CN109112225A - Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise - Google Patents
Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise Download PDFInfo
- Publication number
- CN109112225A CN109112225A CN201811003754.7A CN201811003754A CN109112225A CN 109112225 A CN109112225 A CN 109112225A CN 201811003754 A CN201811003754 A CN 201811003754A CN 109112225 A CN109112225 A CN 109112225A
- Authority
- CN
- China
- Prior art keywords
- promise
- fermentation process
- beautiful
- screened
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the presence of microorganism or the determination techniques fields of type, key microorganisms in a kind of beautiful natural fermentation process of promise are disclosed to screen and the method for screening, using microorganism in the fermented juice of the beautiful fermentation process of promise as object, extracts the macro genome of bacterium in fermented juice and purify；After expanding variable region sequences, the reading of bacterium variable region sequences is completed using high-flux sequence, Quality Control is carried out to sequence using wide bioinformatics platform and completes species annotating, is determined the microbial species to play a crucial role in the beautiful natural fermentation process of promise.The present invention is easy, quickly and accurately qualitative, quantitative screens the core microorganism in the beautiful fermentation process of promise, detection resolution is high, the key microorganisms strain that the advantage established in fermentation can quickly be screened comprehensively, have " forward direction promotes ", for subsequent " carrying out artificial infection, controlledly spontaneous fermentation " beautiful to promise, to shorten fermentation period, the purposive yield for improving core compound, it is ensured that stablizing for fermentation quality provides reference data.
Description
Technical field
The invention belongs to the determination techniques fields more particularly to a kind of beautiful spontaneous fermentation of promise of the presence of microorganism or type
The method that key microorganisms are screened and screened in journey.
Background technique
Currently, the prior art commonly used in the trade is such thatNuo Li (Noni, Morinda citrifolia Linn) is again
Claim Morinda, morinda citrifolia, beach wood Morinda offcinalis How, Nuo Ni, genus rubia section morinda citrifolia category medicinal plant.It is flat to be mainly distributed on South Pacific
The ground such as all island in ocean, Australia, Southeast Asia and China Hainan Island, the Xisha Islands and Taiwan.Its fruit be spend more polymerization and
At collective fruits, Polynesian is used as food, drug has history in about 2000.In various internal and external researchs
In, the product from Morinda has shown that antithrombotic, hypoglycemic, anticancer, antibacterial activity, and removes free radical, adjusts
Cholesterol stimulates immune system, adjusts the multiple functions such as cell function and purification blood, a variety of to cancer, diabetes, gout etc.
Disease has preferable preventive and therapeutic action.Nuo Li is made of the nutrient for plants more than 227 kinds or more: mainly including a variety of ammonia
Base acid, vitamin, polyphenol compound (main component scopoletin), polysaccharide, organic acid, protein, alkaloid (main component
Iridoid).The beautiful Related product of promise has been produced in american commerce metaplasia early in the 1990s, is launched at present
Product category it is various, comprising: fermented juice, fresh juicing, fruit powder and fruit tea etc., Related product has become Europe and North America
Common functional health-care drink, wherein being in great demand the most with fermented juice, health-care efficacy is also declared most preferably.The beautiful fermented juice of promise is most
Common production method is that the processing method based on traditional Polynesian is improved: being original with mature Noni fruit
Material, the bad fruit such as removal scar, rotten, cleans natural air drying through artificial or machine, then fruit is put into sealing container and is protected from light
Spontaneous fermentation (2 months time was differed to 1 year).Correlative study shows: harvest stages are different, the nutrient composition content during promise is beautiful
Also it is not quite similar, phytochemical content is the abundantest when full maturity, and fermented juice yield and nutritional ingredient are also relatively high.
Production process does not add any chemicals, collects the fermentation liquid gradually oozed out naturally from pulp, or the side such as to squeeze, be centrifuged
Formula separates fruit juice with pulp, up to fermented juice after filtering.Morinda Citrifolia juice after fermentation can effectively increase useful microbial body
The quality and quantity of (enzyme, yeast and mushroom etc.) has stronger function than original fruit juice to enhance the bioactivity of effective component
Effect.Traditional fermentation methods are although simple and easy, but the process-cycle is long, the control of fermentation period difficulty, is unable to control microbial contamination, produces
Product quality is unstable.The beautiful fermentation of promise is substantially the multiple-microorganisms such as fruit itself endophyte, surface bacterium, environmental microorganism
Mutually regulation, coefficient as a result, flora is abundant in fermented juice, bacterium is mutually complicated, forms an ingredient multiplicity and has
The Tiny ecosystem of several functions composition, fermentation process not only include the interaction between microorganism itself, and there is also micro-
Biology and substrate between and the interaction between substrate and substrate.Since Fermentation Process of Parameter changes complexity, therefore ferment
Process is highly prone to living contaminants.Therefore, it to have breakthrough on the beautiful fermentation process of promise, just first has to make the beautiful nature of promise clear
Complicated biological community structure relationship in fermentation process is screened out to play in the beautiful natural fermentation process of promise on this basis and be dominated
The microorganism of effect and its ratio, the pass that the advantage established in fermentation is then screened by Selective agar medium, has " forward direction promotes "
Key microorganism fungus kind (dominant bacteria), to Noni fruit " carrying out artificial infection, controlledly spontaneous fermentation ", to shorten fermentation period.
Or after first raw material is sterilized, seeded with pure bacterium or combination strain (the crucial flora of leading fermentation) purposive raising core compound
Yield, it is ensured that the stabilization of fermentation quality.To reduce the probability of harmful microorganism pollution, shorten fermentation time, it is most important
That can ensure that product maintains constant quality, solve industry technology bottleneck, for it is whole promoted industry technology level establish it is solid
Theoretical basis, provide technological guidance for the healthy and sustainable development of the beautiful industry of promise, while being further research Morinda Citrifolia juice fermentation
Regulation Mechanism provides new thinking.And the method for traditional microbiologic population's identification based on pure culture is due to culture medium and culture
The limitation of condition only can partially disclose the microbial diversity in micro-ecological environment sample, can not objective system explaination group
Fall composition.It the appearance of two generation microarray datasets and reaches its maturity and is filled with one for the research in microecosystem microbiologic population field
Fresh blood is based on micro- life along with the rapid development of second generation sequencing technologies and advancing by leaps and bounds for bioinformatics technique
The research of aspect product biology community structure has become possibility.In known microecosystem, during the beautiful dynamic fermentation of promise
Biological community structure compare, human intestine's flora wants complicated more.However even if in such complicated microecosystem
In, the metagenomics technology based on two generation microarray datasets still can be coped with easily and accurately be dissected.Macro genome refers to spy
Determine the summation of whole microorganism hereditary substances in environment.Metagenomics research and utilization sequencing technologies are to all micro- in environmental sample
The genome of biology is measured, and form and function and metabolic pathway with analyzing the gene of micropopulation, interpretation micropopulation
The diversity and abundance of body explore the relationship between microorganism and environment and host, excavate and what research was new has specific function
Gene etc..It being compared with the traditional method, the macro genome research based on high-flux sequence is not necessarily to construct clone library, this avoids
In library construction process using host strain to sample clone caused by system deviation, simplify experimental implementation, improve
Efficiency is sequenced, to be greatly promoted the development of metagenomics.Therefore this technology is applied to its in the beautiful fermentation process of promise
The dynamic studies of fermented juice biological community structure are feasible.
In conclusion problem of the existing technology is:Traditional fermentation methods are although simple and easy, but the process-cycle
Length, the control of fermentation period difficulty, to be unable to control microbial contamination, product quality unstable.
Solve the difficulty and meaning of above-mentioned technical problem:And the method for traditional microbiologic population's identification based on pure culture
Due to the limitation of culture medium and condition of culture, the microbial diversity in micro-ecological environment sample only can be partially disclosed, it can not
The explaination Community composition of objective system.Nuo Li is the characteristic fruit in Hainan Province of China, is a kind of edible and has medical value
Tropical plants, the nutrition and health care with superelevation is worth, and Nuo Li is used to treat various diseases and have as health food super
Cross more than 2000 years history.The beautiful product of the main promise of our province at present is the beautiful fermented juice of promise, therefore the product quality of the beautiful fermented juice of promise
Just becoming influences the vital factor of the beautiful industry of Hainan Province's promise.But the current beautiful fermentation of promise also rests on traditional natural fermentation method
Level, therefore the obtained beautiful fermented juice quality of promise is irregular, and color distinction is larger.Therefore orientation is sent out using key microorganisms
Ferment method invents a whole set of the fast and accurately examination of key microorganisms and screening technique in the beautiful fermentation process of promise, is subsequent multiple
The research base allotted and can improve the beautiful fermented juice quality of promise, reduce production cost, the probiotics for being easily industrialized production
Plinth, therefore meaning is especially great.
Summary of the invention
In view of the problems of the existing technology, the present invention provides key microorganisms in a kind of beautiful natural fermentation process of promise to discriminate
Other and screening method.
The invention is realized in this way a kind of method that key microorganisms are screened and screened in beautiful natural fermentation process of promise,
Key microorganisms are screened in the beautiful natural fermentation process of promise and the method for screening is with micro- life in the fermented juice of the beautiful fermentation process of promise
Object is object, after frozen-thawed broken wall, extracts the macro genome DNA of bacterium and purifying in fermented juice with CTAB method；PCR amplification
After 16S rDNA sequence, the reading of bacterium variable region sequences is completed using high-flux sequence, using wide bioinformatics platform to sequence
Column carry out Quality Control and complete species annotation, play a crucial role from the beautiful natural fermentation process of promise determining in the level of microbial species
Microbial species.
Further, in the beautiful natural fermentation process of the promise key microorganisms screen and screening method the following steps are included:
Step 1 obtains bacterium macro genome DNA simultaneously using the method for frozen-thawed from the beautiful fermented juice enriched microorganism of promise
Purifying；
Step 2, PCR amplification complete the area using universal primer using the bacterial 16 S area rDNA V3-V4 as purpose segment
The amplification and purifying of segment；
Step 3 completes the sequence read of amplified fragments using the high-flux sequence instrument based on two generations sequencing principle；
Step 4 interprets measurement sequence by wide bioinformatics platform, completes the annotation of microbial species, simultaneously
Statistics Application method screens the microbial species to play a crucial role in the beautiful fermentation process of promise；
Step 5 screens the key microorganisms screened out using MRS selective medium.
Further, the step 1 specifically includes: increasing frozen-thawed before extraction and ceramic bead impact crumbling method is added to release
Cellular content is put, the extraction of CTAB method is reapplied, is examined after obtaining DNA with agarose gel electrophoresis and micro ultraviolet specrophotometer
Survey DNA mass.
Further, the step 2 specifically includes:
1) design of sample specific barcode primer: using primer-design software, and the variable region design sample V3-V4 is special
Property bar code high-flux sequence primer；
2) area bacterial 16 S rDNA V3-V4 expands: using mentioned microorganism macro genome DNA as template, with above-mentioned design
Sample specific barcode high-flux sequence primer carries out PCR amplification.
Further, the step 3 specifically includes: after PCR product is through electrophoresis detection and quantitatively, measuring using two generation high passes
Sequence platform carries out bridge-type PCR high-flux sequence to sample, and each sample ensures 20,000 high quality sequence of acquisition, can cover sample
The base sequence of 99% or more microbial information is constituted in product.
Further, the step 4 specifically includes: it is based on wide bioinformatics platform, long progress Quality Control is read to primitive sequencer,
It removes low quality sequence and retains high quality sequence；The high quality sequence of selection is compared with ncbi database Plays sequence
It is right, identify microbial species；Run Analysis of Microbial Diversity program, through selection representative series and with reference bacterium library ratio
To rear acquisition species diversity information, based on the beta diversity between Unifrac evolutionary distance analysis sample and different time is explained
The Community composition dynamic change of point sample；Weight and the variation rule of each strain are calculated using random forest machine learning model
Rule, screens out key microorganisms in fermentation process.
Further, the step 5 specifically includes: being carried out using MRS selective medium to the key microorganisms screened out
Screening.
In conclusion advantages of the present invention and good effect are as follows:The present invention provides one kind from the angle of microecosystem
Quickly screen the method for microorganism dynamic change in the beautiful fermentation process of promise.It is with microorganism in its fermented juice in the beautiful fermentation process of promise
Object, by extracting the macro genome DNA of bacterium and purifying in fermented juice.After expanding its 16S rDNA V3-V4 region sequence, answer
The reading that the trivial sequence of bacterial 16 S rDNA V3-V4 is completed with high throughput sequencing technologies, using wide bioinformatics platform to sequence
It carries out Quality Control and completes species annotation, based on the beta diversity between Unifrac evolutionary distance analysis sample and explain between difference
The Community composition dynamic change of time point sample, on this basis by the statistical method beautiful spontaneous fermentation of objective announcement promise comprehensively
The microbial species to play a crucial role in the process.The present invention can be easy, fast on the basis of cultivating without microorganism fungus kind
Speed, accurately qualitative, quantitative screen the core microorganism in the beautiful fermentation process of promise, intuitively to microorganism in the beautiful fermentation process of promise
Dynamic change is analyzed, and the method detection resolution is high, can quickly screen the advantage established in fermentation comprehensively, have " just
To promoting " key microorganisms strain (dominant bacteria), for subsequent " carry out artificial infection, controlledly spontaneous fermentation " beautiful to promise,
To shorten fermentation period, the purposive yield for improving core compound, it is ensured that stablizing for fermentation quality provides reference data.
The method of the present invention is as shown in table 1 compared with conventional method
Detailed description of the invention
Fig. 1 is the method stream that key microorganisms are screened and screened in the beautiful natural fermentation process of promise provided in an embodiment of the present invention
Cheng Tu.
Fig. 2 is the macro gene DNA electrophoretogram of microorganism in the beautiful fermented juice of promise provided in an embodiment of the present invention.
Fig. 3 is microorganism 16S rDNA V3-V4 region sequence PCR electrophoresis in the beautiful fermented juice of promise provided in an embodiment of the present invention
Figure.
Fig. 4 is that abundance figure is sequenced in microbial diversity in the beautiful fermented juice of promise provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Noni fruit of the present invention to acquisition from Hainan Region is fermented, and natural fermentation process can be precisely quickly finished
The examination and screening of middle key microorganisms provide microorganism preferred strain reference, shorten the beautiful nature of promise for the beautiful fermentation process of promise
The time of fermentation, and technological guidance is provided for the beautiful fermentation preliminary working industrialized development of promise.
As shown in Figure 1, in the beautiful natural fermentation process of promise provided in an embodiment of the present invention key microorganisms screen and screening
Method the following steps are included:
S101: from the beautiful fermented juice enriched microorganism of promise, bacterium macro genome DNA and pure is obtained using the method for frozen-thawed
Change；
It is variable to complete this using universal primer using the bacterial 16 S area rDNA V3-V4 as purpose segment for S102:PCR amplification
The amplification and purifying of area's segment；
S103: the sequence read of amplified fragments is completed in application based on the high-flux sequence instrument of two generations sequencing principle；
S104: measurement sequence is interpreted by wide bioinformatics platform, the annotation of microbial species is completed, answers simultaneously
The microbial species to play a crucial role in the beautiful fermentation process of promise are screened with statistical method；
S105: the key microorganisms screened out are screened using MRS selective medium.
Key microorganisms are screened in the beautiful natural fermentation process of promise provided in an embodiment of the present invention and the method for screening is specifically wrapped
Include following steps:
1, in the beautiful fermented juice of promise microorganism macro genome DNA extraction:
The key of application macro genomics technologies microorganisms group is that microorganism macro genome DNA extracts matter in sample
The superiority and inferiority of amount.Grope by early period and test, present invention discover that if it is desired to comprehensively obtaining all microorganisms of sample as far as possible
Inhereditary material needs to increase before extraction more strong frozen-thawed and adds ceramic bead impact crumbling method release cell content
Object is then extracted using CTAB method, uses agarose gel electrophoresis and micro UV spectrophotometer measuring DNA matter after obtaining DNA
Amount.
2, bacterium variable region expands in the beautiful fermented juice of promise:
The beautiful fermentation process mesocuticle microorganism of promise is mainly made of bacterium, and the directed toward bacteria present invention is quasi- to expand its 16S rDNA
V3-V4 region sequence carries out diversity analysis.1) primer-design software, design the design of sample specific barcode primer: are used
The area sample V3-V4 specific barcode high-flux sequence primer.2) area bacterial 16 S rDNA V3-V4 expands: with mentioned microorganism
Macro genome DNA is template, carries out PCR amplification with the sample specific barcode high-flux sequence primer of above-mentioned design.
3, pcr amplification product purifying, quantitative and pyrosequencing:
After PCR product is through electrophoresis detection and quantitatively, bridge-type PCR high pass is carried out to sample using two generation high-flux sequence platforms
Sequence is measured, each sample ensures 20,000 high quality sequence of acquisition, can cover the alkali of 99% or more microbial information in sample
Basic sequence is constituted.
4, wide bioinformatics platform analyzes microbial diversity:
Based on wide bioinformatics platform, Quality Control is carried out using the raw letter analysis software of open source is long to primitive sequencer reading first, is gone
Except low quality sequence retains high quality sequence.Analysis of Microbial Diversity program then is run on self-built raw letter platform, is passed through
It chooses representative series and obtains species diversity information after comparing with reference bacterium library, sample is analyzed based on Unifrac evolutionary distance
Beta diversity between product and the Community composition dynamic change for explaining different time points sample.Based on the above results, using random gloomy
Woods machine learning model calculates the weight and changing rule of each strain, screens out key microorganisms in fermentation process.
5, the screening of key microorganisms:
The key microorganisms screened out are screened using MRS selective medium.
Application effect of the invention is explained in detail below with reference to concrete application embodiment.
Application Example 1: the examination and screening of key microorganisms in the beautiful fermentation process of Hainan Province Wanning Area promise
1, in the beautiful fermented juice of promise microorganism macro genome DNA extraction:
To acquire 7 parts of mature beautiful fermented juices of promise from Hainan Province Wanning Area as research object, extract micro- in fermented juice
Increase more strong frozen-thawed before biological and add ceramic bead impact crumbling method release cellular content, then applies CTAB method
It extracts, the specific method is as follows: taking the beautiful fermented juice of 1.5mL promise in 1.5mL centrifuge tube, is immediately placed on fully charge in liquid nitrogen, takes
It is put into thawing (about 5min) in 65 DEG C of water-baths after out, multigelation 4 times, is incubated for 4h in 37 DEG C of constant-temperature tables, at room temperature
12000g is centrifuged 10min, collects supernatant and is transferred in another centrifuge tube.Supernatant and isometric chloroform are centrifuged in 12000g
10min (in order to make precipitating, water phase and organic phase layering), Aspirate supernatant, which is transferred in another centrifuge tube, carries out phenol chloroform
2 times, the sodium acetate through 0.1 times of volume, the ice isopropanol precipitating total DNA of 1 times of volume, then 70% ethanol washing precipitates 2 times, returns
It is molten spare.Agarose gel electrophoresis and micro UV spectrophotometer measuring DNA mass are used after obtaining DNA.The DNA gel of acquisition
Electrophoretogram is as shown in Figure 2:
2, the bacterial 16 S area rDNA V3-V4 expands in the beautiful fermented juice of promise:
Microorganism is mainly made of bacterium in the beautiful fermentation process of promise, and the directed toward bacteria present invention is quasi- to expand its 16S rDNA V3-
V4 region sequence carries out diversity analysis.Amplimer: using bacterium V3-V4 area's universal primer as amplimer, primer sequence are as follows:
338F(5′-ACTCCTACGGGAGGCAGCA-3′)；806R(5′-GGACTACHVGGGTWTCTAAT-3′).Amplification system: anti-
Answer 100 μ L:10 × PCR Buffer of system, 10 μ L, 25mmo1/L Mg2+2.0 μ L, 2.5mmol/L dNTPs 8 μ L, 5mmol/
L upstream and downstream primer each 4 μ L, DNA profiling 100ng or so, 5U/ μ L Taq enzyme (Promega) 1.5uL, dd H2O complement to 100 μ
L.Amplification condition: 94 DEG C of denaturation 5min；Recycle 30 times: 94 DEG C of denaturation 1min, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, then 72
DEG C extend 7min, 4 DEG C preservation.PCR electrophoresis is carried out to amplified production, electrophoretogram such as Fig. 3:
3, pcr amplification product purifying, quantitative and pyrosequencing:
After PCR product is through electrophoresis detection and quantitatively, bridge-type PCR high pass is carried out to sample using two generation high-flux sequence platforms
Sequence is measured, each sample ensures 20,000 high quality sequence of acquisition, can cover the alkali of 99% or more microbial information in sample
Basic sequence is constituted.The sequencing abundance of sample is as shown in Figure 4 after sequencing:
4, wide bioinformatics platform analyzes microbial diversity:
Based on wide bioinformatics platform, Quality Control is carried out using the raw letter analysis software of open source is long to primitive sequencer reading first, is gone
Except low quality sequence retains high quality sequence.Analysis of Microbial Diversity program then is run on self-built raw letter platform, is passed through
It chooses representative series and obtains species diversity information after comparing with reference bacterium library, sample is analyzed based on Unifrac evolutionary distance
Beta diversity between product and the Community composition dynamic change for explaining different time points sample.Based on the above results, using random gloomy
Woods machine learning model calculates the weight and changing rule of each strain, screens out key microorganisms in fermentation process.Pass through
It annotates, microbial diversity and its superior microorganism Pseudomonas are as shown in table 2 in the beautiful fermentation process of promise:
Superior microorganism Pseudomonas in the beautiful fermentation process of 2 promise of table
5, the screening of key microorganisms: the key microorganisms screened out are screened using MRS selective medium.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. a kind of method that key microorganisms are screened and screened in beautiful natural fermentation process of promise, which is characterized in that the Nuo Li is certainly
Key microorganisms are screened in right fermentation process and the method for screening is using microorganism in the fermented juice of the beautiful fermentation process of promise as object, liquid
After nitrogen freeze thawing broken wall, the macro genome DNA of bacterium and purifying in fermented juice are extracted with CTAB method；PCR amplification 16S rDNA sequence
Afterwards, the reading that bacterium variable region sequences are completed using high-flux sequence carries out Quality Control simultaneously to sequence using wide bioinformatics platform
Species annotation is completed, the microbial species to play a crucial role from the beautiful natural fermentation process of promise determining in the level of microbial species.
2. key microorganisms are screened in the beautiful natural fermentation process of promise as described in claim 1 and the method for screening, feature exist
In, in the beautiful natural fermentation process of promise key microorganisms screen and screening method the following steps are included:
Step 1 obtains bacterium macro genome DNA and pure using the method for frozen-thawed from the beautiful fermented juice enriched microorganism of promise
Change；
Step 2, PCR amplification complete area's segment using universal primer using the bacterial 16 S area rDNA V3-V4 as purpose segment
Amplification and purifying；
Step 3 completes the sequence read of amplified fragments using the high-flux sequence instrument based on two generations sequencing principle；
Step 4 interprets measurement sequence by wide bioinformatics platform, completes the annotation of microbial species, apply simultaneously
Statistical method screens the microbial species to play a crucial role in the beautiful fermentation process of promise；
Step 5 screens the key microorganisms screened out using MRS selective medium.
3. key microorganisms are screened in the beautiful natural fermentation process of promise as claimed in claim 2 and the method for screening, feature exist
In the step 1 specifically includes: increase frozen-thawed before extraction and adds ceramic bead impact crumbling method release cellular content,
The extraction of CTAB method is reapplied, uses agarose gel electrophoresis and micro UV spectrophotometer measuring DNA mass after obtaining DNA.
4. key microorganisms are screened in the beautiful natural fermentation process of promise as claimed in claim 2 and the method for screening, feature exist
In the step 2 specifically includes:
1) primer-design software, the variable region design sample V3-V4 specificity item the design of sample specific barcode primer: are used
Shape code high-flux sequence primer；
2) area bacterial 16 S rDNA V3-V4 expands: using mentioned microorganism macro genome DNA as template, with the sample of above-mentioned design
Specific barcode high-flux sequence primer carries out PCR amplification.
5. key microorganisms are screened in the beautiful natural fermentation process of promise as claimed in claim 2 and the method for screening, feature exist
In the step 3 specifically includes: after PCR product is through electrophoresis detection and quantitatively, using two generation high-flux sequence platforms to sample
Bridge-type PCR high-flux sequence is carried out, each sample ensures 20,000 high quality sequence of acquisition, can cover 99% or more in sample
The base sequence of microbial information is constituted.
6. key microorganisms are screened in the beautiful natural fermentation process of promise as claimed in claim 2 and the method for screening, feature exist
In the step 4 specifically includes: being based on wide bioinformatics platform, read long progress Quality Control to primitive sequencer, remove low quality sequence
Column retain high quality sequence；The high quality sequence of selection is compared with ncbi database Plays sequence, identifies micro- life
Object species；Analysis of Microbial Diversity program is run, obtains species after choosing representative series and comparing with reference bacterium library
Diversity information based on the beta diversity between Unifrac evolutionary distance analysis sample and explains time point sample between difference
Community composition dynamic change；The weight and changing rule of each strain are calculated using random forest machine learning model, are screened
Key microorganisms during fermenting out.
7. key microorganisms are screened in the beautiful natural fermentation process of promise as claimed in claim 2 and the method for screening, feature exist
In the step 5 specifically includes: being screened using MRS selective medium to the key microorganisms screened out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811003754.7A CN109112225A (en) | 2018-08-30 | 2018-08-30 | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811003754.7A CN109112225A (en) | 2018-08-30 | 2018-08-30 | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109112225A true CN109112225A (en) | 2019-01-01 |
Family
ID=64861602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811003754.7A Pending CN109112225A (en) | 2018-08-30 | 2018-08-30 | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109112225A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109949863A (en) * | 2019-02-18 | 2019-06-28 | 贵州茅台酒股份有限公司 | A method of spirit quality is identified based on Random Forest model |
| CN112608986A (en) * | 2020-12-18 | 2021-04-06 | 河北农业大学 | Method for screening dominant microorganisms in fermentation process of livestock and poultry manure |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
| CN114350574A (en) * | 2022-03-07 | 2022-04-15 | 海南大学 | Novel acetobacter strain AN02 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717828A (en) * | 2009-12-14 | 2010-06-02 | 内蒙古农业大学 | Method for rapidly detecting kinds of lactobacillus in fermented dairy product |
| CN106434914A (en) * | 2016-09-26 | 2017-02-22 | 海南大学 | Key microbial functional genome detection method in pepper peeling process |
| CN107236820A (en) * | 2017-08-03 | 2017-10-10 | 海南大学 | A kind of isolation and identification method of the beautiful endogenetic fungus of promise |
-
2018
- 2018-08-30 CN CN201811003754.7A patent/CN109112225A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717828A (en) * | 2009-12-14 | 2010-06-02 | 内蒙古农业大学 | Method for rapidly detecting kinds of lactobacillus in fermented dairy product |
| CN106434914A (en) * | 2016-09-26 | 2017-02-22 | 海南大学 | Key microbial functional genome detection method in pepper peeling process |
| CN107236820A (en) * | 2017-08-03 | 2017-10-10 | 海南大学 | A kind of isolation and identification method of the beautiful endogenetic fungus of promise |
Non-Patent Citations (3)
| Title |
|---|
| LINZHANG等: "Metatranscriptomic approach reveals the functional and enzyme dynamics of core microbes during noni fruit fermentation", 《FOOD RESEARCH INTERNATIONAL》 * |
| 张哲等: "内蒙古传统发酵驼乳中乳酸菌和酵母菌的分离鉴定及其生物多样性分析", 《中国食品学报》 * |
| 秦文等主编: "《食品科学导论》", 30 September 2011, 中国质检出版社 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109949863A (en) * | 2019-02-18 | 2019-06-28 | 贵州茅台酒股份有限公司 | A method of spirit quality is identified based on Random Forest model |
| CN109949863B (en) * | 2019-02-18 | 2023-05-26 | 贵州茅台酒股份有限公司 | Method for identifying Daqu quality based on random forest model |
| CN112608986A (en) * | 2020-12-18 | 2021-04-06 | 河北农业大学 | Method for screening dominant microorganisms in fermentation process of livestock and poultry manure |
| CN114334003A (en) * | 2021-12-22 | 2022-04-12 | 中国水产科学研究院南海水产研究所 | Fermented golden pomfret deep learning quality discrimination method and system based on single molecule sequencing |
| CN114350574A (en) * | 2022-03-07 | 2022-04-15 | 海南大学 | Novel acetobacter strain AN02 and application thereof |
| CN114350574B (en) * | 2022-03-07 | 2023-04-28 | 海南大学 | Novel acetobacter AN02 strain and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109112225A (en) | Key microorganisms are screened and the method for screening in a kind of beautiful natural fermentation process of promise | |
| Aregbe et al. | Effect of different pretreatment on the microbial diversity of fermented potato revealed by high-throughput sequencing | |
| CN107227278A (en) | A kind of Lactobacillus plantarum A11 and its application | |
| CN109996885A (en) | Exocellular polysaccharide of lactic acid bacteria and application thereof | |
| CN109554318A (en) | Gluconic acid acetobacter and its application in a kind of fermented tea | |
| CN106434914A (en) | Key microbial functional genome detection method in pepper peeling process | |
| Lana et al. | Molecular insights into the effects of rootstocks on maturation of blood oranges | |
| CN110343625B (en) | Saccharomyces cerevisiae strain and application thereof | |
| CN102174414B (en) | Application of new morchella costata M8-13 liquid fermentation substance in development of health-care products and medicaments | |
| de Aguiar Carraro et al. | Identification and characterization of Colletotrichum species associated with anthracnose on persimmon in Brazil | |
| Miles et al. | Novel pathotypes of Elsinoë australis associated with Citrus australasica and Simmondsia chinensis in Australia | |
| Kumla et al. | Survey of edible Amanita in Northern Thailand and their nutritional value, total phenolic content, antioxidant and α-glucosidase inhibitory activities | |
| CN111449239A (en) | Functional food additive of ganoderma lucidum fermented sea buckthorn seed meal and preparation method thereof | |
| CN102523910B (en) | Novel antrodia cinnamomea bacteria for generating high-content triterpenoid | |
| CN102433290B (en) | Strain for producing citrulline and method for biologically synthesizing citrulline with same | |
| CN108379284A (en) | Purposes of the black tiger palm dietary fiber extract in preparing treatment and/or preventing intestinal bacilli illness relevant disease preparation | |
| CN114350527A (en) | Separation and purification method of endophytic fungi in overground part tissue of Vernonia anthelmintica and application of endophytic fungi | |
| CN104830695B (en) | Endophyte with induction Hairy Root Cultures of Salvia miltiorrhiza phenolic acid summation and application thereof | |
| KR20070042384A (en) | Process for preparing ginseng vinegar | |
| CN114395509A (en) | Bacillus WYJ-E14 separated from Curcuma wenyujin and its application in preparing antineoplastic agent | |
| KR101514436B1 (en) | Method of identifying Pseudoalteromonas sp. | |
| CN104593267B (en) | Monascus purpureus and its application in 1 DNJ is prepared | |
| Brugnoli et al. | Zero-and Low-Alcohol Fermented Beverages: A Perspective for Non-Conventional Healthy and Sustainable Production from Red Fruits | |
| CN106399529A (en) | Molecular detection primer for banana cladosporium cucumerinum and detection method | |
| Zhang et al. | Isolating, Identifying, and Analyzing the Biological Characteristics of Pathogens Causing Postharvest Disease in Fresh Radix Astragali |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190101 |