CN117448198A - Bifidobacterium animalis subspecies VB301 and application thereof - Google Patents
Bifidobacterium animalis subspecies VB301 and application thereof Download PDFInfo
- Publication number
- CN117448198A CN117448198A CN202311189581.3A CN202311189581A CN117448198A CN 117448198 A CN117448198 A CN 117448198A CN 202311189581 A CN202311189581 A CN 202311189581A CN 117448198 A CN117448198 A CN 117448198A
- Authority
- CN
- China
- Prior art keywords
- bifidobacterium animalis
- sodium
- animalis subspecies
- lactis
- subspecies
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- IYPNVUSIMGAJFC-HLEJRKHJSA-M sodium;2-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,13r,14s,17r)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)CC1 IYPNVUSIMGAJFC-HLEJRKHJSA-M 0.000 claims description 7
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- GHCZAUBVMUEKKP-UHFFFAOYSA-N ursodeoxycholic acid glycine-conjugate Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)CC2 GHCZAUBVMUEKKP-UHFFFAOYSA-N 0.000 description 1
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- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
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Abstract
The invention discloses a bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.lactis) VB301 and application thereof, and the strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 27998 in the 24 th month of 2023. The bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) VB301 has remarkable bile salt reducing capability, can be used for preparing cholesterol-reducing medicaments, has stronger intestinal tract tolerance capability and antibacterial capability, and has great application prospect in preparing medicaments.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to bifidobacterium animalis subspecies VB301 and application thereof.
Background
Cholesterol is an important lipid in humans and at certain concentrations, it can provide the necessary functions to humans, but excessive intake, cholesterol can accumulate on the walls of blood vessels, forming cholesterol plaques, and thus causing various cardiovascular diseases. At present, cardiovascular and cerebrovascular diseases are main killers for causing death of people, and the trend is rising year by year. Lowering cholesterol levels in the body has therefore become an important means of preventing cardiovascular disease.
In the liver, the main route of cholesterol is the conversion to cholic acid, which is the main bile acid of humans, glycocholic acid and taurocholate derived from cholic acid, which provides an important excretion pathway for cholesterol metabolism, one third of which is achieved by bile acid synthesis. Bile acids can be divided into two categories: free bile acids such as cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, etc.; the other is conjugated bile acid formed by combining free bile acid with glycine or taurine, wherein the conjugated bile acid in human body is mainly 6 kinds, including glycochenodeoxycholic acid (26%), glycocholic acid (26%), glycodeoxycholic acid (22%), taurocholic acid (9%), taurodeoxycholic acid (9%), and taurochenodeoxycholic acid (9%). Bile acid exists in the form of sodium salt or potassium salt, namely bile acid salt, for short, bile salt.
The mechanism proposed by the probiotics for reducing cholesterol at home and abroad comprises: (1) the probiotic adsorbs cholesterol; (2) The combined bile salt is hydrolyzed into free bile salt by the bile salt hydrolase of the probiotics, and the free bile salt is not easy to be absorbed by intestinal tracts due to low solubility, so that the cholesterol and the free bile salt in the body are coprecipitated in an acidic environment, thereby reducing the cholesterol level in serum; (3) The probiotics ferment the carbohydrate of the intestinal tract food source to generate propionic acid, and the propionic acid inhibits the biosynthesis of liver cholesterol; (4) the probiotics inhibit the formation of cholesterol emulsion micelle.
The prior art discloses a plurality of bifidobacterium animalis subspecies, but none of the bifidobacterium animalis subspecies has the capability of simultaneously degrading 6 main binding bile salts of a human body, wherein the 6 bile salts are sodium Taurocholate (TAC), sodium Taurochenodeoxycholate (TCDC), sodium Taurodeoxycholate (TDC), sodium Glycocholate (GC), sodium Glycochenodeoxycholate (GCDC) and sodium Glycodeoxycholate (GDC), and the plurality of bifidobacterium animalis subspecies disclosed in the prior art have no antibacterial effect or have no outstanding antibacterial effect.
Disclosure of Invention
The present invention aims to at least partly solve at least one of the technical problems existing in the prior art.
To this end, the first aspect of the present invention provides a bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) VB301 deposited at the China general microbiological culture collection center under the accession number CGMCC No.27998, month 24 of 2023, which is the subsp. Bifidobacterium animalis subsp. Lactis) VB301.
The animal bifidobacterium subspecies provided by the invention have the capability of degrading 6 kinds of combined bile salts, intestinal tract tolerance capability and antibacterial capability, can be used for preventing and/or relieving and/or treating related diseases caused by high cholesterol on one hand, and can be used for preventing and/or relieving and/or treating related diseases caused by at least one of escherichia coli, staphylococcus aureus, salmonella, listeria, shigella, enterococcus faecalis, clostridium perfringens, such as urinary tract infection, suppurative abdominal infection, septicemia, endocarditis, diarrhea, fever and the like on the other hand.
In a second aspect the invention provides a fermentation supernatant comprising the metabolite of bifidobacterium animalis subspecies VB301 of the first aspect.
In a third aspect the invention provides a bacterial suspension comprising bifidobacterium animalis subspecies lactis VB301 according to the first aspect.
According to a fourth aspect of the present invention there is provided the use of bifidobacterium animalis subspecies lactis VB301 according to the first aspect, the fermentation supernatant according to the second aspect or the bacterial suspension according to the third aspect in the manufacture of a medicament for the prophylaxis and/or treatment of a condition associated with high cholesterol.
According to an embodiment of the present invention, the related diseases caused by high cholesterol include any one of hypercholesterolemia, cardiovascular disease, arteriosclerosis and peripheral vascular disease.
According to a specific embodiment of the present invention, the drug is capable of degrading six bile salts, the six bile salts being sodium taurocholate, sodium taurochenodeoxycholate, sodium taurodeoxycholate, sodium glycocholate, sodium glycochenodeoxycholate, sodium glycodeoxycholate.
In a fifth aspect, the invention provides the use of a bifidobacterium animalis subspecies lactis VB301 according to the first aspect or a bacterial suspension according to the third aspect in the manufacture of a medicament for the treatment of a disease associated with at least one of staphylococcus aureus, escherichia coli, salmonella paratyphi b, listeria monocytogenes, shigella dysenteriae, enterococcus faecalis, clostridium perfringens.
According to a specific embodiment of the present invention, the related diseases caused by staphylococcus aureus, escherichia coli, salmonella paratyphi b, listeria monocytogenes, shigella dysenteriae, enterococcus faecalis, clostridium perfringens include infectious diseases such as urinary tract infection, suppurative abdominal infection, septicemia, endocarditis, diarrhea, fever, etc.
In a sixth aspect, the invention provides a medicament comprising at least one of bifidobacterium animalis subspecies lactis VB301 according to the first aspect, a fermentation supernatant according to the second aspect, and a bacterial suspension according to the third aspect.
According to an embodiment of the invention, the medicament further comprises an excipient and/or carrier.
According to an embodiment of the present invention, the excipient comprises at least one selected from the group consisting of binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, slow-release agents.
According to an embodiment of the present invention, the carrier comprises at least one selected from the group consisting of saccharides, cellulose and derivatives thereof, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, cereal hydrolytic solids.
According to an embodiment of the present invention, the dosage form of the drug includes at least one selected from the group consisting of oral liquid, powder, granule, capsule, tablet, and drop pill.
According to an embodiment of the invention, when bifidobacterium animalis subspecies lactis VB301 acts, it may be present in the form of living cells or in the form of non-living cells.
According to an embodiment of the present invention, the living cells refer to cells having metabolism, reproduction or replication ability of bifidobacterium animalis subspecies VB301, and the non-living cells refer to cells having no metabolism, reproduction and replication ability, including but not limited to dry cells (such as freeze-dried powder).
In addition, the invention provides a microbial agent which contains the bifidobacterium animalis subspecies VB301, the fermentation supernatant fluid or the bacterial suspension, and is used for preventing and/or treating related diseases caused by high cholesterol.
The invention also provides application of the bifidobacterium animalis subspecies VB301 and/or the fermentation supernatant in preparing health-care products and foods, wherein the health-care products or foods are used for preventing and/or treating related diseases caused by high cholesterol.
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:
strain name: VB301
Preservation date: 2023, 7 and 24 days
Preservation unit: china general microbiological culture collection center (CGMCC)
Preservation number: CGMCC No.27998
Drawings
FIG. 1 shows the microscopic morphology of bifidobacterium animalis subspecies of milk of example 1 of the present invention, wherein A in FIG. 1 represents the microscopic morphology of individual bifidobacterium animalis subspecies VB301 and B in FIG. 1 represents the microscopic morphology of clustered bifidobacterium animalis subspecies VB301.
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 by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The terms "comprising," "including," or "comprising" are used herein in an open-ended fashion, i.e., to include what is indicated by the present invention, and not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
"prevent" and "prevent" are used interchangeably herein. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," lactobacillus fermentum or a product containing the same may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.
In this context, the terms "treatment" and "alleviation" both refer to the use of the terms "treatment" and "alleviation" in order to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.
As used herein, "acceptable in a health food" refers to a substance or composition that is edible to humans and that can be tailored to the health food requirements of different countries.
As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.
Herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
Herein, the term "pharmaceutically acceptable excipients" may all include saccharides including mono-or polysaccharides, such as lactose, sucrose, mannitol and sorbitol; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal salts of stearic acid such as magnesium stearate and calcium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; an ethylene glycol polymer; fatty alcohols; and cereal hydrolytic solids as well as other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and the like, which are commonly used in pharmaceutical formulations.
According to a specific embodiment of the invention, the invention proposes a bifidobacterium animalis subspecies lactis (Bifidobacterium animalis subsp. According to the embodiment of the invention, the preservation number of the bifidobacterium animalis subspecies lactis is CGMCC No.27998. The bifidobacterium animalis subspecies of the invention has remarkable bile salt reducing capability, and the bifidobacterium animalis subspecies VB301 has stronger intestinal tract tolerance capability and antibacterial capability.
Herein, "bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) VB301" is synonymous with "bifidobacterium animalis subspecies VB301", "strain VB 301".
Compared with other bifidobacterium animalis subspecies, the bifidobacterium animalis subspecies VB301 obtained by screening can degrade 6 bile salts, wherein the 6 bile salts are sodium Taurocholate (TAC), sodium Taurochenodeoxycholate (TCDC), sodium Taurochenodeoxycholate (TDC), sodium Glycocholate (GC), sodium Glycochenodeoxycholate (GCDC) and sodium Glycodeoxycholate (GDC).
The bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) VB301 of the present invention has a greater intestinal tolerance than other bifidobacterium animalis subspecies. The bacterial strain VB301 has no influence on the survival of thalli after 4 hours of action in 0.03 to 0.3 percent of bovine choline solution, and the survival rate can still reach 91 percent after 4 hours of action in 0.3 percent of bovine choline, which proves that the bacterial strain VB301 has strong tolerance to bovine choline. The bifidobacterium animalis milk subspecies VB301 can survive for 2-4 hours in simulated intestinal fluid with pH value of 8.0, the survival rate of 4 hours still can reach 63%, and the bifidobacterium animalis milk subspecies VB301 has good activity tolerance capability to intestinal environment and can play a role in intestinal tracts for a long time.
The animal bifidobacterium lactis VB301 has stronger antibacterial capability compared with other animal bifidobacterium lactis, and the animal bifidobacterium lactis VB301 has antibacterial effect on 7 pathogenic bacteria, wherein the 7 pathogenic bacteria are escherichia coli, staphylococcus aureus, salmonella, listeria, shigella, enterococcus faecalis and clostridium perfringens.
According to a specific embodiment of the present invention, the present invention provides a fermentation supernatant comprising: a bifidobacterium animalis subspecies VB301 and/or a metabolite of bifidobacterium animalis subspecies VB301. The bifidobacterium animalis subspecies VB301 has remarkable bile salt reducing capability, and the bifidobacterium animalis subspecies VB301 has stronger intestinal tract tolerance capability and antibacterial capability.
The term "fermentation supernatant" refers to a supernatant of a fermentation broth after centrifugation, which may contain bifidobacterium animalis subspecies VB301 or metabolites of bifidobacterium animalis subspecies VB301, or both bifidobacterium animalis subspecies VB301 and metabolites thereof.
According to a specific embodiment of the invention, the invention provides a microbial agent, which contains the bifidobacterium animalis subspecies VB301 and/or the fermentation supernatant. The microbial agent of the invention is used for preventing and/or treating related diseases caused by high cholesterol.
The microbial agent of the present invention may be a microbial liquid agent, including but not limited to fermentation broth, or a microbial solid agent, including but not limited to lyophilized powder.
According to an embodiment of the invention, said bifidobacterium animalis subspecies lactis VB301 is present in the form of living and/or non-living cells.
As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.
Illustratively, the living cells may be immobilized cells. As used herein, "immobilized cells" refer to bifidobacterium animalis subspecies VB301 immobilized on a carrier and capable of performing vital activities such as growth, development, propagation, inheritance, metabolism and the like in a certain spatial range.
Herein, "non-living cells" refers to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dry cells, illustratively, the microbial agent is a lyophilized powder.
As a specific embodiment, the bifidobacterium animalis subspecies lactis VB301 is present as living cells, as dry cells, as immobilized cells, or in any other form.
As a specific embodiment, the dry cell is obtained by freeze-drying the bifidobacterium animalis subspecies lactis VB301.
According to an embodiment of the invention, the invention provides a medicine or health-care product or food, wherein the medicine or health-care product or food contains bifidobacterium animalis subspecies VB301. Preferably, the medicament or health-care product or food is a viable bacteria preparation for reducing cholesterol. The medicine, health care product or food can be various preparations of the single active bifidobacterium animalis subspecies VB301 or can be matched with other active ingredients for use, so long as the activities are not affected; further, the active ingredients of the medicine or the health-care product or the food can have functional complementation or promotion effect, for example, the bifidobacterium animalis subspecies VB301 and other probiotics can be combined to prepare a composite probiotic tablet, which can be used for preventing and/or relieving and/or treating related diseases caused by high cholesterol on one hand, and can be used for preventing and/or relieving and/or treating related diseases caused by at least one of escherichia coli, staphylococcus aureus, salmonella, listeria, shigella, enterococcus faecalis and clostridium perfringens, including infectious diseases, and the specific method can be based on the activity and the components of the composite probiotic tablet, and is not limited herein. Optionally, the medicament or health product or food further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable auxiliary material, a carrier or auxiliary material acceptable in food, and a carrier or auxiliary material acceptable in health food. In the preparation of the viable bacteria preparation, a carrier or an auxiliary material is usually added, so long as the added carrier or auxiliary material and the bifidobacterium animalis subspecies VB301 do not inhibit each other or have adverse side effects. The medicine or health product or food can be powder, tablet, beverage, capsule.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 Strain harvesting and identification
Collecting 10 parts of fresh feces of healthy infants (3-5 years old) and 10 parts of fresh feces of adults, 3 parts of breast milk, taking 0.5g of feces and 1mL of breast milk, respectively adding into 5mL of bifidobacterium BS liquid culture medium, carrying out enrichment culture for 12-16 hours at 37 ℃ and then vibrating and homogenizing to prepare bacterial suspension, taking 1mL of bacterial suspension, carrying out gradient dilution to prepare diluted bacterial suspensions with different gradients, coating the bacterial suspension into an MRS agar plate containing 50 mug/mL mupirocin and 5% serum (V/V), carrying out anaerobic culture for 48-72 hours at 37 ℃, picking up 130 colony strains with different forms, streaking and purifying, extracting DNA, carrying out PCR amplification by using specific primers, sequencing for 16srDNA, simultaneously storing glycerol tubes corresponding to the strains, extracting DNA, and carrying out full sequence sequencing identification of 16s rDNA. And (3) performing BLAST comparison on the 16s sequencing result to finally identify and separate 18 bifidobacteria, namely 1 bifidobacterium adolescentis, 11 bifidobacterium animalis subspecies lactis, 2 bifidobacterium bifidum, 2 bifidobacterium breve and 2 bifidobacterium longum, and the results are shown in Table 1.
Table 1: bacterial strain collection and identification results
The 18 strains were each tested for their ability to degrade 6 bile salts. The 6 bile salts are sodium Taurocholate (TAC), sodium Taurochenodeoxycholate (TCDC), sodium Taurochenodeoxycholate (TDC), sodium Glycocholate (GC), sodium Glycochenodeoxycholate (GCDC) and sodium Glycodeoxycholate (GDC), respectively.
6 different bile salts are respectively added into BBL liquid culture medium, and the concentration of the bile salts is 2-4mmol/L. And taking out the activated glycerol tubes of each strain from the refrigerator at the temperature of minus 80 ℃, and inoculating the glycerol tubes into BBL liquid culture medium according to the inoculum size of 1-3% after thawing. Standing at 37 ℃, anaerobic culturing for 24 hours in an anaerobic workstation to obtain an activated bacterial liquid, taking 1mL of culture liquid, centrifuging, taking supernatant, performing HPLC (high performance liquid chromatography) detection on the content of each bile salt, evaluating the bile salt degradation capability of each strain by calculating the degradation rate of the bile salt, wherein the higher the degradation rate of the bile salt is, the stronger the bile salt hydrolysis capability is, and the result is shown in Table 2.
Bile salt degradation rate = (initial bile salt content-bile salt content after culture)/initial bile salt content 100%;
HPLC detection method of bile salts: chromatographic column: uitimate AQ-C18 (5 um, 4.6X1250 mm); flow rate: 1.0mL/min; detection wavelength: 204nm; column temperature: 45 ℃; mobile phase a phosphate buffer, mobile phase B acetonitrile, a: b=56:44 (V: V).
Table 2: comparison of 6 bile salts degradation Capacity of each Strain
As can be seen from Table 2, the A8 strain can degrade 6 bile salts simultaneously and has remarkable degradation capability, the A8 strain is named VB301 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 24 th month of 2023, the preservation address is North Chen West Lu No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.27998.
The strain VB301 is characterized by the following biological characteristics: anaerobic culture of the strain on MRS agar culture medium at 37deg.C for 48-72 hr, good strain growth, milky white colony, opaque, round, smooth surface, and colony diameter of 1-2mm; the strain has positive gram staining, and the cell morphology under a microscope is in a short rod shape, as shown in figure 1, and is distributed singly (A in figure 1) or in pairs (B in figure 1), and the two ends of the strain are elliptical, and the strain is in a branched rod shape, does not move and has no spores.
The sequence of strain VB301 is:
GTGACAAATGGGGGGCGGCAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGGGGAGCCGTCTAAAGGAGTTTCG(SEQ ID NO:1)。
example 2 evaluation of intestinal tolerability
Inoculating the activated VB301 bacterial suspension into BBL culture medium according to 1% inoculum size, standing at 37 ℃, anaerobically culturing for 24 hours in an anaerobic workstation, taking 9 parts of 1mL bacterial liquid, centrifugally collecting bacterial cells, adding 1mL of water into 1 part of the bacterial liquid, uniformly mixing, counting as a control, respectively placing the rest samples into a pancreatic juice system of 1mL of water, 1mL of artificial intestinal juice (pH 8.0), 1mL of 0.03% bovine choline solution, 1mL of 0.1% bovine choline solution and 1mL of 0.3% Niu Danjian solution, uniformly mixing, sampling viable bacteria count for 0h, 2h and 4h, and calculating survival rates for 2h and 4h, wherein the results are shown in Table 3.
Survival rate calculation: survival = B/a 100% where a is 1ml water, count after mixing; b is the counting result after sample treatment.
Table 3: survival of bifidobacterium animalis subspecies lactis VB301 in different culture systems
Note that: viable count unit: hundred million cfu/mL
The results in Table 3 show that the strain VB301 has no influence on the survival of the bacterial cells when being acted for 4 hours in 0.03 to 0.3 percent of the bovine choline solution, and the survival rate can still reach 91 percent when being acted for 4 hours in 0.3 percent of the bovine choline, which indicates that the strain VB301 has strong tolerance to the bovine choline. The bifidobacterium animalis milk subspecies VB301 can survive for 2-4 hours in simulated intestinal fluid with pH value of 8.0, and the survival rate of 4 hours can still reach 63%, so that the bifidobacterium animalis milk subspecies VB301 has good activity tolerance capability on intestinal environments.
Example 3: evaluation of bacteriostatic ability against pathogenic bacteria
Testing antibacterial capacity by double-layer plate culture method, wherein BBL culture medium is used as lower layer culture medium, 2 μl of animal bifidobacterium subspecies VB301 bacterial suspension, bacterial strain A3 bacterial suspension and water are inoculated on BBL solid culture medium, mature single colony is formed after anaerobic culture for 1 day at 37deg.C, and culture medium containing indicator bacteria (Staphylococcus aureus ATCC 6538, escherichia coli 8099, salmonella paratyphi CMCC50094, listeria monocytogenes ATCC19114, shigella dysenteriae CMCC51252, enterococcus faecalis ATCC29212 and Clostridium perfringens ATCC 13124) is poured into upper layer, and the final concentration of indicator bacteria is 10 mL/dish 6 After CFU/mL is solidified, the bacteria are placed under the proper growth conditions of each indicator bacteria and are respectively cultivated for 16-18 hours, and whether the bacteria inhibition zone and the size of the bacteria inhibition zone exist around the VB301 single colony or not is observed and recorded, and whether the activity of the indicator bacteria is inhibited or not is judged.
Table 4: VB301 evaluation of bacteriostatic ability of 7 pathogenic bacteria (diameter of zone of inhibition, mm)
The results are shown in Table 4, the animal bifidobacterium lactosub-species VB301 has antibacterial effect on 7 pathogenic bacteria, and the other animal bifidobacterium lactosub-species A3 strain has antibacterial activity on 2 pathogenic bacteria, so that the strain can treat related infectious diseases of 7 pathogenic bacteria, including urinary tract infection, suppurative abdominal infection, septicemia, endocarditis, diarrhea, fever and the like.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. The bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) VB301 is characterized in that the bifidobacterium animalis subsp (Bifidobacterium animalis subsp. Lactis) VB301 has a preservation number of CGMCC No.27998.
2. A fermentation supernatant comprising a metabolite of bifidobacterium animalis subspecies lactis VB301 as claimed in claim 1.
3. A bacterial suspension comprising bifidobacterium animalis subspecies lactis VB301 as claimed in claim 1.
4. Use of bifidobacterium animalis subspecies VB301, the fermentation supernatant of claim 2 or the bacterial suspension of claim 3 in the manufacture of a medicament for the prevention and/or treatment of diseases associated with high cholesterol;
optionally, the hypercholesterolemia-related disease comprises any one of hypercholesterolemia, cardiovascular disease, arteriosclerosis, and peripheral vascular disease;
optionally, the drug is capable of degrading six bile salts, the six bile salts being sodium taurocholate, sodium taurochenodeoxycholate, sodium taurodeoxycholate, sodium glycocholate, sodium glycochenodeoxycholate, sodium glycodeoxycholate.
5. Use of a bifidobacterium animalis subspecies VB301 or a bacterial suspension as claimed in claim 3 in the manufacture of a medicament for the treatment of a disease associated with at least one of staphylococcus aureus, escherichia coli, salmonella paratyphi b, listeria monocytogenes, shigella dysenteriae, enterococcus faecalis, clostridium perfringens.
6. A medicament comprising at least one of bifidobacterium animalis subspecies lactis VB301 as claimed in claim 1, a fermentation supernatant as claimed in claim 2 and a bacterial suspension as claimed in claim 3.
7. The medicament according to claim 6, characterized in that it further comprises excipients and/or carriers.
8. The medicament according to claim 7, wherein the excipient comprises at least one selected from the group consisting of binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and sustained-release agents.
9. The medicament according to claim 7, wherein the carrier comprises at least one selected from the group consisting of saccharides, celluloses and derivatives thereof, calcium phosphates, alkaline earth metal salts of stearic acid, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolytic solids.
10. The drug according to claim 6, wherein the dosage form of the drug comprises at least one selected from the group consisting of oral liquid, powder, granule, capsule, tablet, and drop pill.
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