CN110616163A - Enterococcus faecalis capable of reducing concentration of trimethylamine and preparation method and application thereof - Google Patents

Enterococcus faecalis capable of reducing concentration of trimethylamine and preparation method and application thereof Download PDF

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CN110616163A
CN110616163A CN201810636528.6A CN201810636528A CN110616163A CN 110616163 A CN110616163 A CN 110616163A CN 201810636528 A CN201810636528 A CN 201810636528A CN 110616163 A CN110616163 A CN 110616163A
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trimethylamine
enterococcus faecalis
concentration
reducing
enterococcus
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CN110616163B (en
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周宏伟
陈怡然
曾念宜
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Southern Medical University Zhujiang Hospital
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Southern Medical University Zhujiang Hospital
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

The invention relates to enterococcus faecalis capable of reducing trimethylamine concentration and a preparation method and application thereof. Enterococcus faecalis capable of reducing trimethylamine concentration and has a accession number of GDMCC NO 60361. The enterococcus faecalis capable of reducing the concentration of trimethylamine is proved to be capable of decomposing the trimethylamine in vivo by taking the trimethylamine as a carbon source and obviously reducing the concentration of the trimethylamine oxide in blood. The enterococcus faecalis capable of reducing the concentration of trimethylamine can be applied to the preparation of medicines for treating kidney diseases, medicines for treating trimethylaminuria and medicines for preventing or treating cardiovascular diseases.

Description

Enterococcus faecalis capable of reducing concentration of trimethylamine and preparation method and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to enterococcus faecalis capable of reducing trimethylamine concentration and a preparation method and application thereof.
Background
Trimethylamine (TMA) in human intestinal tract is mainly from food containing choline or trimethylamine structure, such as phosphatidylcholine, betaine, l-carnitine, etc., and is metabolized into TMA under the action of trimethylamine lyase produced by intestinal microorganisms, TMA enters liver through portal circulation in intestinal tract, and is produced into trimethylamine oxide (TMAO) under the action of flavin oxygenase (FMOs) in liver, and then excreted from kidney.
Plasma concentrations of TMAO in healthy humans were maintained at normal levels. Under disease conditions, TMAO accumulates in the body and its serum concentration rises significantly.
In addition, excessive TMAO in the blood vessel can promote atherosclerosis in humans, which can cause cardiovascular disease and may also lead to an increased risk of heart disease.
In addition, for the diseases of trimethylamine metabolic disorder such as trimethylamine urine disease (or called TMAU, TMA-induced uremia, or smelly fish syndrome), TMA in the liver cannot be efficiently converted into TMAO due to gene defect of a class of enzymes (flavin monooxygenase, FMOs) in the patient, and is currently mainly solved by reducing the intake of TMA.
However, there is currently a lack of drugs that are effective in reducing trimethylamine.
Disclosure of Invention
Based on the above, there is a need for enterococcus faecalis capable of reducing trimethylamine concentration, and a preparation method and application thereof.
Enterococcus faecalis capable of reducing trimethylamine concentration and has a accession number of GDMCC NO 60361.
A preparation method of enterococcus faecalis capable of reducing trimethylamine concentration comprises the following steps:
diluting fresh feces of healthy people 103Multiple to 105Doubling to obtain a diluent;
inoculating the diluent to a brain-heart leaching solution agar culture medium by adopting a plate marking method, and carrying out anaerobic culture at 37 ℃ for 5-7 days to obtain a plurality of single colonies; and
and respectively extracting DNA of a plurality of single colonies, amplifying and determining the 16S rDNA sequence of the single colonies, comparing the determination result with the 16S rDNA sequence of the enterococcus faecalis, and screening to obtain the enterococcus faecalis capable of reducing the concentration of trimethylamine.
The application of enterococcus faecalis capable of reducing the concentration of trimethylamine in reducing the concentration of the trimethylamine.
The application of the enterococcus faecalis capable of reducing the concentration of trimethylamine in the preparation of functional food capable of reducing the concentration of the trimethylamine.
The enterococcus faecalis capable of reducing the concentration of trimethylamine is applied to the preparation of medicaments for preventing or treating cardiovascular diseases.
A composition for reducing the concentration of trimethylamine, which comprises the enterococcus faecalis capable of reducing the concentration of the trimethylamine.
A composition for reducing the concentration of trimethylamine oxide in blood comprises the enterococcus faecalis capable of reducing the concentration of trimethylamine.
A medicament for preventing or treating cardiovascular diseases comprises the enterococcus faecalis capable of reducing the concentration of trimethylamine.
A functional food for preventing cardiovascular diseases comprises enterococcus faecalis capable of reducing trimethylamine concentration.
In one embodiment, the functional food is a capsule, a tablet, a lotion, a powder, an oral liquid, or a granule.
A medicament for preventing or treating cardiovascular diseases comprises the enterococcus faecalis capable of reducing the concentration of trimethylamine.
The enterococcus faecalis capable of reducing the concentration of trimethylamine is proved to be capable of degrading the trimethylamine in vivo and further capable of obviously reducing the concentration of the trimethylamine oxide in blood.
Drawings
FIG. 1 is a distribution diagram of the flora in a dilution of feces from a healthy person according to example 1;
FIG. 2 is a graph comparing the relative abundance of Enterococcus (Enterococcus) in the fecal dilution of healthy persons of example 1 in the trimethylamine group and the control group;
FIG. 3 is a graph comparing the relative abundance of Lactobacillus in the stool dilution of healthy persons of example 1 in the trimethylamine group and the control group;
FIG. 4 is a graph comparing the relative abundance of Eggerthella in the stool dilution of a healthy person of example 1 in the trimethylamine group and the control group;
FIG. 5 is a graph comparing the relative abundance of Facklamia in the stool dilution of healthy persons of example 1 in the trimethylamine group and the control group;
FIG. 6 is a graph comparing the relative abundance of Veillonella (Veillonella) in the fecal dilution of healthy persons of example 1 in the trimethylamine and control groups;
FIG. 7 is a graph comparing the relative abundance of Plesiomonas in the trimethylamine group and the control group in the fecal dilution of a healthy person of example 1;
FIG. 8 is a graph comparing the relative abundance of Blauteria (Blautia) in a stool dilution of a healthy person of example 1 in the trimethylamine group and the control group;
FIG. 9 is a graph comparing the relative abundance of Bacillus (Bacillus) in the dilution of stool from healthy persons of example 1 in the trimethylamine group and the control group;
FIG. 10 is a graph comparing the relative abundance of Alkaliphilus in the fecal diluent of healthy persons of example 1 in the trimethylamine group and the control group;
FIG. 11 is a graph showing the relative abundance of Campylobacter (Campylobacter) in the stool dilution of a healthy person in example 1 in the trimethylamine group and the control group;
FIG. 12 is a graph comparing the relative abundance of Rhynchosia loiridis (Vagococcus) in fecal dilution in healthy persons of example 1 in trimethylamine and control groups;
FIG. 13 is a graph comparing the relative abundance of Brevibacterium curvatum (Varibacterium) in the fecal diluent of a healthy person of example 1 in the trimethylamine group and the control group;
FIG. 14 is a graph showing a comparison of the relative abundance of Hydrogenophaga (Hydrogenophaga) in a dilution liquid of feces of a healthy person in example 1 in the trimethylamine group and the control group;
FIG. 15 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of the fecal sample 1 of example 1;
FIG. 16 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 2 of example 1;
FIG. 17 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 3 of example 1;
FIG. 18 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 4 of example 1;
FIG. 19 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of the fecal sample 5 of example 1;
FIG. 20 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 6 of example 1;
FIG. 21 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 7 of example 1;
FIG. 22 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 8 of example 1;
FIG. 23 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 9 of example 1;
FIG. 24 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of the fecal sample 10 of example 1;
FIG. 25 is a graph comparing the total amount of OTUs of the genus enterococcus in the trimethylamine group and the control group of fecal sample 11 of example 1;
FIG. 26 is a graph showing the results of color development of 2, 3, 5-triphenyltetrazolium chloride in various culture solutions of example 6;
FIG. 27 is a graph showing the results of the animal experiment in example 8.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Enterococcus faecalis capable of reducing trimethylamine concentration according to an embodiment was deposited at the Guangdong province culture Collection (GDMCC) at 23/4/2018, address: guangzhou city, Jielizhou 100 large yard No. 59 building No. 5, the deposit number is GDMCCNO 60361, the classification name: enterococcus faecalis TMAO-D. The enterococcus faecalis is derived from human feces.
The enterococcus faecalis can decompose trimethylamine by using the trimethylamine as a carbon source, and can obviously reduce the concentration of the trimethylamine oxide in blood.
Microorganisms belong to products that cannot be clearly characterized by structural and/or compositional features alone, and cannot be fully characterized by features other than the manufacturing process. The claims to this type of product allow characterization by the preparation method, according to the provisions of chapter 4.3, chapter tenth, part ii of the examination guidelines.
One embodiment of the present invention provides a method for preparing enterococcus faecalis capable of reducing trimethylamine concentration, comprising the following steps S110 to S120.
S110, screening fresh excrement of healthy people to obtain the bacterium capable of reducing the trimethylamine concentration.
Specifically, fresh excrement diluents of a plurality of healthy people are respectively inoculated into a plurality of trimethylamine brain heart infusion broth (BHI broth) culture mediums and a plurality of non-trimethylamine brain heart infusion broth (BHI broth) culture mediums, anaerobic culture is carried out to obtain a plurality of cultures, then DNA of the plurality of cultures is respectively extracted, 16S rDNV 4 universal primers are selected for amplification, and amplification products are sequenced. And then analyzing the sequencing data by adopting a BIPES analysis process to obtain the genus capable of reducing the trimethylamine concentration.
Fresh feces of healthy persons means feces removed from persons indicated to be free of digestive tract diseases (preservation time at 4 ℃ C. is less than 24 hours).
Further, the faecal diluent is diluted 10 by fresh faeces of healthy persons3Multiple to 105Is obtained by doubling.
Further, when the fecal diluent is inoculated into a plurality of trimethylamine-added brain heart infusion broth (BHI broth) media and a plurality of trimethylamine-not-added brain heart infusion broth (BHI broth) media, the volume ratio of the fecal diluent to the liquid media is 1: 20 to 28. Preferably 1: 23-26, and more preferably 1: 25.
further, the anaerobic culture conditions were: anaerobic culture is carried out for 5 to 7 days at 37 ℃.
Further preferably, the culture is carried out anaerobically at 37 ℃ for 7 days.
Further, the method of extracting the DNA of the culture may be according to an extraction method well known in the art, for example, extraction using a commercially available kit.
Further, the 16S rDNAV4 universal upstream primer is shown in seq.id No. 1. The 16S rDNA V4 universal downstream primer is shown in SEQ ID No. 2.
Furthermore, 16S rDNA V4 universal primer is selected to amplify DNA of multiple cultures, and the amplification system is 20 mu L, so as to obtain multiple amplification products. The kit comprises 2 mu L of DNA template, 10 mu L of SYBR Green PCR Master Mix, 7.2 mu L of ddH2O 7.2 and 0.4 mu L of 10 mu M upstream primer and 10 mu M downstream primer. The amplification reaction conditions were as follows: (1) pre-denaturation at 94 ℃ for 2 min. (2) Denaturation at 94 ℃ for 30 seconds; annealing at 52 ℃ for 30 seconds; extension at 72 ℃ for 30 seconds; 30 cycles. (3) After extension at 72 ℃ for 5 minutes.
Further, sequencing data were analyzed by a BIPES analysis process, including differential analysis of the flora and Enterococcus distribution of the culture products of healthy human fecal dilutions in brain-heart leachate broth medium supplemented with trimethylamine and without trimethylamine, and it was preliminarily presumed that Enterococcus (Enterococcus) may be a genus capable of reducing the trimethylamine concentration.
Specifically, the flora distribution difference analysis of the culture products of the healthy human excrement diluent in the brain-heart leaching liquid broth culture medium added with trimethylamine and without the trimethylamine comprises the comparative analysis among various bacterial genera and the comparative analysis of the level of a single bacterial genus.
Further, the sequencing results of the culture products of the plurality of healthy human fecal dilutions in the trimethylamine-added brain heart leach solution broth culture medium are integrated to form data of a trimethylamine culture group, the sequencing results of the culture products of the plurality of healthy human fecal dilutions in the trimethylamine-not-added brain heart leach solution broth culture medium are integrated to form data of a control group, and comparative analysis between each genus and the level of a single genus are performed on the data of the trimethylamine culture group and the data of the control group.
OTUs (operational nucleic acid units) are often used in the culture-free analysis of microorganisms, and in general, an OTU can be defined as one OTU if the similarity between sequences, such as different 16S rDNA sequences, is higher than 97%, i.e. each OTU corresponds to a different bacterial (microbial) species. By OTU analysis, the abundance of the microorganism (species level) in the sample can be known.
It should be noted that this step can be omitted if the genus of bacteria capable of reducing the trimethylamine concentration in fresh feces of healthy persons is known.
S120, screening enterococcus faecalis capable of reducing the concentration of trimethylamine.
Specifically, screening enterococcus faecalis capable of reducing the concentration of trimethylamine comprises the operations of S121 to S125.
S121, diluting fresh excrement of healthy people by 103Multiple to 105And (5) doubling to obtain a diluent.
Further, preferably, fresh faeces of healthy persons are diluted 105And (5) doubling to obtain a diluent.
S123, inoculating the diluent to a brain-heart leachate agar medium by adopting a plate marking method, and carrying out anaerobic culture at 37 ℃ for 5-7 days to obtain a plurality of single colonies. The single colony is a colony formed by a single bacterium, the properties of the bacteria on the single colony are the same, the DNA and the cell stage are the same, the scientific research is convenient, and one single colony is generally a round point.
And S125, respectively extracting DNAs of the plurality of single colonies, respectively amplifying and determining 16S rDNA sequences of the plurality of single colonies, comparing the determination result with the 16S rDNA sequence of Enterococcus faecalis (Enterococcus faecalis), and identifying the bacteria with the similarity of more than 97 percent with the 16S rDNA sequence of the Enterococcus faecalis (Enterococcus faecalis) as the Enterococcus faecalis.
The use of an embodiment of the aforementioned enterococcus faecalis for reducing trimethylamine concentration.
The use of an embodiment of the aforementioned enterococcus faecalis for reducing the concentration of trimethylamine oxide in blood.
The use of enterococcus faecalis according to one embodiment for the preparation of a functional food for reducing trimethylamine concentration.
An application of the enterococcus faecalis of an embodiment in preparing a medicament for preventing or treating cardiovascular diseases.
It was confirmed that the enterococcus faecalis described above can reduce the concentration of trimethylamine. Therefore, the enterococcus faecalis can be applied to the reduction of the concentration of trimethylamine, for example, the preparation of a medicament for treating kidney diseases, a medicament for treating trimethylaminuria and a medicament for preventing or treating cardiovascular diseases. Of course, the enterococcus faecalis described above may also be used in the preparation of functional foods.
One embodiment of a composition for reducing trimethylamine concentration comprises enterococcus faecalis as described above.
In one embodiment, the composition for reducing the concentration of trimethylamine is a tablet, an aqueous emulsion, a powder or a paste.
In one embodiment, the composition for reducing the concentration of trimethylamine further comprises probiotics.
Further, the probiotic bacteria are selected from at least one of clostridium butyricum, lactobacillus and bifidobacterium.
The enterococcus faecalis can generate a health effect by being matched with probiotics, so that the microecological balance of a host is improved, and a beneficial effect is played.
Of course, in other embodiments, the trimethylamine concentration reducing composition is not limited to inclusion of probiotics and may include other health benefits.
One embodiment of the composition for reducing the concentration of trimethylamine oxide in blood comprises enterococcus faecalis as described above.
The medicament for preventing cardiovascular diseases of an embodiment comprises the enterococcus faecalis.
The medicament for treating cardiovascular diseases comprises the enterococcus faecalis.
The functional food for preventing cardiovascular diseases according to an embodiment includes the enterococcus faecalis described above.
In one embodiment, the functional food is a capsule, a tablet, a lotion, a powder, an oral liquid, or a granule.
The normal serum TMAO content is maintained at a low level, and when the serum concentration of TMAO is remarkably increased, excessive TMAO in blood vessels can promote atherosclerosis of human bodies and increase the risk of heart disease.
Therefore, the medicine for preventing or treating cardiovascular diseases, which comprises the enterococcus faecalis, can reduce the concentration of TMA, thereby reducing TMAO in blood, and can reduce the risk of cardiovascular diseases or treat cardiovascular diseases.
The following are specific examples.
In the following examples, unless otherwise specified, the experimental procedures without specifying the specific conditions are generally carried out according to conventional conditions, for example, the conditions described in the molecular cloning's Experimental guidelines [ M ] (Beijing: scientific Press, 1992) by Sammbruke, EF Friech, T Mannich, et al (decoded by gold winter goose, Rimeng maple, et al) or the procedures recommended by the manufacturers of the kits. The reagents used in the examples are all commercially available.
Materials used in the examples: fresh feces of healthy people come from the clinical laboratory of Zhujiang Hospital, a bacterial genome DNA magnetic bead extraction kit is purchased from Shenzhen Yirui biotechnology, Inc., Sprague Dawley rats are purchased from southern medical university laboratory animal center, high-fat feed is purchased from Guangdong province medical laboratory animal center, brain heart leachate broth culture medium is purchased from Qingdao Haibo culture medium, Inc., and trimethylamine is purchased from Beijing Bailingwei science and technology, Inc.
Example 1
(1) Randomly selecting fresh feces of 11 healthy people, and configuring 1g of fresh feces into 10 g of fresh feces5The diluent is marked as a fecal sample 1 to a fecal sample 11, and the volume ratio of the fecal diluent to the liquid culture medium is 1: 25 inoculating to brain heart extractive solution containing trimethylamine 4g/LBroth (BHI broth) medium and BHI broth medium without added trimethylamine.
(2) Placing a plurality of the two culture media in an anaerobic box (10% H)2，5％CO2，85％N2) Anaerobic culture at 37 deg.C for 7 days to obtain multiple culture products.
(3) Extracting the DNAs of a plurality of culture products by using a magnetic bead extraction kit for bacterial genomic DNAs of Shenzhen Yirui biotechnology Limited, wherein the specific operation method is carried out according to the instruction of the extraction kit for bacterial genomic DNAs.
(4) And (3) selecting a 16S rDNA V4 universal primer, and respectively amplifying 16S rDNA V4 fragments of a plurality of culture products to obtain a plurality of amplification products. Wherein, the 16S rDNA V4 universal upstream primer is shown in SEQ.ID.NO. 1. The 16S rDNA V4 universal downstream primer is shown in SEQ.ID. NO.2, and the 16S rDNA V4 universal primer is synthesized by Shanghai Biotechnology Co., Ltd. PCR corresponds to the information of the corresponding barcode upstream and downstream primers for each DNA sample. Each amplification system is 20 μ L, which comprises 2 μ L of DNA template, 10 μ L of SYBR Green PCR Master Mix, 7.2 μ L of dd H2O 7.2, and 0.4 μ L of 10 μ M upstream and downstream primers. The amplification reaction conditions were as follows: (1) pre-denaturation at 94 ℃ for 2 min. (2) Denaturation at 94 ℃ for 30 seconds; annealing at 52 ℃ for 30 seconds; extension at 72 ℃ for 30 seconds; 30 cycles. (3) After extension at 72 ℃ for 5 minutes.
(5) Sequencing a plurality of amplification products, and analyzing the distribution difference of the flora and enterococcus of cultures of 11 healthy human excrement diluents in brain heart leaching solution broth culture medium added with trimethylamine and without the trimethylamine by adopting a BIPES analysis process on the sequencing result.
Wherein, the distribution difference analysis of the culture product of the healthy human excrement diluent in the brain heart leaching liquid broth culture medium added with trimethylamine and without added with trimethylamine among the genera is specifically as follows: sequencing results of the culture products of 11 dilutions of feces from healthy persons in the trimethylamine-added brain heart infusion broth culture medium and the trimethylamine-not-added brain heart infusion broth culture medium were analyzed, and the analysis results are shown in fig. 1.
As can be seen from FIG. 1, the enterococcus was more abundant in the trimethylamine-cultured group, indicating that the enterococcus was enriched with trimethylamine.
Wherein, the analysis of the relative abundance difference of the culture products of the healthy human excrement diluent in the brain-heart leaching liquid broth culture medium added with trimethylamine and without trimethylamine at the level of the bacterial genus is specifically as follows: the sequencing results of 11 samples in brain heart leachate broth culture medium with and without trimethylamine added were compared and analyzed. FIGS. 2 to 14 are graphs showing comparison of results between Enterococcus (Enterococcus), hydrogenphagostimula (Hydrogenophaga), Rhynchophorus (Vagococcus), Bacillus (Bacillus), Brevibacterium curvatum (Varibacterium), Blautia (Blautia), Alkaliphilus (Alkaliphilus), Plesiomonas (Plesiomonas), Facklamia, Lactobacillus (Lactobacillus), Egghellla, Campylobacter (Campylobacter) and Veilonella in a control group (trimethylamine was not added to the brain leach broth culture medium) and a trimethylamine culture group (trimethylamine was added to the brain leach broth culture medium).
As can be seen from fig. 2 to 14, the control groups of Enterococcus (Enterococcus), hydrogenphagostimula (Hydrogenophaga), loitering coccus (Vagococcus), Bacillus (Bacillus), brevibacterium (Varibaculum), Blautia (Blautia), alcaliophilus (alkalophilus), paramonas (Plesiomonas), Facklamia, Lactobacillus (Lactobacillus), eggerella, Campylobacter (Campylobacter), and Veillonella (Veillonella) were significantly different from the trimethylamine culture group in relative abundance, and the trimethylamine culture group was higher than the control group.
The method for analyzing the relative abundance of the enterococcus in the culture samples of the trimethylamine group and the control group comprises the following specific steps: sequencing results of culture products of 11 fecal samples after anaerobic culture in brain-heart leachate broth culture medium added with trimethylamine and without trimethylamine were analyzed, and relative abundance of enterococcus was analyzed and compared. The analysis results are shown in fig. 15 to 25.
As can be seen from fig. 15 to 25, the total amount of the enterococcus OTUs in the fresh fecal dilutions was significantly different between the control group (no trimethylamine was added to the brain heart infusion broth culture medium) and the trimethylamine culture group (trimethylamine was added to the brain heart infusion broth culture medium). Of the 11 samples, 9 samples of the trimethylamine culture group had a higher total amount of OTUs than the control group, and were significantly different.
Example 2
(1) Randomly selecting 2 healthy people, and disposing 1g of feces into 105The dilutions were then inoculated separately in 100. mu.l each on XX mL BHI agar medium and placed in an anaerobic chamber (10% H)2，5％CO2，85％N2) And carrying out anaerobic culture at the medium temperature of 37 ℃ for 7 days to obtain a culture to be screened.
(2) And (3) purifying the culture to be screened by adopting a plate-streaking method until only a single colony of one form grows on the BHI agar culture medium.
(3) Respectively picking a plurality of single colonies to extract DNA of the single colonies, respectively amplifying and measuring 16S rDNA sequences of the single colonies, comparing the measurement result with the 16S rDNA sequence of Enterococcus faecalis (Enterococcus faecalis), and screening 1 strain of Enterococcus faecalis, wherein the measurement result is the same as the 16S rDNA sequence of the Enterococcus faecalis (Enterococcus faecalis). The enterococcus faecalis was deposited at the Guangdong province culture Collection (GDMCC) at 23/4/2018, address: guangzhou city, Jielizhou 100 Dazhou 59 floor 5, with the number of GDMCC NO:60361, classified name: enterococcus faecalis TMAO-D.
Example 3
The procedure of example 3 is substantially the same as that of example 2, except that in step (3), 16S rDNA sequences of a plurality of single colonies are determined and then aligned with 16S rDNA sequences of Enterococcus casseliflavus (Enterococcus casseliflavus), and 1 strain of Enterococcus casseliflavus is selected.
Example 4
The procedure of example 4 is substantially the same as that of example 2, except that in step (3), 16S rDNA sequences of a plurality of single colonies are determined, and then the determined sequences are aligned with 16S rDNA sequences of Enterococcus durans (Enterococcus durans) to screen 1 strain of Enterococcus durans.
Example 5
The procedure of example 5 is substantially the same as that of example 2, except that in step (3), 16S rDNA sequences of a plurality of single colonies are determined, and then the determined sequences are compared with 16S rDNA sequences of Enterococcus faecium (Enterococcus faecalis), and 1 strain of Enterococcus faecium is selected.
Example 6
(1) In a 96-well plate, test medium (1 g K per liter test medium) was added in the distribution shown in FIG. 262HPO4、2.6g KH2PO4、1.4g MgSO4·7H2O、0.2g NH4Cl, 0.25g KCl, 1g NaCl, 4g trimethylamine and 1g TTC), positive control medium (each liter of positive control medium comprises 1g K2HPO4、2.6g KH2PO4、1.4g MgSO4·7H2O、0.2g NH4Cl, 0.25g KCl, 1g NaCl, 4g glucose and 1g TTC) and negative control medium (per liter negative control medium comprising 1g K2HPO4、2.6g KH2PO4、1.4g MgSO4·7H2O、0.2g NH4Cl, 0.25g KCl, 1g NaCl and 1g TTC), 200. mu.L per well was added.
(2) Then, enterococcus faecalis obtained by screening in example 2, enterococcus casei obtained by screening in example 3, enterococcus firmus obtained by screening in example 4, and enterococcus faecium obtained by screening in example 5 were inoculated on the 96-well culture plate according to the distribution shown in fig. 26, wherein the OD600 of the bacterial liquid at the time of inoculation was 0.9, and the ratio of the bacterial liquid to the culture liquid in each well was 1: 50.
(3) after 48 hours of anaerobic culture at 37 ℃, the 96-well plate culture broth undergoes a color change. The results are shown in FIG. 26. In fig. 26: PC means positive control, NC means negative control, TM means detection culture, Ef1, Ef2 and Ef3 means parallel test groups of enterococcus faecalis obtained in example 2, that is, Ef1, Ef2 and Ef3 are all enterococcus faecalis obtained in example 2, E4 means enterococcus casei obtained in example 3, E5 means enterococcus durans obtained in example 4, and E6 means enterococcus faecium obtained in example 5. Because the metabolism indicator 2, 3, 5-Triphenyl Tetrazolium Chloride (TTC) is added in the culture solution, the oxidation state of the TTC is colorless, if living bacteria exist, the TTC can be reduced into insoluble red triphenylmethane by hydrogen under the action of bacterial dehydrogenase, so that the culture solution is changed into red from colorless (the actual color at the deep color is red because the picture is a black-and-white photo).
From the results in FIG. 26, it can be seen that enterococcus faecalis is metabolically active in the test medium and the positive control, and it was demonstrated that the enterococcus faecalis obtained in example 2 can decompose trimethylamine using trimethylamine as an energy source.
Example 7
Enterococcus faecalis of example 2, enterococcus casei of example 3, enterococcus firmus of example 4, and enterococcus faecium of example 5 were inoculated into a plurality of inorganic salt solutions each containing trimethylamine of 400. mu.g/mL as a sole carbon source, and the concentration and the amount of the inoculated solution were the same as those of example 6. Then, after anaerobic culture at 37 ℃ for 24 hours, the concentration of trimethylamine in each culture solution was measured by picric acid colorimetry. The results are shown in Table 1, wherein Enterococcus faecalis1, Enterococcus faecalis2 and Enterococcus faecalis3 are all the Enterococcus faecalis obtained in example 2.
TABLE 1
As is clear from Table 1, the trimethylamine concentration (average 209.03. mu.g/mL) in the culture broth containing enterococcus faecalis was significantly lower than that in the culture broth containing enterococcus casseliflavus, enterococcus firmus and enterococcus faecium (average 311.4. mu.g/mL). Further, the enterococcus faecalis obtained in example 2 was able to degrade trimethylamine.
Example 8
13 female Sprague Dawley rats of similar body weight and 12 weeks of age purchased at the southern medical university laboratory animal center were randomly divided into an experimental group (7) and a control group (6). The enterococcus faecalis liquid (OD600 ═ 0.9) obtained in example 2 was taken, and then rats in the experimental group were intervened by gavage, and gavage was performed once a day, with 1.5 ml per gavage. Rats in the control group were gavaged daily with an equal amount of PBS. All rats were gavaged daily with 53mg/kg choline chloride. All rats were fed free high-fat diet (high-fat diet purchased from Guangdong provincial medical laboratory animal center). All rat plasma was collected on day 1 and day 14 of the experiment using the ocular venous blood collection method, respectively (12 hours after fasting). All plasma samples were then centrifuged at 3000g for 20 minutes at 4 ℃ and serum was collected. TMAO concentration was determined by liquid mass spectrometry. The concentration of TMAO concentration is shown in fig. 27.
As can be seen in fig. 27, the plasma TMAO levels in the rats of the experimental group were significantly lower after 14 days of intervention, indicating that enterococcus faecalis is able to degrade TMA in vivo.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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Claims (10)

1. Enterococcus faecalis capable of reducing trimethylamine concentration, which is characterized in that the deposit number is GDMCC NO: 60361.
2. A preparation method of enterococcus faecalis capable of reducing trimethylamine concentration is characterized by comprising the following steps:
diluting fresh feces of healthy people 103Multiple to 105Doubling to obtain a diluent;
inoculating the diluent to a brain-heart leaching solution agar culture medium by adopting a plate marking method, and carrying out anaerobic culture at 37 ℃ for 5-7 days to obtain a plurality of single colonies; and
and respectively extracting DNA of a plurality of single colonies, amplifying and determining the 16S rDNA sequence of the single colonies, comparing the determination result with the 16S rDNA sequence of the enterococcus faecalis, and screening to obtain the enterococcus faecalis capable of reducing the concentration of trimethylamine.
3. Use of enterococcus faecalis according to claim 1 or obtainable by the process according to claim 2 for reducing the concentration of trimethylamine.
4. Use of enterococcus faecalis according to claim 1 or obtainable by the process according to claim 2 for the preparation of a functional food for reducing the concentration of trimethylamine.
5. Use of enterococcus faecalis according to claim 1 or obtainable by the process according to claim 2 for the preparation of a medicament for the prevention or treatment of cardiovascular diseases.
6. A composition for reducing the concentration of trimethylamine, which comprises the enterococcus faecalis of claim 1 or the enterococcus faecalis produced by the production method of claim 2.
7. A composition for reducing the concentration of trimethylamine oxide in blood, which comprises the enterococcus faecalis of claim 1 or the enterococcus faecalis produced by the production method of claim 2.
8. A functional food for preventing cardiovascular diseases, comprising the enterococcus faecalis of claim 1 or the enterococcus faecalis produced by the production method of claim 2.
9. The functional food for preventing cardiovascular disease according to claim 8, wherein the functional food is a capsule, a tablet, a lotion, a powder, an oral liquid, or a granule.
10. A medicament for preventing or treating cardiovascular diseases, comprising enterococcus faecalis according to claim 1 or the enterococcus faecalis prepared by the preparation method according to claim 2.
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