CN115433688A - Bacillus faecium and application thereof - Google Patents
Bacillus faecium and application thereof Download PDFInfo
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- CN115433688A CN115433688A CN202110624416.0A CN202110624416A CN115433688A CN 115433688 A CN115433688 A CN 115433688A CN 202110624416 A CN202110624416 A CN 202110624416A CN 115433688 A CN115433688 A CN 115433688A
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- enteritis
- elongatus
- cgmcc
- bacillus
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Abstract
The invention provides application of farmer dung bacillus (Faecalibacterium longum) in ulcerative enteritis, and also provides a composition for treating and preventing the ulcerative enteritis and related diseases thereof, wherein the composition comprises medicines, beverages, foods or animal feed compositions and the like, the farmer dung bacillus is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 6 months and 13 months in 2016, the preservation number is CGMCC 1.5208, and the classification names are as follows: faecalibacterium longissimus longum. The coprobacterium alhenicum has obvious treatment and prevention effects on ulcerative enteritis, and is particularly characterized in that the coprobacterium alhenicum has the effects of improving the apparent state of a mouse with the ulcerative enteritis, reducing the disease activity index of the mouse and reducing the inflammatory response of the mouse, and the effect is obviously superior to that of the existing commercial medicine for treating ulcerative enteritis.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to coprobacterium elongatum and application thereof, and more particularly relates to coprobacterium elongatum, application of coprobacterium elongatum in preventing and treating ulcerative enteritis, application of coprobacterium elongatum in preparing food, medicines, health-care products or compositions, medicines, food, coprobacterium elongatum food compositions, coprobacterium elongatum capsules and preparations.
Background
Ulcerative Colitis (UC) is one of Inflammatory Bowel Diseases (IBD), a chronic Inflammatory bowel disease with unknown pathogenesis. Clinical pathological studies suggest that UC is mainly associated with individual susceptibility genes and intestinal mucosal immune response induced by intestinal microorganisms, and symptoms of UC mainly include persistent abdominal pain, diarrhea and mucous bloody stool, and the disease is repeated with the onset of UC at any age stage.
At present, clinically, medicines for treating UC mainly comprise salicylic acids, glucocorticoid and immune preparations. Salicylic acid medicines can better inhibit prostaglandin synthesis and remove oxygen radicals so as to achieve the purpose of relieving inflammatory reaction, and common salicylic acid western medicines for clinically treating UC are mainly sulfasalazine (SASP) and mainly aim at patients with mild, moderate and chronic UC; glucocorticoids are the first choice for patients with severe or fulminant UC, such as betamethasone; immunosuppressive agents such as cyclosporin can inhibit UC by inhibiting the production of T-cell IL-2, affecting the progress of the immune response.
The existing three medicines aiming at the UC can relieve the UC to a certain extent, but have certain side effects, and the side effects of salicylic acid are rash, hepatotoxicity, leukopenia, anemia and the like caused by digestive tract reaction, headache, reticulocyte increase, spermatopenia, anaphylactic reaction and the like. Glucocorticoid can cause side effects such as metabolic disturbance of an organism, water retention and the like, and can only be used as an emergency medicine and cannot be taken for a long time. The immunosuppressant treatment has large drug dependence and long treatment period, is easy to cause renal toxicity and secondary infection, and can only be used as an auxiliary treatment means.
At present, there is a great need in the art for effective and less side-effect methods and medicaments for the treatment and prevention of UC.
Given the relationship of microorganisms to enteritis, microorganisms will continue to be the subject of enteritis concern and research to better understand their mechanism of action and potential role in treating and preventing enteritis.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventor of the application surprisingly discovers that the selected faecal bacteria (Faecalibacterium longum, which is preserved in the common microorganism center of China general microbiological culture Collection center at 6 and 13 months in 2016 under the accession number CGMCC No. 1 and the accession number CGMCC No. 1.5208 (in the application, the faecal bacteria strain is called as the faecal bacteria of the same species CM 04-06) in the south of the Yangtze district in Beijing of China, the effect of the faecal bacteria CM04-06 on the inhibition of the weight reduction, the increase of Disease Activity Index (DAI) and the colon shortening of UC mice induced by the faecal bacteria (DExts Sulfate Sodium, DSS) is obviously superior to that of the conventional commercial ulcerative enteritis drugs.
Therefore, in the first aspect of the invention, the invention provides the use of the coprobacterium elongatum (Faecalibacterium longum) in the aspect of treating and preventing enteritis, wherein the coprobacterium elongatum is preserved in the common microorganism center of the China general microbiological culture Collection management Committee in 2016, 6 and 13 days, the preservation address is No. 3 Hospital No. 1 of Western Lu of Shangyang district in Beijing City, china, the preservation number is CGMCC 1.5208, and the classification and the designation are as follows: faecalibacterium longissimus longum. The curative and preventive effects of the coprobacterium elongatum CM04-06 on the UC mice induced by DSS according to the embodiment of the invention on the symptoms of inhibiting the weight loss, the DAI increase and the colon shortening of the UC mice are obviously better than those of the existing commercial ulcerative enteritis medicaments.
In the second aspect of the invention, the invention provides the use of the coprobacterium elongatum (Faecalibacterium longum) in the preparation of drugs, health products, foods or compositions, wherein the coprobacterium elongatum is deposited in the common microorganism center of the China Committee for culture Collection of microorganisms at 2016, 6 months and 13 days, the deposition address is No. 3 of the West Lu No. 1 Hospital of the Korean district, beijing, china, the deposition number is CGMCC 1.5208, and the classification and the nomenclature is as follows: faecalibacterium longum for use in at least one of: treating and preventing enteritis, inhibiting weight loss and DAI increase induced by enteritis, and improving intestinal tract change.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to an embodiment of the invention, the enteritis is ulcerative enteritis.
According to an embodiment of the invention, the ulcerative colitis is ulcerative colitis.
According to an embodiment of the invention, the change in the intestine is a shortening of the colon tissue.
The coprobacterium elongatum CM04-06 provided by the embodiment of the invention has good effects of inhibiting enteritis-induced weight loss, disease Activity Index (DAI) increase and improving enteritis-induced intestinal tract changes, and the medicines, health care products, foods and compositions prepared by using the coprobacterium elongatum CM04-06 provided by the embodiment of the invention have obvious effects of treating and preventing ulcerative colitis.
According to an embodiment of the invention, of said coprobacterium elongatumThe concentration is 1.0 × 10 6 CFU/mL～1.0×10 9 CFU/mL. Further, the medicament, health product or food prepared by using the coprinus elongatus CM04-06 has better effects on treating, preventing or relieving ulcerative enteritis, inhibiting weight reduction induced by enteritis, increasing DAI and improving intestinal tract changes.
In a third aspect of the invention, a composition is provided. According to an embodiment of the invention, the composition comprises: the aforementioned faecal bacteria (Faecalibacterium longum) is deposited in the general microbiological center of the China Committee for Culture Collection of Microorganisms (CCM) at 2016, 6 and 13 days, with the deposition address of No. 3 Hospital No. 1 Xilu, north Chen Yang district, beijing, china, the deposition number of CGMCC 1.5208, and the classification name: a Faecalibacterium longissimus L. The composition comprising the coprobacterium elongatum CM04-06 according to the embodiments of the present invention is superior to existing ulcerative enteritis drugs in inhibiting enteritis-induced weight loss, DAI elevation, and improving intestinal changes.
In a fourth aspect of the invention, a medicament is presented. According to an embodiment of the invention, the medicament comprises: the bacillus alvarezii (Faecalibacterium longum) is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2016 (6 months and 13 days), the preservation address is No. 3 of the Xilu No. 1 of the Chaozhong district in Beijing of China, the preservation number is CGMCC 1.5208, and the classification names are as follows: a Faecalibacterium longissimus L. The medicament containing the coprobacterium elongatum CM04-06 according to the embodiment of the invention is superior to the existing ulcerative enteritis medicaments in inhibiting enteritis-induced weight loss, DAI increase and improving intestinal tract changes.
According to an embodiment of the present invention, the medicament may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the pharmaceutical composition further comprises: pharmaceutically acceptable adjuvants.
According to an embodiment of the invention, the pharmaceutically acceptable auxiliary agent comprises at least one of a stabilizer, a wetting agent, an emulsifier, a binder, an isotonicity agent.
According to an embodiment of the invention, the medicament is in at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, a lyophilized formulation. Furthermore, the drugs according to the embodiments of the present invention are convenient to administer and suitable for maintaining the optimum activity of fecal bacillus longissimus.
According to the embodiment of the present invention, the administration dosage of the drug is not particularly limited, and in practical applications, can be flexibly selected according to the health condition of the administration subject.
According to some embodiments of the invention, the medicament of this embodiment is a probiotic.
In a fifth aspect of the invention, the invention provides a coprobacterium elongatum capsule. According to an embodiment of the invention, the coprobacter elongatus capsule comprises: the mass ratio of the coprophilous trichobezoar lyophilized powder to the maltodextrin is 1: a Faecalibacterium longissimus L. The capsule containing the coprobacter elongatus CM04-06 according to the embodiment of the invention is superior to the existing ulcerative enteritis drugs in inhibiting enteritis-induced weight loss, DAI increase and improving intestinal tract changes.
According to an embodiment of the present invention, the aforementioned coprinus elongatus capsule may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the coprobacter elongatus capsule further comprises: 0.5g of lyophilized powder of coprinus elongatus and 0.5g of maltodextrin.
According to the embodiment of the invention, the coprinus elongatus freeze-dried powder is prepared by mixing 1-500 x 10 viable bacteria 6 CFU/mL of live Bacillus faecalis strain is lyophilized.
According to an embodiment of the present invention, the coprobacter elongatus lyophilized powder includes: 0.8 to 1.2 parts by mass of the living coprinus meretrix, 1.8 to 2.2 parts by mass of lactose, 1.8 to 2.2 parts by mass of yeast powder, 0.8 to 1.2 parts by mass of peptone, 90 to 95 parts by mass of purified water and 0.3 to 1 part by mass of vitamin C can keep higher activity.
In a sixth aspect of the invention, the invention provides a coprobacterium elongatum preparation. According to an embodiment of the invention, the formulation comprises: 4-6 volumes of growth factors, 0.5-1.5 volumes of vitamin C and 95-105 volumes of fermented liquid of the Changji coprobacteria, wherein the Changji coprobacteria is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2016, 6 months and 13 days, the preservation address is No. 3 Xilu No. 1 Hospital in the sunward area of Beijing, china, the preservation number is CGMCC 1.5208, and the classification and the name are as follows: a Faecalibacterium longissimus L. The preparation comprising the coprobacterium elongatum CM04-06 according to the embodiments of the present invention is superior to the existing ulcerative enteritis drugs in inhibiting enteritis-induced weight loss, DAI increase and improving intestinal tract changes.
According to an embodiment of the present invention, the aforementioned coprobacter elongatus preparation may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the growth factor is a crude milk growth factor extract.
According to an embodiment of the present invention, the growth factor is further at least one of a vitamin substance, a purine substance, and a pyrimidine substance.
According to the embodiment of the invention, the growth factor is a crude milk growth factor extract obtained by sequentially mixing skim milk and casein, centrifuging and ultrafiltering.
According to an embodiment of the present invention, the fecal bacillus fermentum is obtained by subjecting the fecal bacillus to anaerobic fermentation treatment at 37 ℃ for 2-3 days.
According to an embodiment of the present invention, the coprobacter elongatus preparation further comprises: a maltodextrin.
In a seventh aspect of the invention, a food product is presented. According to an embodiment of the invention, the food product comprises: the bacillus alhenicus (Faecalibacterium longum) is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2016 (6 months and 13 days), the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, china, the preservation number is CGMCC 1.5208, and the classification and the designation are as follows: a Faecalibacterium longissimus L.
It should be noted that the food product described herein should be understood in a broad sense, and any substance that can be chewed, swallowed or otherwise provide energy to the body can be considered a food product, such as puffed food, dairy products, cookies, chewing gum, ice cream, etc.
According to an embodiment of the present invention, the food may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the food product further comprises: a dietetically acceptable additive. And the food has better mouthfeel.
According to an embodiment of the invention, the food is cow's milk, chewing gum, biscuits, tabletted sweets, solid drinks, etc. Further, the fecal bacillus longissimus CM04-06 can maintain better activity in food and is suitable for playing a role in organisms.
Further, the fecal bacillus longissimus CM04-06 of the present invention can be eaten in combination with food materials used in ordinary foods. For example, cereals and potatoes: cereals including rice, flour, and coarse cereals, and potatoes including potato and sweet potato; animal food including meat, poultry, fish, milk, eggs, etc.; legumes and products thereof, including soybeans and other dried legumes; vegetables and fruits including fresh beans, rhizomes, leafy vegetables, solanum melongena, etc.; the pure heat energy food includes animal and vegetable oil, starch, edible sugar, wine, etc.
In an eighth aspect of the invention, a food composition is provided. According to an embodiment of the invention, the food product comprises: 0.3-0.8 mass portion of living bacillus of the coprinus alvarezii, 85-95 mass portions of milk, 8.5-9.5 mass portions of white sugar and 0.3-0.8 mass portion of vitamin C, wherein the coprinus alvarezii is preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2016 year 6 and 13 days, the preservation address is No. 3 of the national institute No. 1 of Xilu of the Xinjiang province of the facing yang district in Beijing, china, the preservation number is CGMCC 1.5208, and the classification names are as follows: a Faecalibacterium longissimus L.
According to an embodiment of the present invention, the food composition may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the content of the coprobacter elongatus in the food composition is 1 to 100 x 10 6 CFU/g can maintain higher activity.
According to the embodiment of the invention, the food composition is prepared by sequentially mixing, homogenizing, sterilizing and cooling the milk and the white sugar, wherein the homogenizing is carried out under the condition of 20Mpa, the sterilizing is carried out for 5-10 minutes under the condition of 90 ℃, the temperature after the cooling treatment is 40-43 ℃, the vitamin C is added into a cooling treatment product, and the coprinus elongatus is inoculated into the mixture of the cooling treatment product and the vitamin C. Further, the fecal bacillus longissimus CM04-06 can maintain a good activity in the food composition and is suitable for the body to exert its effect.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Preservation information:
the strain name: faecalibacterium longum
The preservation date is as follows: 2016 (6 months and 13 days)
The preservation unit: china general microbiological culture Collection center
The preservation number is: CGMCC 1.5208
Address: xilu No. 1 Hospital No. 3 of Chao Chaoyang district, beijing City, china
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a neighbor-joining evolutionary tree of C.alvarezii CM04-06 plotted using MegaX software, according to an embodiment of the present invention;
FIG. 2 is a graph showing the results of the change in body weight of mice after intervention with fecal bacillus longissimus CM04-06 according to an embodiment of the present invention; and
FIG. 3 is a graph showing the results of changes in mouse DAI score after intervention of C.longissimus CM04-06 in mice according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative and intended to explain the present invention and should not be construed as limiting the present invention.
In the following examples, a coprobacter elongatus CM04-06 strain capable of preventing and treating ulcerative enteritis is screened out through in vitro probiotic property evaluation, and in order to verify the efficacy of the strain in treating and relieving ulcerative enteritis in vivo, the inventor adopts an enteritis mouse model to carry out a functional verification experiment. Dextran Sodium Sulfate (DSS) is used as an inducer to induce diarrhea and intestinal inflammation in mice to establish a enteritis mouse model, and the DSS-induced mouse model is the most widely used animal model for acute ulcerative enteritis.
As can be seen from the following examples, the coprobacterium elongatum CM04-06 (CM 04-06 was collected in the general microbiological center of China Committee for culture Collection of microorganisms at 2016, 6.13.D., the collection address is No. 3 of Xilu No. 1 of Shangyang district, beijing, china, and the collection number is CGMCC 1.5208, which has a good effect of relieving ulcerative enteritis.
The embodiments will be described in detail below.
Example 1 screening and identification of fecal Christina bacterium CM04-06
1. Isolated culture of CM04-06
The coprobacter alvarezii CM04-06 is separated from a fecal sample of a healthy child (male) in Shenzhen city. The environment of the separation culture is strict anaerobic condition, and the specific separation process is as follows: taking about 0.2g of excrement sample in an anaerobic operation box, using 1mL of sterile PBS to perform suspension dispersion, fully shaking and uniformly mixing, then performing gradient dilution coating, wherein the culture medium adopts an anaerobic PYG culture medium, the specific components are shown in a table 1, and the composition of an inorganic salt solution is shown in a table 2:
table 1: PYG medium formula
Table 2: formulation of inorganic salt solution
| Inorganic salt component | Content (1 g/L) |
| CaCl 2 ·2H 2 O | 0.25 |
| MgSO 4 ·7H 2 O | 0.5 |
| K 2 HPO 4 | 1 |
| KH 2 PO 4 | 1 |
| |
10 |
| |
2 |
And (3) culturing the coated flat plate under the anaerobic condition of 37 ℃ for 3-4 days, after bacteria grow on the surface of the flat plate, selecting a single bacterial colony, carrying out streak purification until a pure culture strain is obtained, and carrying out cryopreservation on the separated strain at-80 ℃ by adopting glycerol.
2. Identification of 16S rDNA of fecal bacillus longissimus CM04-06
(1) Genome extraction: culturing the separated strain until the concentration of the strain reaches 10 8 2mL of the bacterial solution was taken at CFU/mL level for genomic DNA extraction.
(2) PCR amplification of 16S rDNA: taking DNA as a template to carry out 16S rDNA amplification, wherein the amplification primer selects a 16S rDNA universal amplification primer: 8F-1492R (5-:
(3) And (3) purification and sequencing: and (3) obtaining a PCR product, carrying out magnetic bead purification, carrying out electrophoresis detection, wherein the position of a 16S rDNA band is about 1.5k, and carrying out 3730 sequencing on the purified product.
(4) 16S rDNA sequence database alignment: 1372bp of 16S rDNA sequence is obtained by sequencing, and the sequence is compared in an EzTaxon-e database to obtain the species classification information of the strain primarily. The bacterium having the highest 16S rDNA homology with CM04-06 in the database was Faecalibacterium prausnitzii ATCC 27768 (hereinafter abbreviated as ATCC 27768), and the similarity was 96.87%. CM04-06 can be determined to be a new species belonging to genus Faecalibacterium based on the 16S rDNA information.
The 16S rDNA sequence of CM04-06 is:
caagtcgaacgagagatgaggagcttgctcttcagatcgagtggcgaacgggtgagtaacgcgtgaggaacctgcctcaaagagg gggacaacagttggaaacgactgctaataccgcataagcccacggctcggcatcgagcagagggaaaaggagtgatccgctttgagatg gcctccgtccgattagctggttggtgaggtaacggcccaccaaggcgacgatcggtagccggactgagaggttgaacggccacattggga ctgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggggaaaccctgatgcagcgacgccgcgtggag gaagaaggtcttcggattgtaaactcctgttgttgaggaagataatgacggtactcaacaaggaagtgacggctaactacgtgccagcagcc gcggtaaaacgtaggtcacaagcgttgtccggaattactgggtgtaaagggagcgcaggcgggagaacaagttggaagtgaaatccatgg gctcaacccatgaactgctttcaaaactgtttttcttgagtagtgcagaggtaggcggaattcccggtgtagcggtggaatgcgtagatatcgg gaggaacaccagtggcgaaggcggcctactgggcaccaactgacgctgaggctcgaaagtgtgggtagcaaacaggattagataccctg gtagtccacaccgtaaacgatgattactaggtgttggaggattgaccccttcagtgccgcagttaacacaataagtaatccacctggggagta cgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcagtggagtatgtggtttaattcgacgcaacgcgaagaacctt accaagtcttgacatcccttgacgaacatagaaatattttttctcttcggagcaaggagacaggtggtgcatggttgtcgtcagctcgtgtcgtg agatgttgggttaagtcccgcaacgagcgcaacccttatggtcagttactacgcaagaggactctggccagactgccgttgacaaaacgga ggaaggtggggatgacgtcaaatcatcatgccctttatgacttgggctacacacgtactacaatggcgttaaacaaagagaagcaagaccgc gaggtggagcaaaactcagaaacaacgtcccagttcggactgcaggctgcaactcgcctgcacgaagtcggaattgctagtaatcgtggat cagcatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagccggggggacccgaagtcggtagtctaac cgcaaggaggac。
3. 16S rDNA evolution analysis of CM04-06
Evolutionary analysis of CM04-06 uses 16S rDNA sequences to align the 16S rDNA sequences of CM04-06 with the EzBioCloud database to obtain species with close affinity to CM04-06, these species are aligned with the 16S rDNA sequences of CM04-06, and then the neighbor-joining evolutionary tree is drawn using MegaX software (FIG. 1). The classification position of CM04-06 can be further determined to be Faecalibacterium according to the evolutionary tree, and the species with the most recent evolutionary relationship is ATCC 27768.
4. Microbiological characteristics of CM04-06
(1) Morphological characteristics: the CM04-06 is cultured for 2-3 days at 37 ℃ in an anaerobic environment, the bacterial colony is yellow white, has high water content, is slightly sticky, is approximately round, is opaque, is flat and has a convex middle part, and the diameter of the bacterial colony is about 2-3mm.
(2) Microscopic characteristics: under 1000 times of microscope amplification, the thallus of CM04-06 is long rod-shaped, gram staining reaction is negative, no spore and flagellum are found, the diameter of the thallus is about 1um, and the length is 4-10 um.
(3) Physiological and biochemical characteristics: the oxidase and catalase reactions are negative, the growth temperature range is 30-45 ℃, the pH value range is 4.0-9.0, and the optimal temperature and pH value are 37 ℃ and 7.0. Can tolerate 3 percent of NaCl. A comparison of physiological and biochemical reactions (including substrate utilization API 20A and enzyme reaction API ZYM) of CM04-06 with the most closely related reference strain ATCC 27768 is shown in Table 3: (+, indicates a positive reaction; -, indicates a negative reaction, and w indicates a weakly positive reaction).
Table 3: biobiochemical characteristics of CM04-06 and ATCC 27768
The comparison of the above-mentioned physiological and biochemical reactions of CM04-06 with reference bacteria shows that CM04-06 is a new species different from known bacteria, because it has significant differences in the utilization of raffinose and trehalose, hydrolysis of escin and gelatin, and the activities of esterase (C4), valine arylaminase, cystine arylaminase, chymotrypsin, naphthol-AS-BI-phosphohydrolase, β -uronic acid glycosidase, α -glucosidase, and N-acetyl-glucosaminase.
5. Analysis of cellular fatty acids
Collecting the cultured CM04-06 and ATCC 27768 cells in stationary phase, extracting fatty acid from cells, and detecting. The contents and differences of fatty acids in the cells of the two strains were analyzed by gas chromatography, and the specific results are shown in table 4.
Table 4: fatty acid content of CM04-06 and ATCC 27768 cells
The analysis of fatty acids showed that CM04-06 was similar in composition to the reference strain ATCC 27768 for the major fatty acids (> 10%), including tetradecyl, hexadecyl, and omega 9 c-octadecyl diunsaturated fatty acids, but with some differences in the specific content.
By combining the CM04-06 phenotype, 16S rDNA evolution analysis, physiological and biochemical reaction and analysis data of cell chemical components, the CM04-06 is a new species of Faecalibacterium, and is named as Faecalibacterium longissimus.
Example 2 secretion of bioactive substances by Faecalibacterium longum CM04-06
1. Detection of Short Chain Fatty Acids (SCFA)
(1) Sample preparation: taking 1mL of CM04-06 bacterial liquid cultured for 48h, centrifuging for 5min at 12000r/min, and sucking supernatant for later use.
(2) Determination of SCFA: the determination of the short-chain fatty acid adopts an external standard method, and acetic acid, propionic acid, butyric acid and valeric acid are selected to make a standard curve. An Agilent gas chromatograph (GC-7890B, agilent) is adopted, HP-INNOWax (Cross-Linked PEG) and a capillary column with the size of 30m multiplied by 0.25mm multiplied by 0.25um are selected for analysis, the detector is a hydrogen flame ion detector, and GC parameters are set as the column temperature: 180-200 ℃; temperature of the gasification chamber: 240 ℃; detecting the temperature: at 210 ℃; sample introduction amount: 2 mu L of the solution; carrier gas flow: n is a radical of 2 50mL/min; hydrogen flow rate: 50mL/min; air flow rate: 600-700 mL/min.
(3) As a result: the measurement results show that the SCFA yield is as follows: formic acid (7.62 mmol/L), acetic acid (44.8 mmol/L), butyric acid (40.03 mmol/L).
2. Detection of organic acids
(1) Sample preparation: detection of SCFA as described above
(2) Determination of organic acids: the detection standard substance of the organic acid is selected from: 3-methylbutyric acid, valeric acid, quinic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-ascorbic acid. Still adopting Agilent gas chromatograph (GC-7890B, agilent), selecting 122-5532G DB-5ms (40 m multiplied by 0.25mm multiplied by 0.25 um) as chromatographic column, and adopting the following column temperature: 270 to 290 ℃; sample inlet temperature: 250 ℃; the gas flow rate was 0.86mL/min.
(3) As a result: the organic acid yield was determined as detailed in table 5.
Table 5: yield of organic acid of fecal bacillus longissimus CM04-06
| Organic acids | 3-methyl butyric acid | Valeric acid | Quinic acid | Lactic acid | Oxalic acid | Malonic acid |
| Content (mmol/L) | 0.26 | 0.49 | 0 | 30.53 | 0 | 0 |
| Organic acids | Benzoic acid | Maleic acid | Succinic acid | Fumaric acid | Malic acid | Adipic acid |
| Content (mmol/L) | 1.79 | 0 | 0.88 | 0 | 0 | 0.60 |
| Organic acids | Tartaric acid | Shikimic acid | Citric acid | Isocitric acid | L-ascorbic acid | |
| Content (mmol/L) | 0 | 0 | 0. | 0 | 0.11 |
Example 3 treatment of UC mice with Faecalibacterium longissimus CM04-06
The mouse models selected in this example were: a DSS-induced ulcerative enteritis mouse model is characterized in that C57bl/6 mice (purchased from Guangdong province medical experimental animal center) are adopted, the mice are 8 weeks old and 20g +/-2 g in weight, the feeding environment of the mice is SPF grade, the mice are adaptively fed for 1 week for DSS induction, and 2.5% DSS (molecular weight of 36000-50000) is continuously drunk by the mice for 7 days. The therapeutic effect of C.longissimus CM04-06 and VSL # 3 was compared using VSL #3 (purchased from Sigma Tau, USA) as a positive control.
The test mice were 48 in total and randomly divided into 4 groups of 15, including a control group (control group), a DSS-induced model group (each gavage with 0.2ml PBS per day), a c.alhenicola CM04-06 treatment group and a VSL # 3 treatment group.
The treatment process of the bacillus faecalis CM04-06 is as follows: anaerobically culturing 24 hr of Bacillus faecium CM04-06 bacteria solution, centrifuging to collect bacteria, suspending with PBS, adjusting bacteria concentration to 1 × 10 9 CFU/mL, each mouse was gavaged with 200. Mu.L of fecal bacillus longissimus CM04-06 per day.
The VSL # 3 process was: suspension was carried out with PBS, likewise adjusted to a concentration of 1X 10 9 CFU/mL, 200. Mu.L of gavage per mouse per day.
The treatment adopts a preventive administration mode, the stomach is perfused with probiotics three days before the DSS treatment, the weight, diet and drinking conditions of the mice are recorded every day, the fecal characters and fecal occult blood conditions of the mice are observed at the same time, the DAI scores of the mice are calculated on the 1 st day, the 3 rd day, the 5 th day and the 7 th day of the DSS treatment respectively, and the DAI scores are detailed in a table 6.DSS treatment lasted 7 days with daily gavage of 200 μ L of probiotic and PBS. After the experiment was completed, the mice were sacrificed and all mice were bled, decapped, colons were removed, photographed, weighed, and the colons length was measured. The colon tissue was stored in a-80 ℃ freezer and paraformaldehyde.
Table 6: DAI index scoring sheet
| Weight loss (%) | Stool character | Occult/weak bloody stool | Integral of |
| 0 | Is normal | Is normal | 0 |
| 1-5 | 1 | ||
| 5-10 | Loosening | Positive |
2 |
| 10-15 | 3 | ||
| >15 | Thin stool | Bloody stool with |
4 |
Stool characteristics in the table: normal stool-formed stool; loose stool-pasty, semi-formed stool that does not adhere to the anus; loose stool-watery stool that can adhere to the anus. Wherein the hematochezia condition is as follows: the hematochezia of the normal mice is positive; the naked eye is red or brown in blood; occult blood positive is an unobvious macroscopic stool and is detected by using tetramethyl benzidine. The DAI index is equal to the sum of the three integrals of body weight, stool behavior and fecal occult blood/weak ocular bloody stool.
And (3) test results:
(1) The body weight changes of the mice before and after treatment are shown in table 7 below and fig. 2:
table 7: effect of CM04-06 on mouse body weight
As can be seen from table 7 and fig. 2, the body weight of the mice in the control group maintained a tendency of slow increase, the body weight of the mice in the DSS-induced model group gradually decreased, the decrease in body weight was more significant at day 3 (P < 0.05), and the difference between the two was more significant at day 5 (P < 0.01). Whereas treatment with CM04-06 and VSL # 3 reduced the weight loss in UC mice, the inhibition of weight loss in CM04-06 and VSL # 3 mice on days 5 and 7, respectively, was significantly different relative to the model group (a P < 0.05). The above results indicate that the two groups of probiotics CM04-06 and VSL # 3 can inhibit the weight loss of mice caused by UC. The weight values of the groups on the 7 th day are compared to find that the weight of the mice in the CM04-06 group is slightly higher than that of the VSL # 3, the weight reduction of the mice is more remarkably inhibited (a-solidup-P is less than 0.01), and the capacity of inhibiting the weight reduction of the UC mice by the CM04-06 group is better than that of the VSL # 3.
(2) Change in mouse DAI score before and after treatment
DSS-induced ulcerative enteritis mice induced changes in DAI index due to weight loss, stool behavior, and changes in hematochezia status, with specific results shown in table 8 and fig. 3:
table 8: effect of CM04-06 on mouse DAI score
| | Day | 1 | |
|
|
| Control group | 1.0±0.5 | 1.2±0.6 | 1.1±0.6 | 1.3±0.5 | |
| Model set | 1.2±0.4 | 3.5±1.1** | 6.8±1.6** | 8.9±1.9** | |
| CM04-06 | 1.1±0.5 | 3.1±1.2 | 5.2±1.5 ▲▲ | 7.0±1.8 ▲▲ | |
| |
1.2±0.6 | 3.3±1.1 | 5.8±1.8 ▲▲ | 7.4±1.6 ▲ |
Table 8 and figure 3 data show that control mice maintained a steady low DAI score, whereas DSS-induced mice developed a cascade of pathologies resulting in a gradual rise in DAI score, with model mice having a very significant DAI score compared to the control group starting at day 3 (P < 0.01) and model mice reaching a maximum DAI score at day 7. Probiotic intervention inhibited the rise in DAI score, with some degree of control of DAI score at day 5 and day 7 in CM04-06 and VSL # 3 mice compared to the model group (P <0.05, P < 0.01). From the results on day 7, the DAI scores of the mice of the CM04-06 group are slightly lower than those of the VSL # 3 group, and the DAI scores of the mice of the CM04-06 group and the model group are extremely different (a-solidup P is less than 0.01), thereby indicating that the effect of the CM04-06 group in inhibiting the increase of the DAI scores of the UC mice is better than that of the VSL # 3.
(3) Changes in colon Length
The colon tissue of the UC model mouse is changed mainly because the colon tissue is shortened due to the occurrence of ulcer and inflammation, the colon length of the mouse is dissected and measured after the treatment is finished, and the experimental result is shown in Table 9.
Table 9: effect of CM04-06 on Colon Length in mice
| Grouping | Colon Length (cm) |
| Control group | 7.16±0.82 |
| Model set | 4.58±0.98** |
| VSL # Group 3 | 5.43±0.90 ▲ |
| CM04-06 group | 5.70±0.85 ▲ |
Table 9 results show that colon tissue shortening was more severe in mice (model group) 7 days after DSS induction, and the difference was very significant compared to control group (. About.p < 0.01). Intervention of probiotics CM04-06 and VSL # 3 can obviously inhibit shortening of colon of mice, and the difference between the two groups of CM04-06 and VSL # 3 is obvious compared with that of a model group (a P is less than 0.05). From the data in Table 9, it was found that the colon length of the mouse in CM04-06 group was longer than that of the mouse in VSL # 3 group, indicating that the colon shortening ability of UC mouse controlled by CM04-06 was slightly stronger than that of VSL # 3.
Example 4 food composition containing fecal bacillus longissimus CM04-06
Table 10: food composition raw material formula
| Raw materials | Mass percent (%) |
| Bacillus faecalis CM04-06 | 0.5 |
| Milk | 90.0 |
| White sugar | 9.0 |
| Vitamin C | 0.5 |
Mixing milk and white sugar according to the formula proportion shown in Table 10, stirring to mix completely, preheating, homogenizing under 20Mpa, sterilizing at 90 deg.C for 5-10 min, cooling to 40-43 deg.C, adding vitamin C as protective agent, inoculating 1-100 × 10 6 CFU/g of Bacillus faecalis CM04-06 strain, and food composition containing the same
Example 5 preparation of capsules containing fecal bacillus longissimus CM04-06
Table 11: capsule raw material formula
| Raw materials | Mass percent (%) |
| Bacillus faecium CM04-06 | 1.0 |
| Lactose | 2.0 |
| Yeast powder | 2.0 |
| Peptone | 1.0 |
| Purified water | 93.5 |
| Vitamin C | 0.5 |
Mixing lactose, yeast powder and peptone with purified water uniformly according to the formula proportion shown in Table 11, preheating to 60-65 ℃, homogenizing under 20Mpa, sterilizing at about 90 ℃ for 20-30 minutes, cooling to 36-38 ℃, mixing with protective agent vitamin C, inoculating live Bacillus faecalis CM04-06 viable bacteria (1-500 × 10) 6 CFU/mL), fermenting at 36-38 ℃ until the pH value is 6.0, centrifuging, and freeze-drying until the water content is less than 3 percent, thus preparing the freeze-dried product of the bacillus faecalis CM04-06 strain. Weighing 0.5g of the freeze-dried product of the faecal bacteria Christi CM04-06, mixing with maltodextrin in equal amount, and encapsulating to obtain the pharmaceutical composition containing the faecal bacteria Christi CM04-06.
EXAMPLE 6 preparation of a formulation for the treatment of ulcerative enteritis
1. Preparing bacterial liquid: culturing Bacillus faecalis CM04-06 (1X 10) 9 CFU/mL) is subjected to anaerobic culture, the anaerobic culture medium adopts a PYG culture medium, and the fermentation is carried out for 2 to 3 days at 37 ℃.
2. Preparation of growth factors: mixing skimmed milk and casein, centrifuging, and ultrafiltering to obtain crude extract of milk growth factor (containing vitamins, purine, pyrimidine, etc.).
3. Preparation of a medicament formulation: adding 5 volumes of growth factor and 1 volume of protective agent vitamin C into 100 volumes of CM04-06 fermented bacterial liquid, stirring thoroughly, mixing well, and adding starch adjuvant (such as maltodextrin) to prepare into pharmaceutical dosage form.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. Use of faecal bacteria (Faecalibacterium longum), deposited at the general microbiological culture collection center of the China Committee for culture Collection of microorganisms at 2016, 6 and 13 with the deposit number CGMCC 1.5208, for the treatment and prevention of enteritis.
2. Use of a Faecalibacterium longissimus (Faecalibacterium longum) deposited at the china community collection of microorganisms with the accession number CGMCC 1.5208 at 2016, 6/13 in the manufacture of a medicament, health product, food product, or composition for at least one of:
treating and preventing enteritis;
inhibiting enteritis-induced weight loss;
inhibiting an increase in disease activity index due to enteritis; and
improving changes in the intestinal tract;
optionally, the change in the intestinal tract is a shortening of the colon tissue.
3. Use according to claim 1 or 2, wherein the enteritis is ulcerative enteritis;
optionally, the ulcerative enteritis is ulcerative colitis;
optionally, the enteritis is caused by DSS.
4. The use according to claim 2, characterized in that the concentration of said coprobacter elongatus is 1.0 x 10 6 CFU/mL～1.0×10 9 CFU/mL。
5. A composition, which comprises a fecal strain of Faecalibacterium longum (Faecalibacterium longum) deposited at the center for China general microbiological culture Collection management Committee (CGMCC) with the collection number of CGMCC 1.5208 at 6/13 of 2016.
6. A medicament, comprising: the bacillus faecalis (Faecalibacterium longum) is preserved in the China general microbiological culture Collection center at 2016, 6 and 13 days, and the preservation number is CGMCC 1.5208;
optionally, the medicament further comprises: pharmaceutically acceptable adjuvants;
optionally, the pharmaceutically acceptable adjuvants include at least one of stabilizers, wetting agents, emulsifiers, binders, isotonic agents;
optionally, the medicament is in at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension and a freeze-dried preparation.
7. A coprobacterium elongatum capsule, comprising: the mass ratio of the coprinus elongatus freeze-dried powder to the maltodextrin is 1,
wherein the coprinus elongatus is preserved in the China general microbiological culture Collection center in 2016, 6 and 13 days, the preservation number is CGMCC 1.5208,
the freeze-dried powder is prepared by mixing live bacteria with the content of 1-500 multiplied by 10 6 The bacterial strain is obtained by freeze-drying CFU/mL live bacillus of the coprobacterium elongatum;
optionally, the capsule comprises 0.5g of coprinus elongatus lyophilized powder and 0.5g of maltodextrin;
optionally, the coprinus elongatus freeze-dried powder comprises: 0.8 to 1.2 parts by mass of the living coprinus meretrix, 1.8 to 2.2 parts by mass of lactose, 1.8 to 2.2 parts by mass of yeast powder, 0.8 to 1.2 parts by mass of peptone, 90 to 95 parts by mass of purified water and 0.3 to 1 part by mass of vitamin C.
8. A coprobacter elongatus preparation comprising: 4 to 6 volumes of growth factor, 0.5 to 1.5 volumes of vitamin C, 95 to 105 volumes of fermented bacterial liquid of the coprinus elongatus,
wherein the coprinus elongatus is preserved in the China general microbiological culture Collection center in 2016, 6 and 13 days, the preservation number is CGMCC 1.5208,
optionally, the growth factor is a crude milk growth factor extract;
optionally, the growth factor comprises at least one of a vitamin, a purine, a pyrimidine;
optionally, the growth factor is a milk growth factor crude extract obtained by sequentially mixing skim milk and casein, centrifuging and ultrafiltering;
optionally, the fecal bacillus lentus zymocyte liquid is obtained after anaerobic fermentation treatment of the fecal bacillus lentus at 37 ℃ for 2-3 days;
optionally, the coprobacterium elongatum preparation further comprises maltodextrin.
9. A food product, comprising: the bacillus faecalis (Faecalibacterium longum) is preserved in the China general microbiological culture Collection center at 2016, 6 and 13 days, and the preservation number is CGMCC 1.5208;
optionally, the food product further comprises: a dietetically acceptable additive;
optionally, the food product is a dairy product, chewing gum, biscuit, tabletted candy or solid beverage.
10. A food composition, comprising: 0.3 to 0.8 mass portion of live bacillus of the coprinus elongatus, 85 to 95 mass portions of milk, 8.5 to 9.5 mass portions of white sugar and 0.3 to 0.8 mass portion of vitamin C,
wherein the coprinus elongatus is preserved in the China general microbiological culture Collection center in 2016, 6 and 13 days, the preservation number is CGMCC 1.5208,
the content of the fecal bacillus longissimus in the food composition is 1-100 multiplied by 10 6 CFU/g；
Optionally, the food composition is obtained by:
1) Mixing the milk and white sugar, homogenizing, sterilizing, cooling,
wherein the homogenization is carried out under the condition of 20MPa,
the sterilization is carried out for 5 to 10 minutes at the temperature of 90 ℃,
the temperature after the cooling treatment is 40-43 ℃,
2) Adding the vitamin C into the cooling treatment product,
3) Inoculating the coprobacterium longipes into the product obtained in the step 2).
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Citations (2)
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| WO2018045492A1 (en) * | 2016-09-06 | 2018-03-15 | 深圳华大基因研究院 | Faecalibacterium longum and application thereof |
| WO2018226690A1 (en) * | 2017-06-05 | 2018-12-13 | The University Of Chicago | Microbiome biomarkers of immunotherapy responsiveness: diagnostic, prognostic and therapeutic uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018045492A1 (en) * | 2016-09-06 | 2018-03-15 | 深圳华大基因研究院 | Faecalibacterium longum and application thereof |
| CN109219656A (en) * | 2016-09-06 | 2019-01-15 | 深圳华大生命科学研究院 | Long dwell bacillus faecalis (Faecalibacterium longum) and its application |
| WO2018226690A1 (en) * | 2017-06-05 | 2018-12-13 | The University Of Chicago | Microbiome biomarkers of immunotherapy responsiveness: diagnostic, prognostic and therapeutic uses thereof |
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