application prospect in the field of alcoholic liver disease prevention and treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the results of liver-to-body ratio measurements in mice in the blank group, model group and FCYS01 group in example 2 (.;. Times.P < 0.01;. Times.P < 0.001);

FIG. 2 shows the results of staining of HE and O sections of liver tissues of mice of the blank group, model group and FCYS01 group in example 2;

FIG. 3 shows the results of expressing colon Occludin protein in mice of blank group, model group and FCYS01 group in example 2;

FIG. 4 shows the results of the measurement of the abundance of microorganisms in the intestinal tracts of mice in the blank group, the model group and the FCYS01 group in example 2, wherein A is a Chao1 index in the alpha diversity of the microorganisms in the intestinal tracts, B is a shannon index in the alpha diversity of the microorganisms in the intestinal tracts, and C is a differential result of the classification of the microorganisms.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Unless otherwise specified, all PBS buffers in the examples were PBS buffer at pH 7.0 and 0.1M.

Solid YPD medium: contains yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 15g/L, bromocresol purple sodium salt 0.1g/L, and water in balance. Liquid YPD medium differs from solid YPD medium only in that no agar and bromocresol purple sodium salt were added.

Male SPF grade C57 mice 7-8 weeks old, body mass (20 ± 2) g: animal center, university of agriculture in china, animal license number: HZAUMO-2021-0010.

Lieber-DeCarli basic control liquid feed TP4030C (about 600mL warm water at 40 ℃,218.80g main material, 0.53g choline, 2.50g vitamin are stirred to uniform liquid, the constant volume is 1000mL, and the mixture is mixed) and Lieber-DeCarli alcohol liquid feed TP4030D (about 600mL warm water at 40 ℃,148g main material, 0.53g choline, 2.50g vitamin are stirred to uniform liquid, 50mL absolute ethyl alcohol is added to about 200mL warm water at 40 ℃, the constant volume is 1000mL, the mixture is mixed), the concentration of the alcohol solution for gastric perfusion is 31.5% (v/v), and the concentration of the dextrin solution for gastric perfusion is 45% (w/v): nantong Telofu feed science and technology, inc.

The content of Alanine aminotransferase (ALT/GPT) in serum is detected by Nanjing-built Alanine aminotransferase kit (C009-2-1). The content of Aspartate aminotransferase (AST/GPT) in serum is detected by Nanjing built Aspartate aminotransferase kit (C009-2-1). The Lipopolysaccharide (LPS) content in serum is detected by using Jiangsu enzyme immune mouse Lipopolysaccharide enzyme linked immunosorbent assay kit (MM-0634M 1). The content of total Triglyceride (TG) in liver is detected by Nanjing-constructed total triglyceride kit (A110-1). The Interleukin-10 (Interleukin-10, IL-10) content in the liver was determined using the enzyme immunoassay mouse Interleukin-10 enzyme linked immunosorbent assay kit (MM-0176M 1) of Jiangsu. The Interleukin-1 beta (Interleukin-1 beta, IL-1 beta) content in the liver is detected by adopting a Jiangsu enzyme immune mouse Interleukin-1 beta enzyme linked immunosorbent assay kit (MM-0040M 1). The Interleukin-22 (Interleukin-22, IL-22) content in the liver was determined using Jiangsu enzyme-immunized mouse Interleukin-22 enzyme linked immunosorbent assay kit (MM-0892M 1). The content of tumor necrosis factor alpha (TNF-alpha) in the liver is detected by adopting a Jiangsu enzyme immune mouse tumor necrosis factor alpha enzyme linked immunosorbent assay kit (MM-0132M 1). The content of mouse Myeloperoxidase (MPO) in the liver is detected by adopting a Jiangsu enzyme immune mouse Myeloperoxidase enzyme linked immunosorbent assay kit (MM-0338M 1).

An Anti-Occludin antibody (Occludin antibody), an internal reference Anti-beta Actin (beta-Actin) and a secondary antibody Goat Anti-Rabbit IgG (HRP) are Abcam company in England.

EXAMPLE 1 isolation, identification and preservation of the Strain

1. Isolation of the Strain

The method comprises the steps of taking samples from a cattle farm in Xinkamura village of Mirabo, deyang, sichuan province, and taking the samples as fresh cattle manure.

And repeatedly carrying out streak culture on the sample by a solid YPD medium plate, selecting a single colony which generates a yellow circle, inoculating the single colony on the solid YPD medium plate, continuously culturing, and repeatedly carrying out streak culture and purification by the solid YPD medium plate to obtain a plurality of pure culture strains.

Selecting gram-positive strain from pure cultured strains, inoculating to liquid YPD medium, adding 20% glycerol, and storing at-80 deg.C in refrigerator.

2. Identification of strains

And performing morphological identification, physiological and biochemical identification and molecular identification on each separated strain, wherein the strain FCYS01 belongs to Bacillus coagulans (Bacillus coagulans).

The morphological identification and physiological and biochemical identification results of the strain FCYS01 are as follows: gram-positive, spore-forming, short rod-shaped thallus, two ends of which are blunt and round, and grow in a single, paired or chain-shaped arrangement; spores are resistant to high temperature; the hydrolyzed starch has cellulase activity and no protease activity, and can produce acid by fermenting glucose, galactose, xylose, fructose, sorbitol, arabinose and maltose.

The strain FCYS01 grows in a uniform turbid state in a liquid YPD culture medium, and the thallus is white and precipitated after being placed for a long time.

The optimum growth temperature of the strain FCYS01 is 37-45 ℃, and the suitable pH is 6.6-7.0.

The 16S rDNA (SEQ ID NO. 1) sequence of strain FCYS01 is shown below:

GCGATGCGCGTGGCTAATACTGCAGGTTCGTTGCGGACCTTTTAAAGCTTGCTTTTAAAAGGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGATCGGGATAACGCCGGGAAACCGGGGCTAATACCGGATAGTTTTTTCCTCCGCATGGAGGAAAAAGGAAAGACGGCTTCTGCTGTCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCGGGTCGTAAAACTCTGTTGCCGGGGAAGAACAAGTGCCGTTCGAACAGGGCGGCGCCTTGACGGTACCCGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGCGGCTCAACCGCAAGCGGTCATTGGAAACTGGGAGGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTGGAGACAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCAGAAAACCATTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGGTAACCTTTACGAACCACCGCCGAAGGACAAGT。

3. preservation of the Strain

Bacillus coagulans FCYS01 (Bacillus coagulans) FCYS01 is preserved in China center for type culture Collection (CCTCC for short, the address is university of China, wuhan and Wuhan) at 10 months and 14 days in 2021, and the preservation number is CCTCC NO: M20211273.

Example 2 therapeutic Effect of Bacillus coagulans FCYS01 on Alcoholic liver disease mice

1. Preparation of the bacterial suspension

The preparation method of the bacterial suspension comprises the following steps: test bacteria were suspended in PBS buffer to a concentration of 1.0X 10 ⁹ CFU/200μL。

The test bacteria are bacillus coagulans FCYS01, and the obtained bacterial suspension is named as FCYS01 bacterial suspension.

2. Packet processing method

45 male C57 mice, randomly divided into 3 groups of 15, were treated as follows:

blank group: feeding the feed with Lieber-Decalli basic control liquid feed from the beginning to the end of the test; from day 8 to day 15 of the experiment, sterile PBS buffer was administered (administration mode: gavage, administration amount: 200 ul/each/day); on day 16, 45% (w/v) dextrin solution was administered (administration manner: gavage, administration amount: volume of gavage solution (μ l) = body weight (g) × 20 per mouse);

model group: feeding with Lieber-Decalci basic control liquid feed from the beginning to the 4 th day of the test, and feeding with Lieber-Decalci alcohol liquid feed from the 5 th day to the end; from day 8 to day 15 of the experiment, sterile PBS buffer was administered (administration mode: gavage, administration amount: 200 ul/each/day); on day 16, 31.5% (v/v) alcohol solution was administered (administration manner: gavage, administration amount: volume of gavage solution per mouse (μ l) = body weight (g) × 20);

bacillus coagulans FCYS01 group (FCYS 01 group for short): feeding with Lieber-Decali basic control liquid feed from the beginning to 4 days, and feeding with Lieber-Decali alcohol liquid feed from 5 days to the end of the experiment; FCYS01 bacterial suspension was administered from day 8 to day 15 of the experiment (administration manner: gavage, administration amount: 200 ul/each/day); on day 16, 31.5% (v/v) alcohol solution was administered (administration manner: gavage, administration amount: volume of gavage solution per mouse (μ l) = body weight (g) × 20);

the test started and ended on days 1 to 16 of the test.

3. Serum-related index detection

On the 16 th morning of the experiment, 8 points of the experiment are filled with 31.5 percent (v/v) alcohol solution in the FCYS01 group and the model group, 45 percent (w/v) dextrin solution in the contrast group, blood is taken from the orbit after 9 hours of filling, the blood is centrifuged for 15min at the temperature of 4 ℃ and 3000r/min, and serum is collected.

And (3) detecting the concentrations of glutamic-pyruvic transaminase (ALT/GPT), glutamic-oxalacetic transaminase (AST/GPT) and Lipopolysaccharide (LPS) in serum.

The results of detection of alanine aminotransferase (ALT/GPT), aspartate aminotransferase (AST/GPT), and Lipopolysaccharide (LPS) are shown in Table 1.

TABLE 1 ALT, AST, LPS concentration test results in serum

Group of	ALT(U/g)	AST(U/g)	LPS(ng/g)
				Blank group	0.11±0.04	0.21±0.04	1.64±0.40
Model set	0.45±0.09****	0.34±0.06**	2.41±0.54***
				FCYS01 group	0.28±0.07###	0.22±0.08#	1.68±0.43###

Note: # or, P <0.05; # # or #, P <0.01; # # or #, P <0.001; # # # # # or #, P <0.0001.

Compared with a blank group, the levels of ALT, AST and LPS in the serum of the mouse in the model group are obviously increased, and compared with the model group, the levels of ALT, AST and LPS in the serum of the mouse in the FCYS01 group are obviously reduced, which shows that the bacillus coagulans FCYS01 can obviously improve the levels of ALT, AST and LPS in the serum of the model mouse with alcoholic liver diseases and prevent liver diseases.

4. Liver-related index detection

After serum was taken on day 16 of the experiment, the mice were sacrificed and dissected and livers were collected. The whole liver was weighed and the liver to body ratio of the mice was calculated by recording.

Detecting the content of total Triglyceride (TG), interleukin 1 beta (IL-1 beta), interleukin 10 (IL-10), interleukin 22 (IL-22), tumor necrosis factor alpha (TNF-alpha) and Myeloperoxidase (MPO) in the liver.

The results of the liver-to-body ratio measurements in mice are shown in FIG. 1.Ctrl is blank, etOH is model, FCYS01 is FCYS01. Compared with a blank group, the liver body ratios of the mice in the model group are obviously increased, and compared with the model group, the liver body ratios of the mice in the FCYS01 group are obviously reduced, which shows that the bacillus coagulans FCYS01 can obviously improve the hepatomegaly of the mice in the alcoholic liver disease model and prevent the occurrence of liver diseases.

The results of detection of total Triglycerides (TG), interleukin 1 beta (IL-1 beta), interleukin 10 (IL-10), interleukin 22 (IL-22), tumor necrosis factor alpha (TNF-alpha), myeloperoxidase (MPO) are shown in Table 2.

TABLE 2 results of assay of TG, IL-1 beta, IL-10, IL-22, TNF-alpha, MPO content in liver

Compared with the blank group, the total Triglyceride (TG), interleukin 1 beta (IL-1 beta), interleukin 22 (IL-22), tumor necrosis factor alpha (TNF-alpha) and Myeloperoxidase (MPO) of the model group of mice are all obviously increased, and compared with the model group, the liver of the FCYS01 group of mice is obviously reduced than the total Triglyceride (TG), the interleukin 1 beta (IL-1 beta), the interleukin 22 (IL-22), the tumor necrosis factor alpha (TNF-alpha) and the Myeloperoxidase (MPO). The interleukin 10 (IL-10) was significantly reduced in the model group mice compared to the blank group, and the interleukin 10 (IL-10) was significantly increased in the FCYS01 group mice compared to the model group. The bacillus coagulans FCYS01 is proved to have the inhibiting effect on inflammatory factors such as IL-1 beta, IL-22, TNF-alpha and the like in the pathological change of an alcoholic liver model, have the promoting effect on the anti-inflammatory factor IL-10, have the inhibiting effect on the cell factor TG and have the inhibiting effect on the cell infiltration factor myeloperoxidase, thereby inhibiting or slowing down the formation of alcoholic liver diseases.

5. Histological morphology observation

After serum was taken on day 16 of the experiment, mice were sacrificed and dissected and livers were collected. Liver sections were sectioned, stained with hematoxylin-eosin (HE) and Oil Red O (Oil Red O), and the liver lesions were observed by optical microscopy and photographed at 400 x photomicrographs.

The results of staining mouse liver tissue HE and oil red O sections are shown in FIG. 2.Ctrl is blank, etOH is model, FCYS01 is FCYS01. The blank mice have normal liver cell morphology, complete liver lobule structure, no liver cell degeneration, necrosis or inflammatory cell infiltration and no obvious lipid deposition; the liver cells of the model group mice are degenerated, the volume is increased, the cytoplasm is filled with balloon-like fat vacuoles, and significant lipid deposition occurs; FCYS01 group mice liver cell morphology tends to be normal, and compared with the model group, lipid deposition is obviously improved. The results of the HE and oil red O section staining indicate that the alcohol feed can cause liver lipid accumulation, hepatocyte degeneration, volume increase, fat vacuole of cytoplasm, inflammatory cell infiltration and finally alcoholic liver disease; the bacillus coagulans FCYS01 can reduce the formation of fat vacuoles, reduce the deposition of liver fat, relieve the degeneration of liver cells and finally relieve the process of the alcoholic liver disease of mice.

6. Western Blotting detection

After serum was taken on day 16 of the experiment, the mice were sacrificed and dissected to collect the colon, total protein was extracted according to the method of the BCA protein quantification kit instructions, and the expression of the colon Occludin protein was detected by Western Blot.

The result of Western Blot protein expression is shown in FIG. 3. Compared with a blank group, the expression level of Occludin in the colon of the mice in the model group is obviously reduced; compared with the model group, the expression level of Occludin in the colon of mice in the FCYS01 group is obviously increased and is consistent with that in the blank group.

The Western Blot detection result shows that: the alcoholic diet can down-regulate the expression of intestinal connexin Occludin; the bacillus coagulans FCYS01 can restore the down-regulation of connexin Occludin caused by alcohol diet, which indicates that the bacillus coagulans FCYS01 can possibly repair intestinal tract barriers, thereby preventing and treating liver injury caused by alcohol.

7. Detection of abundance of beneficial and harmful bacteria

After serum was taken on day 16 of the experiment, the mice were sacrificed and dissected, the cecum contents were collected, liquid nitrogen-frozen, DNA extracted and high throughput sequencing was performed by bio-information technology ltd, kinoform origin. The microbial diversity in the intestinal tract of each group of mice was examined by the area of the standard bacterium 1696 V3-V4.

The results of the 16S microbial diversity assay are shown in FIG. 4. The Chao1 index in the intestinal microbial alpha diversity of the model group was significantly reduced compared to the normal group; the Chao1 index and Shannon index in the intestinal microbial alpha diversity of FCYS01 group were significantly increased compared to the model group. Supplementation with b.coagulans FCYS01 was shown to enhance the abundance (fig. 4A) and diversity (fig. 4B) of the intestinal microflora. Compared with a blank group, the content of harmful bacteria Escherichia-Shigella in the caecum content of the mice in the model group is obviously increased, and the content of beneficial bacteria Akkermansia and Lachnospiraceae is obviously reduced; compared with the model group, the content of Escherichia-Shigella in the caecum content of mice in the FCYS01 group is remarkably reduced, the content of beneficial bacteria Akkermansia and Lachnospiraceae is remarkably increased, and the expression level of the beneficial bacteria is consistent with that of the blank group.

The results of the 16S microbial diversity test show that: the alcohol diet can adjust the content of harmful bacteria in intestinal tract up and adjust the content of beneficial bacteria down; the bacillus coagulans FCYS01 can improve the abundance and diversity of intestinal microflora, restore the up-regulation of harmful bacteria content caused by alcohol diet and the down-regulation of beneficial bacteria content, and indicate that the bacillus coagulans FCYS01 can prevent and treat liver injury caused by alcohol by restoring the intestinal microflora.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> university of agriculture in Huazhong

<120> Bacillus coagulans and application thereof in treatment of alcoholic liver disease

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1454

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcgatgcgcg tggctaatac tgcaggttcg ttgcggacct tttaaagctt gcttttaaaa 60

ggttagcggc ggacgggtga gtaacacgtg ggcaacctgc ctgtaagatc gggataacgc 120

cgggaaaccg gggctaatac cggatagttt tttcctccgc atggaggaaa aaggaaagac 180

ggcttctgct gtcacttaca gatgggcccg cggcgcatta gctagttggt ggggtaacgg 240

ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacat tgggactgag 300

acacggccca aactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg agtgaagaag gccttcgggt cgtaaaactc tgttgccggg 420

gaagaacaag tgccgttcga acagggcggc gccttgacgg tacccggcca gaaagccacg 480

gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540

gggcgtaaag cgcgcgcagg cggcttctta agtctgatgt gaaatcttgc ggctcaaccg 600

caagcggtca ttggaaactg ggaggcttga gtgcagaaga ggagagtgga attccacgtg 660

tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcggc tctctggtct 720

gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780

cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc gccctttagt gctgcagcta 840

acgcattaag cactccgcct ggggagtacg gccgcaaggc tgaaactcaa aggaattgac 900

gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960

caggtcttga catcctctga cctccctgga gacagggcct tccccttcgg gggacagagt 1020

gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac ccttgacctt agttgccagc attcagttgg gcactctaag gtgactgccg 1140

gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200

tacacacgtg ctacaatgga tggtacaaag ggctgcgaga ccgcgaggtt aagccaatcc 1260

cagaaaacca ttcccagttc ggattgcagg ctgcaacccg cctgcatgaa gccggaatcg 1320

ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380

cgtcacacca cgagagtttg taacacccga agtcggtgga ggtaaccttt acgaaccacc 1440

gccgaaggac aagt 1454

Claims (6)

1. A Bacillus coagulans (Bacillus coagulans) FCYS01 is preserved in China center for type culture Collection with preservation number of CCTCC NO: M20211273, preservation address of university of Wuhan, china, preservation date of 2021 year, 10 months and 14 days.

2. The use of the bacillus coagulans FCYS01 according to claim 1 in the preparation of a medicament for the prevention and/or treatment of alcoholic liver disease.

3. The use as claimed in claim 2, wherein the active ingredient of the medicament is the Bacillus coagulans FCYS01.

4. The use according to claim 3, wherein the number of Bacillus coagulans FCYS01 in the medicament is 1.0 x 10 ⁹ CFU。

5. The use of claim 2, wherein the medicament is in a pharmaceutically acceptable dosage form.

6. The use according to claim 5, wherein the dosage form is a powder, injection, capsule, tablet or oral liquid.

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