CN117903965A - Acremonium muciniphilum VB202 and application thereof - Google Patents
Acremonium muciniphilum VB202 and application thereof Download PDFInfo
- Publication number
- CN117903965A CN117903965A CN202311658889.8A CN202311658889A CN117903965A CN 117903965 A CN117903965 A CN 117903965A CN 202311658889 A CN202311658889 A CN 202311658889A CN 117903965 A CN117903965 A CN 117903965A
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- microorganism
- strain
- fermentation broth
- bacterial suspension
- acremonium
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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Abstract
The invention discloses a mucin-philin Acremonium (AKKERMANSIA MUCINIPHILA) VB202 and application thereof, and the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 28295 in the year 2023, month 8 and 29. The mucin-philin Acremonium provided by the invention has strong antibacterial capability, can be used for replacing antibiotics, has good blood sugar reducing capability and intestinal tract tolerance capability, and can be applied to preparing blood sugar control products.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a mucin-philin Acremonium VB202 and application thereof.
Background
Acremonium muciniphilum (AKKERMANSIA MUCINIPHILA, AKK) is a novel intestinal microorganism first found in healthy human feces in 2004, derrien, etc., akkermansia belonging to the family Oncomelanocortidae, belonging to the genus Oncomelanocortidae, is colonized on the intestinal mucosa layer by taking mucin as the only nitrogen source and carbon source. AKKERMANSIA MUCINIPHILA is widely distributed in the intestinal tracts of healthy infants and adults, is stably planted in the intestinal tracts after 1 year of birth and accounts for 1% -3% of the total number of intestinal microorganisms of the adults, and is an important component of intestinal flora. The research shows that the content of the bacteria in the intestinal tracts of patients with various diseases is obviously reduced, and the content of the bacteria is related to the disease conditions, so the bacteria are hopeful to be developed into next generation probiotics after bifidobacteria and lactobacillus (O'Toole PW,Marchesi JR,Hill C.Next-generation probiotics:the spec-trum from probiotics tolive biotherapeutics[J].Nat Microbiol,2017,2:17057).
Different types of pathogenic bacteria not only can cause food safety problems, but also can infect humans and animals to cause related diseases and even threaten life, and at present, antibiotics or chemical synthetic drugs are mainly adopted to inhibit or kill the pathogenic bacteria so as to realize effective control. However, as the drug resistance of pathogenic bacteria is continuously enhanced, irreversible damage such as residue in chemical drug bodies, environmental pollution and the like frequently occurs, and serious challenges are presented to the sustainable development of human health and socioeconomic performance. The 194 th bulletin issued by the agricultural rural department of China in 7 months in 2019 indicates that the research of completely prohibiting the addition of antibiotics and antibiotic substitutes in the feed of China from 1 month 1 day in 2020 becomes increasingly urgent. The probiotics can generate relevant metabolites with inhibition effect on pathogenic bacteria in the growth and propagation process, and can improve the intestinal acid environment of animal organisms, optimize the dynamic balance of intestinal microorganisms, stimulate the intestinal mucosa immunity of the organisms, inhibit the colonization of harmful bacteria and improve the immunity of the organisms, thereby improving the production performance and the health level of the animals. Has wide development prospect in the natural prevention and control aspect of pathogenic bacteria.
At present, many studies are conducted on the correlation between the AKK content and various disease conditions, but few studies are conducted on the antibacterial properties, so that it is highly desirable to provide AKK bacteria with antibacterial properties.
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 microorganism, which is akkermansia muciniphila (AKKERMANSIA MUCINIPHILA) VB202, deposited in the China general microbiological culture collection center (ccm) under the accession number CGMCC No.28295, at 8/29 of 2023.
The mucin-philin Acremonium VB202 provided by the invention has better antibacterial capability, intestinal tract tolerance capability and blood sugar reducing capability, can inhibit the growth of harmful bacteria such as helicobacter pylori, listeria monocytogenes and Shigella on one hand, helps the organism to restore the balance of microbiome without causing drug resistance, can be used as a safer antibiotic substitute, and has the blood sugar reducing effect on the other hand, and can be used for preparing blood sugar control products.
According to an embodiment of the invention, the microorganism has a 16S rDNA sequence as shown in SEQ ID NO. 1.
In a second aspect, the invention provides a fermentation broth obtained by fermentation of a microorganism according to the first aspect.
In a third aspect the invention provides a bacterial suspension comprising a microorganism according to the first aspect.
In a fourth aspect, the invention provides the use of a microorganism according to the first aspect, a fermentation broth according to the second aspect or a bacterial suspension according to the third aspect for the preparation of a medicament, feed, additive for inhibiting pathogenic bacterial activity.
According to an embodiment of the present invention, the pathogenic bacteria are selected from at least one of helicobacter pylori, listeria monocytogenes, shigella.
In a fifth aspect, the invention provides the use of a microorganism according to the first aspect, a fermentation broth according to the second aspect or a bacterial suspension according to the third aspect for the preparation of a medicament, feed, additive for controlling or reducing blood glucose.
In a sixth aspect, the invention provides a composition comprising at least one of a microorganism according to the first aspect, a fermentation broth according to the second aspect, and a bacterial suspension according to the third aspect.
According to an embodiment of the invention, the composition further comprises an excipient and/or carrier.
According to an embodiment of the present invention, the excipient includes at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a slow-release agent.
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, and cereal hydrolytic solids.
According to an embodiment of the present invention, the formulation of the composition includes at least one selected from the group consisting of oral liquid, powder, granule, capsule, tablet, and drop pill.
Compared with the prior art, the invention has the beneficial effects that:
The mucin-philin Acremonium VB202 provided by the invention has stronger intestinal tract tolerance and antibacterial capacity, can help organisms restore the balance of microbiome by inhibiting the growth of harmful bacteria, can not cause the generation of drug resistance, and can be used as a safer antibiotic substitute. In addition, the strain also has the function of reducing blood sugar, does not produce gas, has the function of synergizing anti-inflammatory, can be used for preparing blood sugar control products and the like, and has wider application prospect.
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: acremonium muciniphilum AKKERMANSIA MUCINIPHILA VB202,202
Preservation date: 2023, 8, 29
Preservation unit: china general microbiological culture Collection center (Beijing Kogyo district beichen Xiyu No.1, 3)
Preservation number: CGMCC No.28295.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a photograph of a colony of strain VB202 provided by the invention on a culture medium;
FIG. 2 shows a gram of strain VB202 provided by the invention;
FIG. 3 shows a microscopic photograph of strain VB202 provided by the present invention;
FIG. 4 shows a phylogenetic tree of strain VB202 provided by the present invention;
FIG. 5 shows the acid resistance test results of strain VB202 provided by the invention;
FIG. 6 shows the results of the bile salt tolerance test of strain VB202 provided by the invention;
FIG. 7 shows the results of the intestinal juice test of strain VB202 provided by the invention;
Fig. 8 shows the results of the hypoglycemic test of the strain VB202 provided by the invention.
Detailed Description
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.
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 endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other 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.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but 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.
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.
Herein, the term "carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those of skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
According to an embodiment of the present invention, there is provided a microorganism, which is Acremonium muciniphilum (AKKERMANSIA MUCINIPHILA) VB202 deposited at China general microbiological culture Collection center, with a deposit number of CGMCC No.28295, at about 29/2023/8. The mucin-philin Acremodelling bacteria VB202 provided by the invention has strong antibacterial capability, can be used for replacing antibiotics, and has good blood sugar reducing capability and intestinal tract tolerance capability. The hypoglycemic drugs in the current market have certain side effects such as gas production, intestinal inflammation and the like, and the mucin-philin Acremonium VB202 provided by the invention has the effect of reducing blood sugar, has the effects of no gas production and synergistic anti-inflammatory effect, and can be applied to the preparation of blood sugar control products.
Herein, "akkermansia muciniphila (AKKERMANSIA MUCINIPHILA) VB202" is synonymous with "akkermansia muciniphila VB202", "strain VB 202".
The akkermansia muciniphila VB202 provided by the invention has strong inhibition capability on three pathogenic bacteria, wherein the pathogenic bacteria are helicobacter pylori, listeria monocytogenes and shigella respectively.
The akkermansia muciniphila VB202 provided by the invention also has good blood sugar reducing capability and stronger intestinal tract tolerance capability. Specifically, the effect of the mucin Acremonium VB202 provided by the invention on the survival of the thalli is small when the mucin Acremonium VB202 acts in 0.03-0.3% of bovine choline solution for 4 hours, which proves that the strain VB202 has very strong tolerance to bovine choline. The gastric juice can survive for at least 4 hours in simulated gastric juice with pH of 3.0-6.0, can survive for 4 hours in simulated intestinal juice with pH of 6.8, and the survival rate of 4 hours reaches 73.56%, which shows that the Alkermansia muciniphila VB202 has good activity tolerance capability on the gastrointestinal tract environment and can play a role in the intestinal tract for a long time.
According to a specific embodiment of the present invention, there is provided a fermentation broth fermented from the aforementioned microorganism.
According to a specific embodiment of the present invention, there is provided a bacterial suspension comprising the aforementioned microorganism.
According to a specific embodiment of the invention, the invention provides the use of the aforementioned microorganism, fermentation broth or bacterial suspension for the preparation of a medicament, feed, additive for inhibiting pathogenic bacterial activity or for controlling or reducing blood glucose.
According to a specific embodiment of the present invention, there is provided a composition comprising at least one of the aforementioned microorganisms, fermentation broths or bacterial suspensions.
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 collection and identification of strains
1. Isolation and purification of strains
The invention provides a mucin-philin Acremonium VB202 which is separated from healthy adult feces, and specifically comprises the following steps: adding 0.5g of feces into 10mL of PBS solution containing 0.05% cysteine to obtain stock solution, concentrating the stock solution to 10 5 times, respectively inoculating into AKK enrichment medium, placing into an anaerobic tank at 37 ℃ for enrichment culture for 4d, and inoculating with 10%. The DNA of the strains with different dilutions is extracted, gene amplification is carried out by adopting 16S full-length primer 27F (SEQ ID NO:2:5 '-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO:3:5 '-TACGGCTACCTTGTTACGACTT-3'), PCR amplified products are detected by using 1.5% agarose gel, a tube with positive PCR identification result and highest dilution degree is selected, and the positive tube is stored in BHI+10% glycerol protectant and is stored at-80 ℃.
Wherein the enrichment medium comprises the following components (in W/V): BHI medium 3.85%, mucin 0.25%, L-cysteine 0.05%, and sterilizing at 115deg.C for 20min.
2. Identification of strains
(1) Morphological characteristics:
bacterial strain VB202 is anaerobically cultured on an isolated culture medium at 37 ℃ for 4-5 days to form circular colonies, the diameters of the colonies are 1-3mm, the surfaces of the bacterial colonies are glossy, the bacterial colonies are milky white, the middle of the bacterial colonies are light yellowish, and the bacterial colony morphology is shown in figure 1.
Wherein the isolation medium composition is (in W/V): BHI medium 3.85%, mucin 0.25%, L-cysteine 0.05%, agar 1.8%, and sterilizing at 115deg.C for 20min.
(2) Physiological and biochemical characteristics:
Clean slides were taken for gram staining and microscopic examination to observe the microscopic morphology of the strain, as shown in fig. 2 and 3. The use of different carbon sources is shown in Table 1, and the use of different nitrogen sources is shown in Table 2. The results show that the strain can grow by utilizing carbon sources such as glucose, fructose and maltose, can also grow by utilizing nitrogen sources such as tryptone, polypeptone and hydrolyzed casein, has slower effect in soybean meal, soybean meal and yeast extract powder FM860, has the later effect similar to that of the soybean peptone OX and the yeast powder 601, and simultaneously does not utilize monosodium glutamate in the growth of the strain, and the malt extract powder has an inhibition effect on thalli.
Table 1: carbon source utilization of strain VB202
| Carbon source | VB202 growth conditions | Carbon source | VB202 growth conditions |
| Availability starch | + | Xylitol | + |
| Corn starch | + | Glucose | ++++++ |
| Glutinous rice flour | + | Glycerol | + |
| Maltodextrin | + | Lactose and lactose | +++ |
| Sucrose | ++ | Fructose | +++++ |
| Mannitol (mannitol) | + | Maltose | ++++ |
Note that: +: the carbon source can be utilized.
Table 2: nitrogen source utilization condition of strain VB202
| Nitrogen source | Growth conditions | Nitrogen source | Growth conditions |
| Soybean powder | +++ | Soytone OX | ++++ |
| Tryptone | +++++ | Hydrolyzed casein | +++++ |
| Bean pulp powder | +++ | Monosodium glutamate | - |
| Polypeptone | +++++ | Yeast powder 601 | ++++ |
| Cottonseed fine powder | ++ | Yeast peptone 103 | ++++ |
| Corn protein powder | ++ | Peptide powder | ++++ |
| Malt extract powder | -- | Bactopeptone | ++++ |
| Corn starch | ++ | Urea | + |
| Yeast leaching powder FM860 | +++ | Ammonium sulfate | + |
Note that: +: the nitrogen source can be utilized, -: the nitrogen source cannot be utilized.
(3) 16S rDNA Gene analysis
The obtained positive tube is concentrated to 10 5 times by PBS solution, coated on AKK separation culture medium, placed in an anaerobic box at 37 ℃ for 4 to 5 days, selected circular single colony with the diameter of 1mm is inoculated in AKK primary screening culture medium for culture, and 16S rDNA sequencing analysis is carried out. BLAST comparison of sequencing results is performed, specifically using a method based on k-mer frequency fingerprinting. A length 16 k-mer is first extracted from each sequence and counted for occurrences to generate a k-mer frequency fingerprint. The cosine distance is then used to calculate the similarity between the k-mer frequency fingerprints of the two sequences. From these distance values, a phylogenetic tree was constructed, as shown in fig. 4. The obtained strain was found to have a close relationship with Akkermansia_ muciniphilaATCC _BAA-835 and then formed a larger population with Akkermansia_ MASSILIENSIS MARSEILLE-P6666 and PJKB _sGP22, showing that the obtained strain has a relatively close relationship with these strains, so that the strain was identified as a strain of Ackermansis mucin, VB202, which was deposited in China general microbiological culture Collection center, CGMCC No.28295, at 8/29 of 2023.
Wherein the primary screening culture medium comprises the following components (in W/V): BHI medium 3.85%, L-cysteine 0.05%, and sterilizing at 115℃for 20min.
The 16S rDNA sequence of the strain is:
CCGACATTGGGGGGGTGGAAAGGACATGCAGTCGAACGAGAGAATTGCTAGCTTGCTAATAATTCTCTAGTGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCCCCGAGAGCGGGATAGCCCTGGGAAACTGGGATTAATACCGCATAGTATCGAAAGATTAAAGCAGCAATGCGCTTGGGGATGGGCTCGCGGCCTATTAGTTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGACGGGTAGCCGGTCTGAGAGGATGTCCGGCCACACTGGAACTGAGACACGGTCCAGACACCTACGGGTGGCAGCAGTCGAGAATCATTCACAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGGAATGAAGGTCTTCGGATTGTAAACCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCAAGCGTTGTTCGGAATCACTGGGCGTAAAGCGTGCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAGGCGCGGGCTCAACCCGCGGACGGCACATGATACTGCGAGACTAGAGTAATGGAGGGGGAACCGGAATTCTCGGTGTAGCAGTGAAATGCGTAGATATCGAGAGGAACACTCGTGGCGAAGGCGGGTTCCTGGACATTAACTGACGCTGAGGCACGAAGGCCAGGGGAGCGAAAGGGATTAGATACCCCTGTAGTCCTGGCAGTAAACGGTGCACGCTTGGTGTGCGGGGAATCGACCCCCTGCGTGCCGGAGCTAACGCGTTAAGCGTGCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGAAATTGACGGGGACCCGCACAAGCGGTGGAGTATGTGGCTTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTAATGAACAACATGTGAAAGCATGCGACTCTTCGGAGGCGTTACACAGGTGCTGCATGGCCGTCGTCAGCTCGTGTCGTGAGATGTTTGGTTAAGTCCAGCAACGAGCGCAACCCCTGTTGCCAGTTACCAGCACGTGAAGGTGGGGACTCTGGCGAGACTGCCCAGATCAACTGGGAGGAAGGTGGGGACGACGTCAGGTCAGTATGGCCCTTATGCCCAGGGCTGCACACGTACTACAATGCCCAGTACAGAGGGGGCCGAAGCCGCGAGGCGGAGGAAATCCTAAAAACTGGGCCCAGTTCGGACTGTAGGCTGCAACCCGCCTACACGAAGCCGGAATCGCTAGTAATGGCGCATCAGCTACGGCGCCGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACATCATGGAAGCCGGTCGCACCCGAAGTATCTGAAGCCAACCGCAAGGAGGCAGGTCCCTCAGGGTTGTCTTG(SEQ IDNO:1).
Example 2 preparation of fermentation broth and physiological and Biochemical Properties of Strain VB202
The glycerol tube preserved with the strain VB202 is coated on an AKK separation culture medium after being diluted in a gradient way, cultured for 4-5 days at 37 ℃, single colony with the diameter of 1-3mm is selected, inoculated on an AKK enrichment culture medium, the inoculated amount is 10 7 CFU/mL, cultured for 24-30 hours at 37 ℃ under the condition of 60-80rpm, when the pH value of the seed solution is 5.5-6.8, the OD value is 0.8-1.5 and the AKK bacterial amount is 5X 10 8～2×109 CFU/mL, 3% inoculated amount is inoculated on a fermentation tank, cultured for 30-48 hours at 37 ℃ under the condition of 60rpm, and the physiological and biochemical characteristic analysis is carried out, and the detection results are shown in tables 3-4.
Table 3: physiological and biochemical characteristics of strain VB202
Note that: positive for + negative for-negative.
Table 4: physiological and biochemical characteristics of strain VB202
| Authentication item | VB202 | Authentication item | VB202 |
| Raffinose | + | MR test | - |
| Ribitol | + | Peptone water | - |
| Melibiose | + | Urease enzyme | - |
| Sorbitol | + | Hydrogen sulfide | - |
| Inositol (inositol) | + | Citrate salt | - |
| Mannitol (mannitol) | + | Semisolid agar | - |
| Phenylalanine (Phe) | - | Arginine (Arg) | + |
| VP test | - | Ornithine | - |
Note that: positive for + negative for-negative.
Example 3 evaluation of the ability of Strain VB202 to withstand the gastrointestinal tract
Preparation of artificial simulated gastric juice: preparing 1% sodium chloride solutions with pH of 1, 2, 3, 4, 5 and 6 respectively, sterilizing at 121deg.C for 30min, and adding pepsin according to 0.3%;
artificial intestinal juice: r22156-500ml, pH 6.8, shanghai-derived leaf organism;
Manually simulating pancreatic juice: firstly, preparing 1% sodium chloride solution, adding 0.03%, 0.1% and 0.3% of bovine choline respectively, sterilizing at 121deg.C for 30min, and adding 0.1% of trypsin.
Preparation of bacterial suspension: taking counted bacterial suspension (the viable bacteria count is required to reach about 10 9-1010 CFU/ml), thawing, and adding the bacterial suspension into the split-packed artificial simulated gastric juice, intestinal juice and pancreatic juice, wherein the adding amount is as follows: after mixing 10. Mu.L of bacterial suspension and 990. Mu.L of simulated liquid, anaerobic culture is carried out at 37 ℃, viable bacteria counts are sampled at 0h, 2.5h and 4h respectively, and survival rates of 2.5h and 4h are calculated.
The results of the acid resistance test are shown in FIG. 5. Wherein, strain VB202 dies instantly in simulated gastric fluid at pH 1; all died in simulated gastric fluid at pH 2 for 2.5 hours and survived stably in simulated gastric fluid at pH 3-6 for 4 hours. The results of the cholate resistance test are shown in FIG. 6. Wherein, the strain VB202 can stably survive in 4 hours in a 0.03-0.3% bovine choline system. The results of the intestinal juice test are shown in fig. 7. Wherein, the survival rate of the strain VB202 in simulated intestinal fluid for 2.5 hours is about 84.28 percent and the survival rate of the strain VB202 in simulated intestinal fluid for 4 hours is about 73.56 percent. Taken together, it is demonstrated that strain VB202 has a very good active tolerability to the gastrointestinal tract environment.
EXAMPLE 4 bacteriostasis test of Strain VB202
The antibacterial capacity of the strain is tested by adopting a double-layer plate culture method, and whether the strain VB202 has the antibacterial capacity is evaluated according to the growth condition of upper pathogenic bacteria. The specific method comprises the following steps:
After thawing the activated strain VB202 frozen tube, inoculating the frozen tube into AKK separation culture medium, and performing anaerobic culture at 37 ℃ for 4-5 days, wherein the strain inoculation amount is 2 μl/point. Cooling the sterilized upper layer culture medium to 40-50deg.C, adding different pathogenic bacteria (helicobacter pylori ATCC26695, listeria monocytogenes ATCC19114, shigella CMCC 51252) into the culture medium (final concentration of pathogenic bacteria in the culture medium is about 10 6 CFU/ml), mixing, adding the culture medium containing pathogenic bacteria into the single bacteria plate according to 7 ml/dish, culturing respectively according to different pathogenic bacteria growth conditions after solidification, observing whether there is a bacteriostasis zone and the size of the bacteriostasis zone around the single colony of VB202, and judging whether there is bacteriostasis capability, and the result is shown in Table 5.
As shown in Table 5, the mucin-philin Acremodelling bacterium VB202 has the effect of inhibiting helicobacter pylori, listeria monocytogenes and Shigella.
Wherein, the culturing conditions of helicobacter pylori are as follows: fetal bovine serum Broth medium was microaerophilically cultured at 37℃for 3 days. The culture conditions of listeria monocytogenes are: LB medium, aerobic culture at 37℃for1 day. The culture conditions of shigella are: LB medium, aerobic culture at 37℃for1 day.
Table 5: evaluation of bacteriostatic ability of strain VB202 on three pathogenic bacteria
EXAMPLE 5 hypoglycemic test of Strain VB202
A model animal dbdb male with 12 weeks old gene-deficient type 2 diabetes is selected, after the model animal is adapted to one week, blood is collected by cutting the tail, blood glucose is measured once a week, fasting blood glucose is measured once a week, 30 mice with fasting blood glucose value larger than 11.1mmol/L are selected after feeding for one month, the mice are divided into 2 groups, 10 groups and one group of control, one group is given with fresh viable bacteria VB202 each time, the dosage is 10 10 CFU, the administration is carried out once every 12 hours, and the fasting blood glucose is measured once a week. As shown in FIG. 8, strain VB202 has a better effect of reducing fasting blood glucose in a mouse model.
In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," 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 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. A microorganism is characterized in that the microorganism is Alkermansia muciniphila (AKKERMANSIA MUCINIPHILA) VB202, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 28295 in the 8 th month of 2023.
2. The microorganism of claim 1, wherein the microorganism has a 16S rDNA sequence as set forth in SEQ ID NO. 1.
3. A fermentation broth, characterized in that it is fermented by a microorganism according to claim 1 or 2.
4. A bacterial suspension comprising the microorganism of claim 1 or 2.
5. Use of a microorganism according to claim 1 or 2, a fermentation broth according to claim 3 or a bacterial suspension according to claim 4 for the preparation of a medicament, feed, additive for inhibiting pathogenic bacterial activity.
6. The use according to claim 5, wherein the pathogenic bacteria are selected from at least one of helicobacter pylori, listeria monocytogenes, shigella.
7. Use of a microorganism according to claim 1 or 2, a fermentation broth according to claim 3 or a bacterial suspension according to claim 4 for the preparation of a medicament, feed, additive for controlling or reducing blood glucose.
8. A composition comprising at least one of the microorganism of claim 1 or 2, the fermentation broth of claim 3, and the bacterial suspension of claim 4.
9. The composition of claim 8, further comprising an excipient and/or carrier.
10. The composition according to claim 9, wherein the excipient comprises at least one selected from the group consisting of binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, slow-release agents;
Optionally, the carrier comprises at least one selected from saccharides, cellulose 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;
Optionally, the formulation of the composition comprises at least one selected from oral liquid, powder, granules, capsules, tablets and dripping pills.
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| CN202311658889.8A CN117903965A (en) | 2023-12-05 | 2023-12-05 | Acremonium muciniphilum VB202 and application thereof |
| PCT/CN2024/108440 WO2025118641A1 (en) | 2023-12-05 | 2024-07-30 | Akkermansia muciniphila vb202 and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119662483A (en) * | 2024-12-27 | 2025-03-21 | 武汉微康益生菌研究院有限公司 | Acremonium muciniphilum Akk with blood sugar reducing capability, application, product and method thereof |
| WO2025118641A1 (en) * | 2023-12-05 | 2025-06-12 | 杭州微致生物科技有限公司 | Akkermansia muciniphila vb202 and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964650B (en) * | 2018-09-27 | 2022-10-11 | 上海上药信谊药厂有限公司 | Bacterial strain for preventing and treating metabolic diseases and application thereof |
| KR102197180B1 (en) * | 2018-10-11 | 2020-12-31 | 주식회사 고바이오랩 | Akkermansia muciniphila and composition for controlling appetite or preventing, improving, relieving and treating metabolic disease comprising the same |
| KR102128287B1 (en) * | 2019-08-23 | 2020-06-30 | 주식회사 엔테로바이옴 | NEW Akkermansia muciniphila EB-AMDK19 strain AND uses thereof |
| JP2023542447A (en) * | 2020-04-03 | 2023-10-10 | デュポン ニュートリション バイオサイエンシス エーピーエス | Compositions containing bacterial strains to improve metabolic health |
| CN113322202B (en) * | 2021-05-31 | 2022-03-01 | 君维安(武汉)生命科技有限公司 | Ackermanella, culture method and application thereof |
| CN117903965A (en) * | 2023-12-05 | 2024-04-19 | 杭州微致生物科技有限公司 | Acremonium muciniphilum VB202 and application thereof |
-
2023
- 2023-12-05 CN CN202311658889.8A patent/CN117903965A/en active Pending
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2024
- 2024-07-30 WO PCT/CN2024/108440 patent/WO2025118641A1/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025118641A1 (en) * | 2023-12-05 | 2025-06-12 | 杭州微致生物科技有限公司 | Akkermansia muciniphila vb202 and use thereof |
| CN119662483A (en) * | 2024-12-27 | 2025-03-21 | 武汉微康益生菌研究院有限公司 | Acremonium muciniphilum Akk with blood sugar reducing capability, application, product and method thereof |
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| WO2025118641A1 (en) | 2025-06-12 |
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