CN107043714A - A kind of probiotics and preparation method thereof - Google Patents
A kind of probiotics and preparation method thereof Download PDFInfo
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- CN107043714A CN107043714A CN201611146623.5A CN201611146623A CN107043714A CN 107043714 A CN107043714 A CN 107043714A CN 201611146623 A CN201611146623 A CN 201611146623A CN 107043714 A CN107043714 A CN 107043714A
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- probiotics
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- 239000006041 probiotic Substances 0.000 title claims abstract description 57
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 241000894006 Bacteria Species 0.000 claims abstract description 57
- 239000007788 liquid Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 18
- 102000001621 Mucoproteins Human genes 0.000 claims description 16
- 108010093825 Mucoproteins Proteins 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 238000010790 dilution Methods 0.000 claims description 8
- 239000012895 dilution Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 241000702460 Akkermansia Species 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 5
- 210000004369 blood Anatomy 0.000 claims description 5
- 235000019219 chocolate Nutrition 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 238000012795 verification Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 235000013406 prebiotics Nutrition 0.000 claims description 2
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- 229910052760 oxygen Inorganic materials 0.000 claims 2
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- 229930003231 vitamin Natural products 0.000 abstract description 21
- 235000013343 vitamin Nutrition 0.000 abstract description 21
- 239000011782 vitamin Substances 0.000 abstract description 21
- 150000003722 vitamin derivatives Chemical class 0.000 abstract description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 18
- 230000004060 metabolic process Effects 0.000 abstract description 12
- 230000000813 microbial effect Effects 0.000 abstract description 7
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- 230000007812 deficiency Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 241000894007 species Species 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
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- 238000012360 testing method Methods 0.000 abstract description 3
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- 238000000746 purification Methods 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 229960002477 riboflavin Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
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- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical class [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
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- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003471 Vitamin B2 Natural products 0.000 description 1
- OMEBXAYMRCYOTB-UHFFFAOYSA-N [P].OCC(O)CO Chemical compound [P].OCC(O)CO OMEBXAYMRCYOTB-UHFFFAOYSA-N 0.000 description 1
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- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
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Classifications
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- 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
-
- 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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to microbial technology field, more particularly to a kind of probiotics and preparation method thereof.The invention provides a kind of probiotics, the deposit number of the probiotics is：GDMCC60085.Present invention also offers a kind of preparation method of above-mentioned probiotics, the preparation method is：Step 1: configuration bacterium solution；Step 2: PCR is expanded：Step 3: culture purified bacterium colony.Bent present invention also offers a kind of probiotics solid-state, the probiotics solid-state song is made by above-mentioned probiotics.In the technical scheme that the present invention is provided, it is sequenced and tests through full genome, obtained strain may participate in carbohydrate metabolism and fat metabolism, be a kind of probiotics with good potentiality while vitamin and short chain fatty acids acetic acid can also be produced.Meanwhile, the preparation method that the present invention is provided is simple to operation, is adapted to large-scale promotion application.Solve in the prior art, the technological deficiency that probiotics species is single, separation is complicated.
Description
Technical field
The invention belongs to microbial technology field, more particularly to a kind of probiotics and preparation method thereof.
Background technology
Probiotics, is the class active microorganism beneficial to host, is to be colonized in human body intestinal canal, in reproductive system, can produce
Raw definite health efficacy is so as to the active beneficial microorganism general name for improving host's microecological balance, playing beneficial effect.It is prebiotic
Bacterium plays an important role to host health, mainly including the following aspects：First, digestion of the host to nutriment is helped to inhale
Receive；2nd, the important substances such as vitamin, short chain fatty acids and antioxidant are produced；3rd, by suppressing the growth of harmful bacteria and clear
Help host to resist bacteriovirus infection except the toxin of harmful bacteria generation, lift the resistance of host.
At present, the probiotics that the mankind have found can generally be divided into three classes：Bacillus acidi lactici, Bifidobacterium and Gram-positive
Bacterium coccus.As the understanding to gut flora is more and more deep, majority research shows different bacteriums to different sick peoples
Play different effects, it is proposed that needs are separately cultured out the probiotics of a variety ofization this market demand.2004, by
Muriel Derrien et al. isolate Akkermansia and belong to first plant of bacterium Akkermansia muciniphila, researcher
Functional study to Akkermansia muciniphila finds that this plant of bacterium has improvement fat and patients with NIDDM
Metabolic condition, the effect with losing weight and reducing blood sugar.In the prior art, the bacterium of isolated Akkermansia category only has
This is a kind of by Akkermansia muciniphila, also more complicated for the separation method of such bacterium, limits probiotics
Further genralrlization application.
Therefore, a kind of probiotics and preparation method thereof is developed, for solving in the prior art, probiotics species is single,
Complicated technological deficiency is separated, becomes those skilled in the art's urgent problem to be solved.
The content of the invention
In view of this, the invention provides a kind of probiotics and preparation method thereof, for solving in the prior art, probiotics
The technological deficiency that species is single, separation is complicated.
The invention provides a kind of probiotics, the deposit number of the probiotics is：GDMCC60085.
Present invention also offers a kind of preparation method of above-mentioned probiotics, the preparation method is：
Step 1: configuration bacterium solution：Excrement dilutes to obtain dilution after being mixed with physiological saline, and the dilution is inoculated in viscous egg
In white fluid nutrient medium, bacterium solution is obtained after culture；
Step 2: PCR is expanded：Using the bacterium solution as template, enter performing PCR using for the primer that Akkermansia belongs to, choose
Select the bacterium solution in PCR reacting positive pipes to be inoculated on mucoprotein solid medium, bacterium colony is obtained after culture；
Step 3: culture purified bacterium colony.
Preferably, excrement concentration is 10-6g/ml, the dilution and the mucoprotein Liquid Culture in the dilution
The volume ratio of base is 1:9.
Preferably, the condition cultivated described in step one is：37 DEG C of Anaerobic culturels, the time cultivated described in step one is
4 days.
Preferably, it is described to include for the Akkermansia primers belonged to：Primer one and primer two；
The sequence of the primer one is CAGCACGTGAAGGTGGGGAC, and the sequence of the primer two is
CCTTGCGGTTGGCTTCAGAT。
Preferably, the condition cultivated described in step 2 is：37 DEG C of Anaerobic culturels, the time cultivated described in step one is
4 days.
Preferably, the culture purified bacterium colony obtains method and is：The colony inoculation, will be skilful after being cultivated on chocolate flat board
Colony inoculation on gram force flat board turns out bacterium colony in progress purifying on Colombia's blood plate
Preferably, the preparation method also includes：Sequence verification, the sequence verification method is in the culture purified bacterium colony
Carried out after step.
Bent present invention also offers a kind of probiotics solid-state, the probiotics solid-state song is made by above-mentioned probiotics.
Preferably, during the probiotics solid-state is bent, the number of viable of the probiotics is 109Cfu/g is bent.
In summary, the invention provides a kind of probiotics, the deposit number of the probiotics is：GDMCC60085.This
Invention additionally provides a kind of preparation method of above-mentioned probiotics, and the preparation method is：Step 1: configuration bacterium solution；Step 2:
PCR is expanded：Step 3: culture purified bacterium colony.Present invention also offers a kind of probiotics solid-state is bent, the probiotics solid-state it is bent by
Above-mentioned probiotics is made.In the technical scheme that the present invention is provided, it is sequenced and tests through full genome, obtained strain may participate in carbon water
Compound metabolism and fat metabolism, are that one kind has good dive while vitamin and short chain fatty acids acetic acid can also be produced
The probiotics of power.Meanwhile, the preparation method that the present invention is provided is simple to operation, is adapted to large-scale promotion application.Solve existing
The technological deficiency that in technology, probiotics species is single, separation is complicated.
Biological deposits explanation
Norzea, Classification And Nomenclature：Akkermansia sp., Guangdong Province's microbial bacteria is deposited on October 11st, 2016
Collection is planted, address is 5 building, the compound No. 59 of Xianlie Middle Road, Guangzhou City 100, and deposit number is GDMCC No.60085.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
The embodiment of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can also basis
The accompanying drawing of offer obtains other accompanying drawings.
Fig. 1 be bacterial clump metabolite in short-chain fat acid content block diagram.
Embodiment
The invention provides a kind of probiotics and preparation method thereof, for solving in the prior art, probiotics species is single,
Separate complicated technological deficiency.
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model that the present invention is protected
Enclose.
A kind of probiotics provided in order to which the present invention is described in more detail with reference to embodiment the present invention and its preparation side
Method, is specifically described.
Embodiment 1
The present embodiment is the specific embodiment for preparing probiotics.
1.1 mucoproteins are purified
1.1.1 get the raw materials ready
Weigh 0.2g anhydrous Nas H2PO4、7gNa2HPO4·12H2O and 6gNaCl are dissolved in deionized water, and regulation pH is 7.8,
Constant volume is to 1000ml, and A liquid is made in sterilizing 30min under the conditions of 125 DEG C of high pressure, and normal temperature is preserved.
5.85gNaCl is weighed in deionized water, regulation pH is 7.0, constant volume to 1000ml, gone out under the conditions of 125 DEG C of high pressure
B liquid is made in bacterium 30min, and normal temperature is preserved.
1.1.2 purifying
Weigh in 10~15g mucoprotein powder, the flask for being added to 500mlA liquid, the uniform stirring 2h on magnetic stirring apparatus
Afterwards, it is 7.2 ± 0.2 with 1M sodium hydroxides adjustment pH value of solution, 500~1000ul toluene is added dropwise, continues on magnetic stirring apparatus
Even stirring 18h, 3000rpm centrifuge 10min, collect supernatant into another high flask pressed through, discard precipitation, are added into solution
The absolute ethyl alcohol of precooling, it is about 60% to make its concentration.30min, 3000rpm centrifugation 10min are placed in 4 DEG C of refrigerators, precipitation is collected,
Supernatant is discarded, precipitation is dissolved in 200mlB liquid, after stirring 6h, 3000rpm centrifugation 10min collect supernatant and high pressed through to another
Flask in, discard precipitation, into solution add precooling absolute ethyl alcohol, make its concentration be 60%.Placed in 4 DEG C of refrigerators
30min, 3000rpm centrifuge 10min, collect precipitation, discard supernatant, precipitation is dissolved in 100ml distilled water, 4 DEG C of sealing preserves.
It is prepared by 1.2 liquid mucoprotein culture mediums
1.2.1 get the raw materials ready
The acid trace element of configuration：Weigh and/or measure 1.491gFeCl2·4H2O、0.06gH3BO4、
0.068gZnCl2、0.1725gCuCl2·7H2O、0.0635gMnCl2、0.0119gCoCl2·6H2O、0.0235gNiCl2·
6H2O and 4.18mlHCl, is dissolved in distilled water, is settled to 100ml.
Configuration alkalescence trace element：Weigh 0.01729gNa2SeO3、0.0242gNa2MoO4·2H2O and 0.4gNaOH, it is molten
In distilled water, 100ml is settled to.
Configure vitamin liquid：Weigh 0.02g biotins, 0.21g vitamin Bs3, 0.5g vitamin Bs6, 0.1g vitamin Bs2、
0.2g vitamin Bs1, 0.25g vitamin Bs12With pantothenic acid 0.1g, it is dissolved in distilled water, is settled to 100ml.
Weigh and/or measure 1.1gCaCl2、1.0gMgCl2, acidity trace element 1ml and alkaline trace element 1ml, distill
Water constant volume is to 100ml, and 1 liquid, normal temperature sealing preserve is made in sterilizing 30min under the conditions of 125 DEG C of high pressure.
Weigh 5.3gNa2HPO4, 3gNaCl and 4gKH2PO4It is dissolved in distilled water, is settled to 100ml, 125 DEG C of high pressure
2 liquid, normal temperature sealing preserve are made in the 30min that sterilized under the conditions of 30min, distilled water constant volume to 100ml, 125 DEG C of high pressure.
Weigh and/or measure 2gNaHCO3 and vitamin liquid 2ml, distilled water constant volume to 100ml, 0.22 μm of membrane filtration system
Obtain 3 liquid, 4 DEG C of sealing preserves.
Weigh 2.5gNa24 liquid, 4 DEG C of sealing preserves are made in S, distilled water constant volume to 100ml, 0.22 μm of membrane filtration.
1.2.2 culture medium is configured
Take 200ml sterile distilled waters to be contained in the high wide-mouth bottle pressed through, sequentially added in superclean bench 2ml 1 liquid,
2ml 2 liquid, 4 liquid of 1ml 3 liquid and 2ml and mucoprotein solution 30-40ml after purification, often add needs to shake after a kind of liquid
It is even.The liquid subpackage for drawing 5ml with electric pipettor covers tightly lid, 4 DEG C of sealing preserves in 15ml centrifuge tube.
It is prepared by 1.3 solid mucoprotein culture mediums
1.3.1 get the raw materials ready
The acid trace element of configuration：Weigh and/or measure 1.491gFeCl2·4H2O、0.06gH3BO4、
0.068gZnCl2、0.1725gCuCl2·7H2O、0.0635gMnCl2、0.0119gCoCl2·6H2O、0.0235gNiCl2·
6H2O and 4.18mlHCl, is dissolved in distilled water, is settled to 100ml.
Configuration alkalescence trace element：Weigh 0.01729gNa2SeO3、0.0242gNa2MoO4·2H2O and 0.4gNaOH, it is molten
In distilled water, 100ml is settled to.
Configure vitamin liquid：Weigh 0.02g biotins, 0.21g vitamin Bs3, 0.5g vitamin Bs6, 0.1g vitamin Bs2、
0.2g vitamin Bs1, 0.25g vitamin Bs12With pantothenic acid 0.1g, it is dissolved in distilled water, is settled to 100ml.
Weigh and/or measure 1.1gCaCl2、1.0gMgCl2, acidity trace element 1ml and alkaline trace element 1ml, distill
Water constant volume is to 100ml, and 1 liquid, normal temperature sealing preserve is made in sterilizing 30min under the conditions of 125 DEG C of high pressure.
Weigh 5.3gNa2HPO4, 3gNaCl and 4gKH2PO4It is dissolved in distilled water, is settled to 100ml, 125 DEG C of high pressure
2 liquid, normal temperature sealing preserve are made in the 30min that sterilized under the conditions of 30min, distilled water constant volume to 100ml, 125 DEG C of high pressure.
Weigh and/or measure 2gNaHCO3 and vitamin liquid 2ml, distilled water constant volume to 100ml, 0.22 μm of membrane filtration system
Obtain 3 liquid, 4 DEG C of sealing preserves.
Weigh 2.5gNa24 liquid, 4 DEG C of sealing preserves are made in S, distilled water constant volume to 100ml, 0.22 μm of membrane filtration.
1.3.2 culture medium is configured
2g agar is weighed in 200ml distilled water, after the 30min that sterilized under the conditions of 125 DEG C of high pressure, immediately in superclean bench
In the mucoprotein that sequentially adds made from 2ml 1 liquid, 2ml 2 liquid, 1ml 3 liquid, 2ml 4 liquid and step 1.1 after purification it is molten
30~40ml of liquid, often add needs to shake up after a kind of liquid；Pour into sterilized petri dishes, each plate 20ml；Solidification to be cooled is rearmounted
In in aseptic plastic bag, 4 DEG C of sealing preserves.
1.4 bacteria distribution
1.4.1 bacterium solution is configured
The fresh excrement of the mankind is collected, takes 1g excrement to add 9ml physiological saline and mixes, is carried out with 10 times of gradient continuous dilute
Release, it is 10 to take dilution factor-6Fecal suspension 1ml be inoculated in the mucoprotein fluid nutrient medium obtained by 9ml steps 1.2, in 37
DEG C Anaerobic culturel obtains bacterium solution in 4 days.
Human feces used in the present embodiment are gathered from healthy volunteer.
1.4.2 PCR is expanded
Bacterium solution after cultivating 3 days enters performing PCR as template using for the primer that Akkermansia belongs to；Wherein, draw
The sequence of thing one is CAGCACGTGAAGGTGGGGAC, and the sequence of primer two is CCTTGCGGTTGGCTTCAGAT.Select PCR anti-
Answer the bacterium solution in positive pipe to be inoculated in Anaerobic culturel on the mucoprotein solid medium obtained by step 1.3 and go out bacterium colony.
1.4.3 culture purified bacterium colony
Colony inoculation on mucoprotein solid medium is gone out into bacterium colony in Anaerobic culturel on chocolate flat board, chocolate is put down
Colony inoculation on plate turns out bacterium colony in progress purifying on Colombia's blood plate, takes the bacterium colony on Colombia's blood plate to lead to
Cross 16sRNA universal primers and enter performing PCR sequence verification.
For ease of describing, in the embodiment of the present invention, obtained purifying bacterium colony is named as NORZEA37.
Embodiment 2
The present embodiment participates in the specific embodiment of carbohydrate metabolism for the purifying bacterium colony obtained by checking embodiment 1.
2.1 DNA are extracted, are built storehouse and sequencing
Using the low dose of faeces DNA extracts kit of mouse (U.S. base is biological, Guangzhou), according to product description and ability
The all well known technological means of domain those of ordinary skill carries out DNA of bacteria extraction.
It is all ripe according to product description and those of ordinary skill in the art using TruSeq DNA sample reagent preparation boxes
The technological means known, builds double end 150bp, Insert Fragment length 250-300bp DNA libraries.
DNA sequencing is carried out using Illumina Hiseq2500 sequenators, and extracts high-quality sequencing fragment, is carried out
The follow-up works such as bacterial genomes assembling, annotation and icp gene group analysis.
2.2 genomes are assembled
Sequencing fragment is assembled using Velvet microbial genomes composite software, selected successively not in assembling process
Same Kmer parameters (between 35-145), and therefrom choose optimal assembling result.
Obtain after preliminary contig assembling results, contig extensions and scaffold structures are carried out using SSPACE softwares
Frame, then carries out room elimination using GapFiller to scaffold.The contig that length is less than 200bp is finally filtered out, and
Obtain final assembling result.
2.3 Gene correlation
Gene annotation is carried out using Prokka genome analysises software, Prokka is recognized using a series of forecasting tool
The characteristic area of genome and its position, including：1) using the small rRNA of RNAmmer software predictions (such as 5S, 16S and 23S
rRNA)；2) using Aragorn software predictions tRNA；And 3) using PILER-CR prediction CRISPR sequences.Prokka uses multiple
Step to carry out functional annotation to protein coding gene：1) using the encoding histone base in Prodigal software predictions assembling result
Cause and pseudogene site；2) with the function of homologous type strain ATGG BAA-835 protein coding gene then reference model bacterial strain
Annotation；3) remaining protein family is then searched for bacterioprotein information in UniProt and RefSeq databases and is compared.
After Prokka operations are finished, COG (the gene family work(that American National Bioinformatics Institute NCBI is provided is continuing with
Can database) and KEGG (capital of a country gene and genome are encyclopaedical) databases compare annotation gene function.Compare what is required
Parameter is：Exceed the 70% of the mrna length than upper length, and similarity is higher than 35%.Looked for more than in several databases
Genetic marker less than matching is hypothesis albumen.Gene correlation to carbohydrate inversion enzyme is to use CAZy (carbon aquations
Compound kinase database) annotated with same method.
According to Gene correlation result, find NORZEA37 have participate in galactose metabolism, fructose and sweet dew glycometabolism,
The gene of starch and Sucrose Metabolism.Therefore, NORZEA37 may participate in the metabolism of carbohydrate.
Embodiment 3
The present embodiment participates in the specific embodiment of fat composition metabolism for the purifying bacterium colony obtained by checking embodiment 1.
3.1 DNA are extracted, are built storehouse and sequencing
Using the low dose of faeces DNA extracts kit of mouse (U.S. base is biological, Guangzhou), according to product description and ability
The all well known technological means of domain those of ordinary skill carries out DNA of bacteria extraction.
It is all ripe according to product description and those of ordinary skill in the art using TruSeq DNA sample reagent preparation boxes
The technological means known, builds double end 150bp, Insert Fragment length 250-300bp DNA libraries.
DNA sequencing is carried out using Illumina Hiseq2500 sequenators, and extracts high-quality sequencing fragment, is carried out
The follow-up works such as bacterial genomes assembling, annotation and icp gene group analysis.
3.2 genomes are assembled
Sequencing fragment is assembled using Velvet microbial genomes composite software, selected successively not in assembling process
Same Kmer parameters (between 35-145), and therefrom choose optimal assembling result.Obtain after preliminary contig assembling results,
Contig extensions and scaffold frameworks are carried out using SSPACE softwares, then scaffold is carried out using GapFiller empty
Position is eliminated.Finally filter out length and be less than 200bp contig, and obtain final assembling result.
3.3 Gene correlation
Gene annotation is carried out using Prokka genome analysises software, Prokka is recognized using a series of forecasting tool
The characteristic area of genome and its position, including：1) using the small rRNA of RNAmmer software predictions (such as 5S, 16S and 23S
rRNA)；2) using Aragorn software predictions tRNA；And 3) using PILER-CR prediction CRISPR sequences.Prokka uses multiple
Step to carry out functional annotation to protein coding gene：1) using the encoding histone base in Prodigal software predictions assembling result
Cause and pseudogene site；2) with the function of homologous type strain ATGG BAA-835 protein coding gene then reference model bacterial strain
Annotation；3) remaining protein family is then searched for bacterioprotein information in UniProt and RefSeq databases and is compared.
After Prokka operations are finished, COG (the gene family work(that American National Bioinformatics Institute NCBI is provided is continuing with
Can database) and KEGG (capital of a country gene and genome are encyclopaedical) databases compare annotation gene function.Compare what is required
Parameter is：Exceed the 70% of the mrna length than upper length, and similarity is higher than 35%.Looked for more than in several databases
Genetic marker less than matching is hypothesis albumen.Gene correlation to carbohydrate inversion enzyme is to use CAZy (carbon aquations
Compound kinase database) annotated with same method.
According to Gene correlation result, it is found that NORZEA37 has and participate in fatty acid metabolism, glyceride metabolism, glycerine phosphorus
The gene of fat and sphingolipid metabolism.Therefore, NORZEA37 may participate in the metabolism of lipid.
Embodiment 4
The present embodiment can produce the specific embodiment of vitamin for the purifying bacterium colony obtained by checking embodiment 1.
4.1 DNA are extracted, are built storehouse and sequencing
Using the low dose of faeces DNA extracts kit of mouse (U.S. base is biological, Guangzhou), according to product description and ability
The all well known technological means of domain those of ordinary skill carries out DNA of bacteria extraction.
It is all ripe according to product description and those of ordinary skill in the art using TruSeq DNA sample reagent preparation boxes
The technological means known, builds double end 150bp, Insert Fragment length 250-300bp DNA libraries.
DNA sequencing is carried out using Illumina Hiseq2500 sequenators, and extracts high-quality sequencing fragment, is carried out
The follow-up works such as bacterial genomes assembling, annotation and icp gene group analysis.
4.2 genomes are assembled
Sequencing fragment is assembled using Velvet microbial genomes composite software, selected successively not in assembling process
Same Kmer parameters (between 35-145), and therefrom choose optimal assembling result.
Obtain after preliminary contig assembling results, contig extensions and scaffold structures are carried out using SSPACE softwares
Frame, then carries out room elimination using GapFiller to scaffold.The contig that length is less than 200bp is finally filtered out, and
Obtain final assembling result.
4.3 Gene correlation
Gene annotation is carried out using Prokka genome analysises software, Prokka is recognized using a series of forecasting tool
The characteristic area of genome and its position, including：1) using the small rRNA of RNAmmer software predictions (such as 5S, 16S and 23S
rRNA)；2) using Aragorn software predictions tRNA；And 3) using PILER-CR prediction CRISPR sequences.Prokka uses multiple
Step to carry out functional annotation to protein coding gene：1) using the encoding histone base in Prodigal software predictions assembling result
Cause and pseudogene site；2) with the work(of homologous type strain ATGG BAA-835 protein coding gene then reference model bacterial strain
Can annotation；3) remaining protein family is then searched for bacterioprotein information in UniProt and RefSeq databases and is compared.
After Prokka operations are finished, COG (the gene family work(that American National Bioinformatics Institute NCBI is provided is continuing with
Can database) and KEGG (capital of a country gene and genome are encyclopaedical) databases compare annotation gene function.Compare what is required
Parameter is：Exceed the 70% of the mrna length than upper length, and similarity is higher than 35%.Looked for more than in several databases
Genetic marker less than matching is hypothesis albumen.Gene correlation to carbohydrate inversion enzyme is to use CAZy (carbon aquations
Compound kinase database) annotated with same method.
Gene correlation finds that NORZEA37 has and participates in vitamin B1 (thiamine), vitamin B2 (riboflavin), dimension
Raw element B3 (nicotinic acid), vitamin B5 (pantothenic acid), vitamin B6, vitamin B7 (biotin) and the FA (base of folic acid synthesis
Cause.Therefore, NORZEA37 may participate in the synthesis of vitamin.
Embodiment 5
The present embodiment can produce the specific implementation of short chain fatty acids acetic acid for the purifying bacterium colony obtained by checking embodiment 1
Example.
5.1 DNA are extracted, are built storehouse and sequencing
Using the low dose of faeces DNA extracts kit of mouse (U.S. base is biological, Guangzhou), according to product description and ability
The all well known technological means of domain those of ordinary skill carries out DNA of bacteria extraction.
It is all ripe according to product description and those of ordinary skill in the art using TruSeq DNA sample reagent preparation boxes
The technological means known, builds double end 150bp, Insert Fragment length 250-300bp DNA libraries.
DNA sequencing is carried out using Illumina Hiseq2500 sequenators, and extracts high-quality sequencing fragment, is carried out
The follow-up works such as bacterial genomes assembling, annotation and icp gene group analysis.
5.2 genomes are assembled
Sequencing fragment is assembled using Velvet microbial genomes composite software, selected successively not in assembling process
Same Kmer parameters (between 35-145), and therefrom choose optimal assembling result.
Obtain after preliminary contig assembling results, contig extensions and scaffold structures are carried out using SSPACE softwares
Frame, then carries out room elimination using GapFiller to scaffold.The contig that length is less than 200bp is finally filtered out,
And obtain final assembling result.
5.3 Gene correlation
Gene annotation is carried out using Prokka genome analysises software, Prokka is recognized using a series of forecasting tool
The characteristic area of genome and its position, including：1) using the small rRNA of RNAmmer software predictions (such as 5S, 16S and 23S
rRNA)；2) using Aragorn software predictions tRNA；And 3) using PILER-CR prediction CRISPR sequences.Prokka uses multiple
Step to carry out functional annotation to protein coding gene：1) using the encoding histone base in Prodigal software predictions assembling result
Cause and pseudogene site；2) with the function of homologous type strain ATGG BAA-835 protein coding gene then reference model bacterial strain
Annotation；3) remaining protein family is then searched for bacterioprotein information in UniProt and RefSeq databases and is compared.
After Prokka operations are finished, COG (the gene family work(that American National Bioinformatics Institute NCBI is provided is continuing with
Can database) and KEGG (capital of a country gene and genome are encyclopaedical) databases compare annotation gene function.Compare what is required
Parameter is：Exceed the 70% of the mrna length than upper length, and similarity is higher than 35%.Looked for more than in several databases
Genetic marker less than matching is hypothesis albumen.Gene correlation to carbohydrate inversion enzyme is to use CAZy (carbon aquations
Compound kinase database) annotated with same method.
Gene correlation finds that NORZEA37 has the gene for participating in acetic acid synthesis.Therefore, NORZEA37 may participate in short
The synthesis of chain fatty acid.
Short chain fatty acids in 5.4 gas chromatographies identification opsonigenous substance
105Cfu NORZEA37 are inoculated in 500ml mucoprotein fluid nutrient medium, contain 1g in every liter of deionized water
The L- cysteine hydrochlorides of mucoprotein, the brain heart infusion of 37g pigs and 5mg, are placed in anaerobic culture box and cultivate 87 hours.
Collect culture supernatant and carry out following handle：Media samples about 180~200mg is weighed to be placed in 2mL EP pipes, plus
Enter 1mL distilled water suspension samples.Concussion 10min is vortexed again into homogenate, is then centrifuged (5000 × g) 10min at 4 DEG C, is taken supernatant
0.22 μm of filter membrane is crossed, 50% sulfuric acid of 1/10 volume is added into supernatant, 1min is shaken up.The ether for adding 2 times of volumes is light
Light mix (not vortex) extracts centrifugation (1000 × g) 5min at 20min, latter 4 DEG C, takes supernatant liquor to be analyzed.Use Agilent
7890 gas chromatographs are detected
It is specific to detect that parameter is, the specification of chromatographic column：DB-FFAP, column length 30m, internal diameter 0.32mm, thickness 0.25um,
230 DEG C of injector temperature, 250 DEG C of detector temperature, split ratio 10:1.The specific method of temperature programming is：100 DEG C of holdings
0.5min, rises to 170 DEG C with 8 DEG C/min and maintains 0.5min.Finally rise to 220 DEG C with 20 DEG C/min and maintain 2min.
Short-chain fat acid content in experiment gained bacterial clump metabolite see Fig. 1.
It can show that the bacterium obtained by technical scheme that the present invention is provided may participate in carbon from 2~embodiment of embodiment 5
Hydrate is metabolized and fat metabolism, while vitamin and short chain fatty acids acetic acid can also be produced, is further estimated, this
The obtained bacterium of invention, is a kind of probiotics with fat-reducing and anti-oxidation function.
In summary, the invention provides a kind of probiotics, the deposit number of the probiotics is：GDMCC60085.This
Invention additionally provides a kind of preparation method of above-mentioned probiotics, and the preparation method is：Step 1: configuration bacterium solution；Step 2:
PCR is expanded：Step 3: culture purified bacterium colony.Present invention also offers a kind of probiotics solid-state is bent, the probiotics solid-state it is bent by
Above-mentioned probiotics is made.In the technical scheme that the present invention is provided, it is sequenced and tests through full genome, obtained strain may participate in carbon water
Compound metabolism and fat metabolism, are that one kind has good dive while vitamin and short chain fatty acids acetic acid can also be produced
The probiotics of power.Meanwhile, the preparation method that the present invention is provided is simple to operation, is adapted to large-scale promotion application.Solve existing
The technological deficiency that in technology, probiotics species is single, separation is complicated.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of probiotics, it is characterised in that the deposit number of the probiotics is：GDMCC60085.
2. a kind of preparation method of probiotics as claimed in claim 1, it is characterised in that the preparation method is：
Step 1: configuration bacterium solution：Excrement dilutes to obtain dilution after being mixed with physiological saline, and the dilution is inoculated in mucoprotein liquid
In body culture medium, bacterium solution is obtained after culture；
Step 2: PCR is expanded：Using the bacterium solution as template, enter performing PCR using for the primer that Akkermansia belongs to, select
Bacterium solution in PCR reacting positive pipes is inoculated on mucoprotein solid medium, and bacterium colony is obtained after culture；
Step 3: culture purified bacterium colony.
3. preparation method according to claim 2, it is characterised in that excrement concentration is 10 in the dilution-6G/ml, institute
The volume ratio for stating dilution and the mucoprotein fluid nutrient medium is 1:9.
4. preparation method according to claim 2, it is characterised in that the condition cultivated described in step one is：37 DEG C are detested
Oxygen culture, the time cultivated described in step one is 4 days.
5. preparation method according to claim 2, it is characterised in that described to include for the Akkermansia primers belonged to：
Primer one and primer two；
The sequence of the primer one is CAGCACGTGAAGGTGGGGAC, and the sequence of the primer two is
CCTTGCGGTTGGCTTCAGAT。
6. preparation method according to claim 2, it is characterised in that the condition cultivated described in step 2 is：37 DEG C are detested
Oxygen culture, the time cultivated described in step one is 4 days.
7. preparation method according to claim 2, it is characterised in that the culture purified bacterium colony obtains method and is：The bacterium
Fall to be inoculated on chocolate flat board cultivate after, the colony inoculation on chocolate flat board is purified on Colombia's blood plate
Turn out bacterium colony.
8. preparation method according to claim 2, it is characterised in that the preparation method also includes：Sequence verification, it is described
Sequence verification method is carried out after the culture purified bacterium colony step.
9. a kind of probiotics solid-state is bent, it is characterised in that the probiotics solid-state song is as the probiotics system described in claim 1
.
10. probiotics solid-state according to claim 9 is bent, it is characterised in that described prebiotic during the probiotics solid-state is bent
The number of viable of bacterium is 109Cfu/g is bent.
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